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+{"text": "NIH3T3 cells transfected with c-H-ras and/or c-myc genes were examined for differences in drug sensitivity. The five transfectants used were N8, NIH3T3-nm-1, pT22-3-nm-2, pP1-4 and pT22-3. They were transfected with pKOneo alone, pKOneo and c-myc, pKOneo and c-myc plus activated c-H-ras, normal c-H-ras and activated c-H-ras genes, respectively. The IC50s of cisplatin, 4-hydroperoxycyclophosphamide, adriamycin, melphalan, and CPT-11 were significantly higher for NIH3T3-nm-1 abd pT22-3-nm-2 than for the parental NIH3T3 and N8 cells. Transfection with normal and activated C-H-ras oncogenes only led to increases in the IC50s of alkylating agents. There was no significant difference between the IC50s of N8 and those of NIH3T3 parental cells to any of these anticancer agents. These results strongly suggest that the expression of the c-myc gene plays a role in the acquisition of drug resistance. The c-myc gene may therefore provide us with an important clue in determining the mechanism of drug resistance."}
+{"text": "Previous studies using the yeast two-hybrid assay (Y2H) have identified cyclin L1 (CCNL1) and Ewing sarcoma breakpoint region 1 protein (EWSR1) as being interacting partners of tuftelin-interacting protein 11 (TFIP11). All three proteins are functionally related to the spliceosome and involved in pre-mRNA splicing activities. The spliceosome is a dynamic ribonucleoprotein complex responsible for pre-mRNA splicing of intronic regions, and is composed of five small nuclear RNAs (snRNAs) and \u03bc140 proteins. TFIP11 appears to play a role in spliceosome disassembly allowing for the release of the bound lariat-intron. The roles of CCNL1 and EWSR1 in the spliceosome are poorly understood. Using fluorescently-tagged proteins and confocal microscopy we show that TFIP11, CCNL1 and EWSR1 frequently co-localize to speckled nuclear domains. These data would suggest that all three proteins participate in a common cellular activity related to RNA splicing events. YLR424W gene in S. Cerevisiae) and shares about 30% amino acid similarity with mammalian TFIP11 proteins. The N-terminal region of TFIP11 contains a G-patch, which is a highly conserved domain of many RNA-processing proteins. In yeast, Ntr1 has been shown to interact directly with Prp43, an ATP-dependent RNA helicase -dCTP using the Prime-It II Random Prime Labeling Kit . Northern blot analysis was performed using standard methodologies [Northern Blot analyses were done using the FirstChoice Northern Human Blot 2 for multiple organs . Full-length cDNAs for human TFIP11, CCNL1 and EWSR1 were purchased from Origene . The 3\u2019 terminal regions of TFIP11, CCNL1 and EWSR1 cDNAs were released with Eco RV and Xba I, Xba I and Apa I, Xba I (which cut 2 times) respectively to give partial cDNAs of \u223c 420 bp, \u223c 380 bp and \u223c 500 bp which were gel purified and subsequently used as the template DNA for the radiolabeled probe. Note that for all three partial cDNAs, the Xba I site at the 3\u2019 terminus was part to the multicloning site of the host vector. The complete, 1.8 kb human \u03b2-actin (ACTB) cDNA was purchased from Clontech  and used as a control. Double-stranded cDNAs were labeled with [\u03b1-The TFIP11 cDNA, covering the entire open reading frame (ORF), was released from the construct TFIP11-N1 , and subATGGCGTCCGGGCCTC; and the reverse primer 3\u2019-GGCGCCTGTGCCTGCCATGTCCTG. The CCNL1 start codon is underlined. The PCR product was directionally subcloned into the vector pcDNA3.1/CT-GFP-TOPO  and the resulting plasmid is called CCNL1-N1.Full-length human CCNL1 cDNA was purchased from Origene . The entire ORF of CCNL1, covering 526 amino acids and minus its stop codon, was amplified by PCR using the forward primer 5\u2019-GATATCAAGACTCTCGAGGAGAAAATGGCGTCCACGGATTACAG; and reverse 5\u2019-TCCGCGGGTAGGGCCGATCTCTGCGCTCC which included an Xho I and a Sac II restriction site respectively (underlined). The purified PCR product was then directionally subcloned into the vector pDsRed-Express-N1 (Clontech) at the Xho I and Sac II multicloning site. The resulting plasmid is called EWSR1-Red-N1. The Myc-tagged EWSR1 vector was prepared by removing the EWSR1 cDNA from EWSR1-Red-N1 using Xho I and Sac II restriction enzymes, and subcloning this cDNA into pcDNA3.1/myc-His plasmid (Invitrogen Corporation) at the Xho I and Sac II multicloning site. This construct is called EWSR1-Myc.For EWSR1, a full-length human cDNA was generated by RT-PCR using total RNA from a human lung tumor . The primers used for PCR were forward 5\u2019-TAll plasmid construct cDNA inserts were subcloned by standard methodologies , and seqHEK293, HeLa, and LS8 cells  were maiCells were fixed with 4% paraformaldehyde for 5 minutes at room temperature (RT), permeabilized with 1% Triton X-100 for 10 minutes at RT, and washed with phosphate buffered saline (PBS). Cells were then incubated with primary antibodies for 1 hour at RT, washed with PBS three times, and incubated with secondary antibodies for 1 hour at RT. After incubation, cells were washed with PBS three times and mounted with VECTASHIELD medium . Confocal images were captured as previously described [Transfected cells were lysed with RIPA buffer . The cells were collected and sheared by passing through a 22-guage needle repeatedly. The cell suspension was collected by centrifugation at 14,000 rpm for 10 minutes. After centrifugation, the protein concentration of the supernatant was measured using a Bio-Rad protein assay kit . The cell lysate was pre-cleared by incubation with protein G agarose beads  for 1 hour at 4\u00b0C which was included to reduce the non-specific background. After pre-clearing, the cell lysate was incubated for 2 hours at 4\u00b0C with an anti-FLAG antibody conjugated to agarose beads (Sigma-Aldrich) using constant motion. Immunoprecipitates were then collected by centrifugation at 2,500 rpm for 5 minutes and washed three times with high-salt wash buffer (1M NaCl). The final pellet was resuspended in 2x SDS loading buffer, boiled for 4 minutes, and stored at \u221220\u00b0C. For Western blot analysis, immunoprecipitates and cell lysates were resolved by SDS-PAGE and transferred to Immobilon-P membrane . The membranes were incubated with anti-FLAG or anti-Myc antibodies. The protein-antibody complexes were visualized by enhanced chemiluminescence .Northern blot analysis was used to characterize TFIP11, CCNL1 and EWSR1 mRNA expression profiles in multiple tissues. In all tissues tested, a signal was observed corresponding, in size, to the predominantly expressed mRNA transcript . For TFIThe splicing factor SC35 is a well-characterized molecular marker for the nuclear speckles. Nuclear speckles are subnuclear compartments for most, but not all, splicing factors. To visualize the subcellular localization of CCNL1 and EWSR1, compared to SC35 nuclear speckles, fusion constructs were generated with CCNL1 fused to green fluorescent protein (CCNL1-N1) and EWSR1 fused to red fluorescent protein (EWSR1-Red-N1). Each construct was transfected into LS8 cells individually. Consistent with previous studies , GFP-tagCCNL1-N1 and EWSR1-Red-N1 were used to study the subcellular localization within the cell nucleus, and related to the spatial nuclear expression pattern of endogenous TFIP11. HeLa cells transfected with either CCNL1-N1 or EWSR1-Red-N1 were immuno-labeled with the anti-TFIP11 antibody , i. TFIPin vitro, coimmunoprecipitation data demonstrated the interaction between TFIP11 and EWSR1 , which is a component of Cajal bodies . Cajal bin silico) will be able to establish which of these splicing proteins are redundant, and which are absolutely essential. Identifying protein-protein interactions between the various splicing factors, and relating these to their spatiotemporal expression profiles in vivo, will lead to a better understanding of the molecular mechanism of RNA splicing.Our results indicate the similar spatial expression pattern between TFIP11 and CCNL1, and a less-well defined spatial overlap between TFIP11 and EWSR1. These finding are complementary to previously published Y2H data showing protein-protein interactions between TFIP11 and CCNL1, and between TFIP11 and EWSR1. The functional relationship of these three proteins remains to be determined. While the functional spliceosome appears to consist of \u223c140 proteins, future proteomic analyses (experimental and"}
+{"text": "C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, \u2018digital mRNA counting\u2019 is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation. The emergence of next-generation sequencing (NGS) and the RNA-seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression and effectively deals with the problems noted above. Mortazavi et al. 2 intercrosses derived from these strains; included in these datasets are data from the striatum The current study compares RNA-Seq with two microarray platforms (Affymetrix MOE 430 2.0 and Illumina MouseRef-8 v2.0) to detect differential striatal gene expression between the B6 and DBA/2J (D2) inbred mouse strains. There are several reasons for comparing the B6 and D2 strains. First, the B6 is the reference strain for the mouse genome and the D2 strain is one of several strains that are being completely sequenced as part of the Sanger Mouse Genome project. Also there are extensive gene expression datasets comparing the B6 and D2 strains and both recombinant inbreds and FNa\u00efve, adult, male B6 and D2 strain mice were used in the RNA-Seq and Illumina microarray experiment while both genders were utilized in the Affymetrix microarray experiment. This animal study was reviewed and approved by the Portland Veterans Affairs Medical Center Institutional Animal Care and Use Committee under protocol ID VA1509. Animals were sacrificed by cervical dislocation and the brains removed. The striatum was dissected as follows. A 1.5-mm coronal slice of tissue was removed for which the caudal boundary was the optic chiasm. The slice was laid rostral surface down on an ice-cold Petri dish, and the material surrounding the striatum was carefully removed. From the caudal to rostral surface, the striatum decreases in size and thus an angled cut was used to remove additional non-striatal material. After dissection, the tissue was either flash frozen in liquid nitrogen or stored in RNAlater . Total RNA was isolated using TRIzol\u00ae reagent  using a one-step guanidine isothiocyanate procedure. RNA samples were evaluated by ultraviolet spectroscopy for purity and concentration  and were assessed further for RNA integrity on the Agilent Bioanalyzer . All samples had an RNA Integrity Number (RIN) of 8 or better.Total RNA from 21 male mice (10 B6 and 11 D2) was provided to the Oregon Health & Science University Massively Parallel Sequencing Shared Resource facility Dataset S1. The single nucleotide (SNP) correction of the NCBI m37 genome assembly was performed using a custom perl script using SNPs obtained for the D2 strain from the Sanger Mouse Genomes Project All reads were realigned to the NCBI m37 version of the mouse genome assembly using the Bowtie High quality samples containing 2 \u00b5g of total RNA were sent to the Oregon Health & Science University Gene Microarray Shared Resource facility for labeling and hybridization on microarray chips. The procedures used precisely followed Affymetrix's or Illumina's specifications, respectively An independent group of 20 mouse samples  were assessed using the Illumina MouseRef-8 v2.0 array. These include an independent group of 5 D2 and 5 B6 mice, and a group of 7 B6 and 7 D2 samples that were also analyzed using RNA-seq. The Illumina array data were analyzed with the lumi Bioconductor package within R Table S1, including total reads and uniquely mapped reads. To facilitate summarization of the RNA-Seq data relative to known gene annotations, we began by assembling a gene-level representation based upon Ensembl build 59 exon annotations as described in the Fig S1, those genes removed tended to have very low read counts compared to the remaining genes. Our subsequent analyses focused on the remaining 16,183 genes.We generated single end RNA-Seq reads from 10 B6 and 11 D2 mice (21 lanes on three Illumina GAIIx flowcells). A high level summary of the reads for each sample is presented in Fig S2).Although the distribution of read counts was variable between lanes and flowcells, these differences could be normalized using an upper quartile scaling procedure In our hands, the single factor Poisson model coupled with a likelihood ratio test proposed previously Dataset S1 (with an accompanying description in Text S1) are the genes, their fold change, a measure of average read count and a flag indicating especially lowly expressed (low read count) genes. An examination of the distribution of total union exon lengths classified by whether or not the gene was determined to be differentially expressed showed that there did not seem to be appreciable gene length bias contrary to what had been seen previously Fig S3). In contrast, if we examine the same plots generated for the single factor Poisson model incorporating the upper quartile scaling procedure read count adjustments in the offset, we see evidence that longer gene regions tend to be biased toward differential expression  indicating that choice of statistical model may be an important factor regarding the influence of gene length on differential expression.After estimating the common dispersion parameter and applying the exact test, a false discovery rate (FDR) controlling Fig S5 the distribution of the D2>B6 differential expression calls is shifted downward relative to the B6>D2 calls in terms of the density of known SNPs present, although the distributions largely overlap. To further address this issue we synthesized a D2 reference sequence to which we realigned the D2 reads. We found that there was a small increase in the number of uniquely mapping reads (less than 0.2% increase in reads for each D2 sample) and minimal increase in multi-mapping reads (less than 0.1% for each D2 sample). Further, after summarization, normalization, and application of the edgeR procedure, we found that 934 (97.5%) of the 958 genes originally identified as DE with B6>D2 remained DE, but status changed for the remaining 24 genes, which were no longer detected as DE indicating that they initially may have been false positives. Status also changed for 36 genes originally showing no DE; with the realigned reads, these were detected as DE with D2>B6, indicating that allele bias may have lead to false negative results. Finally, status changed for 5 genes originally detected as DE D2>B6, which were not found to be differentially expressed. Overall the number of putative false positive and false negative differential expression calls was minimal relative to the 1,727 differentially expressed genes found by RNA-Seq. The genes that changed status are indicated in Dataset S1.In addition to gene length, we assessed the potential impact of genome structure on our results. As documented in Walter et al. Table S2 and complete details are presented in Datasets S2 and S3 (both described in Text S1). For comparing DE across platforms, we used the microarray probeset or probe with the best q-value. Presented in Datasets S2 and S3 are the probesets and probes, their fold change, and complete annotation details.The Affymetrix MOE 430 2.0 microarray contains 45,037 probesets interrogating 18,461 genes annotated in Ensembl .First, we compared the overlap of genes detected by each of the three platforms. 7,211 genes are common to all three platforms Fig. 2. Next we compared the differential expression across platforms. RNA-Seq detected a total of 1,727 DE genes. The largest subset (591 genes) of the RNA-Seq DE genes were only detected as expressed in the RNA-Seq analyses, so evidence for their DE is also exclusive to the RNA-Seq data. 369 and 212 of the genes differentially expressed by RNA-Seq were detected as expressed using the Affymetrix and Illumina microarrays, but only 51% and 31% of these genes were DE in these microarray analyses, respectively. t-test) suggesting that many of the DE genes found only in the RNA-Seq analysis may indeed be true positives.Of the 555 that were compared Fig. 3, The present studies used a gene-level summarization approach, which has been shown to provide more reliable values than single base positions RNA-Seq provided more genes that were \u201cdetected,\u201d that is had reliable signal and were not impacted by SNP or annotation issues, than either the Illumina or Affymetrix microarray platforms. We recognize the difficulty in analytically describing a background level for RNA-Seq and for the future recommend additional experiments accurately measuring the abundance of mRNA from genes with a relatively wide range of counts in the model system of choice similar to what was done in earlier studies The largest proportion of RNA-Seq DE genes interrogated by all three platforms was represented by those that were seen to be differentially expressed in RNA-Seq only. We found that these genes tended to have a greater average read count relative to those that agreed with at least one microarray platform, further indicating the utility of RNA-Seq over these two microarray platforms. The 144 genes that are differentially expressed in common with the two microarray technologies may represent instances of relatively homogenous expression across the annotated gene, as the probes from these two microarray platforms should tend to be biased towards the 3\u2032UTR end of the gene. In this respect genes that only agreed with one platform in terms of differential expression or were only seen in RNA-Seq may represent differences in transcript isoform abundances. The variation in each of these categories illustrates an advantage of RNA-Seq compared to microarrays in that, in this case, RNA-Seq calculates DE across the entire gene rather than just at an individual probe(set) location within the gene. By some estimates there are, on average, approximately 2.5 alternative transcripts for each mouse gene Another potentially informative biological assessment of sensitivity across platforms could be done examining the Y-chromosome genes across the platforms. However, review of the Ensembl annotation utilized for the analysis in this study revealed only 20 genes annotated to be on the Y chromosome. Of these, if we examine the probes that passed our filters for SNPs, detection above background, and unique Ensembl mappings that were used in our analyses, this leaves only 7 probes on the Illumina array interrogating 5 genes on the Y chromosome and no probesets on the Affymetrix array. We do note that of the 5 genes detected by Illumina array, 4 of those were also detected by RNA-seq . However, with so few annotated genes and such poor coverage on the arrays, assessments using this type of assay would not be informative at this time.Additionally, many choices of experimental protocols currently exist for RNA-Seq, each with their own benefits and consequences for downstream analysis. For example to remove highly abundant rRNA molecules, either enrichment of poly-A containing sequences or depletion of the rRNAs has been used de novo identification of unannotated expressed regions Although many methods for analyzing RNA-Seq data currently exist and more continue to be produced, the most basic and fundamental question that can be asked is whether a gene/transcript is differentially expressed between two groups. Gene level differential expression then forms the basis for further experiments directed at identifying and quantifying transcript isoforms between samples Figure S1Distrubution of counts from genes that were removed relative to those remaining. The distribution of log2 mean read counts per gene categorized by whether the gene had a zero count in at least one lane for both the B6 and D2 strains (right) or either had no zero counts or zero counts in at least one lane for one of the strains (left).(TIF)Click here for additional data file.Figure S2Distribution of genes scaled by the total count per lane. Boxplots of the distribution of counts per gene scaled by the total unique read count (in megabases) for each lane.(TIF)Click here for additional data file.Figure S3Relationship between gene length and gene significance for q-values computed by edgeR. Shown in A) are the distribution of log2 gene lengths, in terms of union exon bases relative to whether that gene was determined to be differentially expressed at a q-value<.01 (DE) or not (Non DE). Shown in B) is the distribution of q-values for the gene lengths categorized by quartile of length.(TIF)Click here for additional data file.Figure S4Relationship between gene length and gene significance for q-values computed using the single factor Poisson model. Shown in A) are the distribution of log2 gene lengths, in terms of union exon bases relative to whether that gene was determined to be differentially expressed at a q-value<.01 (DE) or not (Non DE). Shown in B) is the distribution of q-values for the gene lengths categorized by quartile of length.(TIF)Click here for additional data file.Figure S5Relationship between the number of SNPs per kilobase of gene length and gene differential expression direction. The boxplot on the left (Not DE) is the distribution of SNPs per kilobase for non differentially expressed genes. The middle boxplot (DE B6>D2) shows the distribution for those genes that are both differentially expressed with the normalized count for B6 being higher than D2. Similarly the boxplot on the right shows the distribution for those genes that are differentially expressed with D2 showing a higher normalized read count than B6.(TIF)Click here for additional data file.Table S1Summary of read mapping statistics. Shown are the statistics in terms of reads for each lane of each flowcell (batch). The strain column references whether the sample was derived from the C57BL6/J (B6) or DBA2/J (D2) inbred strain of mouse. Total reads refers to the total number of generated reads for each lane. The remaining columns reference whether the read was uniquely placed to a location in the genome (Uniquely mapped), whether it mapped to more than one location (Multi-mapped) or whether it failed to map (Non-mapped). All realignments were performed using Bowtie (XLS)Click here for additional data file.Table S2Microarray data agreement within platform when one or more probesets or probes exist for a gene. Numbers in table indicate number of genes. Differentially expressed (DE) at q<0.01; fold change (FC); probeset refers to both probeset for Affymetrix or probe for Illumina microarray; same direction of fold change means that the same strain showed higher gene expression; \u2018some\u2019 means at least one.(XLS)Click here for additional data file.Text S1Readme file for (DOC)Click here for additional data file.Dataset S1RNA-Seq results of all genes detected and comparisons to Affymetrix and Illumina microarray data.(TXT)Click here for additional data file.Dataset S2Microarray results of all probesets detected by Affymetrix MOE 430 2.0 array in B6 and D2 mouse striatum.(TXT)Click here for additional data file.Dataset S3Microarray results of all probesets detected by Illumina MouseRef-8 v2.0 array in B6 and D2 mouse striatum.(TXT)Click here for additional data file."}
+{"text": "Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 \u00d7 SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host\u2013pathogen interaction with enhancement of R-gene mediated resistance.Stem rust  analysis, provides the opportunity to identify genes involved in transcriptional regulation and simultaneously identifying their downstream targets. This paper describes an eQTL analysis of a barley population segregating for qualitative and quantitative immunity to stem rust (Puccinia graminis f. sp. tritici), a devastating pathogen of Triticeae grain crops. Analysis of treatment-specific effects identified several regulatory loci that alter the expression of many inoculation responsive genes. On chromosome 2H, a trans-eQTL hotspot coincides with an enhancer of adult plant resistance. Notably, the resistance allele for this locus is associated with suppressing the transcription for hundreds of genes, with some of these having been previously associated with plant disease defense. In this respect, conventional wisdom is challenged by these findings.An important step in molecular plant pathology is the identification of the biologically relevant events that are directly involved in mediating resistance to pathogens. Historically, it is known that  Plants respond to invading pathogens with several forms of defense, ranging from the generation of toxic chemicals to programmed cell death Puccinia graminis, has been a serious problem wherever wheat and barley are grown P. graminis germinate within 4 to 8 hours after inoculation (HAI) during nights with dew formation or rainfall Rpg (Resistance to P. graminis) genes, which mediate resistance to particular formae speciales of P. graminis by Rpg genes have been identified, with five specifying resistance to races of P. graminis f. sp. tritici (Pgt) and three to P. graminis f. sp. secalis (Pgs) Pgt known as TTKSK, (commonly referred to as Ug99), initiated a major collaboration to identify resistance genes in germplasm repositories of wheat and barley  Pgt race TTKSK, Steffenson and colleagues identified the Rpg-TTKSK locus on the long arm of chromosome 5H, contributed by the barley cv. Q21861 rpg4 and Rpg5, respectively rpg4 confers immunity to Pgt race QCCJ, while Rpg5 provides dominant/semi-dominant resistance to Pgs isolate 92-MN-90. Sequencing of the genomic region in cv. Morex  found five candidate genes encoding two nucleotide-binding site (NBS), leucine-rich repeat (LRR) proteins, two actin depolymerizing factors , and a protein phosphatase 2C protein (PP2C) Rpg5 co-segregated with the two NBS-LRR, ADF3, and PP2C encoding genes in the susceptible cv. Morex Rpg5. The recessive resistance gene rpg4 has been associated with Adf2 by allele and recombinant sequencing Pgt race QCCJ in the informative recombinants indicates that both Rpg5 and rpg4 may be required to mediate an effective resistance response Rpg-TTKSK mediated resistance to Pgt race TTKSK, but it is hypothesized that both Rpg5 and rpg4 are required Stem rust, caused by the obligate fungal biotroph cis-eQTL) and the weaker distant regulation generated by genes that impact the transcriptional status of other genes (trans-eQTL) trans-eQTL tend to have more moderate effects than cis-eQTL, the functional polymorphisms that permit their discovery often affect more than one gene. For example, if a polymorphism altered activity of a transcription factor or hormone signaling gene, eQTL analysis may trace the regulation of many to hundreds of genes to the locus harboring this polymorphism, which would then be termed a trans-eQTL hotspot trans-eQTL hotspot affecting secondary metabolism has been associated with the AOP (ALKENYL HYDROXALKYL PRODUCING) locus, where cis-eQTL in genes involved in glucosinolate biosynthesis lead to the altered transcriptional and metabolic status of Arabidopsis Recently, several studies have exploited natural variation combined with expression profiling to decipher complex regulatory pathways, and in some cases phenotypic consequences Rpg-TTKSK locus and the polymorphism(s) responsible for its existence, we analyzed the mRNA abundance of 22,792 host genes in each member of the Q21861 \u00d7 SM89010 (QSM) doubled-haploid mapping population subjected to Pgt race TTKSK-inoculation (INOC) and mock-inoculation (MOCK). By integrating the dynamics of eQTL hotspot formation, inoculation-responsive gene expression, and alternative control of eQTL between INOC and MOCK treatments, we describe two forms of transcriptional regulation that are associated with the resistance response. First, we provide evidence for Adf3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene based on a strong cis-eQTL that has its effect magnified by inoculation with Pgt race TTKSK. Second, we report the identification of an inoculation-dependent, trans-eQTL hotspot that governs the expression of hundreds of genes, which under normal conditions, are controlled by additional modular regulators. The position of this chromosome 2H trans-eQTL hotspot is coincident with a quantitative resistance factor that acts as an enhancer of Rpg-TTKSK-mediated resistance in adult plants. Notably, the alleles across this shared genomic position that enhance Rpg-TTKSK-mediated resistance, lead to transcriptional suppression of numerous genes associated with disease defense.To gain insight into the regulatory functions of the Pgt race TTKSK, with Q21861 and SM89010 exhibiting seedling infection types (IT) of 0; and 213\u2212 to 3, respectively P. graminisPgt race MCCF The parents of the QSM population represent resistant and susceptible selections of barley against Rpg-TTKSK locus on chromosome 5H at bin 49 (5H.49) was the major qualitative locus for all infection frequencies and PC1  or susceptibility (rpg-TTKSK) did not identify any other loci that substantially explained residual variation (We used a QSM genetic map generated from transcript-derived markers (TDMs) see  to perfo and PC1 . In addiariation  and S2.Pgt race TTKSK and isolates in this lineage. Reactions of QSM progeny were assessed three times in October and November by estimating the severity of rust infection  on stem and leaf sheath tissue and also lesion size on a semi-quantitative scale  , with negative additive effect estimates (AEE) of SEV by 7.7%, LES by 0.1 units, and IC by 8.4% . The second most prevalent QTL identified among the set of temporal observations was located on chromosome 2H at bin 16 (2H.16). This 2H.16 locus  had negative AEE of SEV by 6.7%, LES by 0.1 units, and IC by 8.5%. QTL analysis using resistant and susceptible QSM sub-populations  revealed that resistance mediated by 2H.16 was only detectable in the resistant sub-population. A two-way ANOVA test also revealed a significant interaction term between 5H.49 and 2H.16, further implicating 2H.16 as an enhancer of Rpg-TTKSK.In parallel with the seedling experiments, infection phenotyping was performed in Njoro, Kenya during the 2008 season using natural inoculum of to 1.00) . An addiRpg-TTKSK. In seedlings, greater than 50% of the phenotypic variance for IF0, IF1, IF3, and PC1 was attributed to Rpg-TTKSK, whereas, Rpg-TTKSK explained 11.5% to 35.2% of the phenotypic variance in adult plants surveyed under field conditions, depending on the trait and date of data collection. In the experiments involving adult plants, the weaker relative contribution of Rpg-TTKSK suggests that it may act so strongly in seedlings that the effect of loci such as 2H.16 may be masked  with an interaction between genotype and treatment was expected, as most variation in gene expression attributed to inoculation is insensitive to the genotype assayed Variation in gene expression between the parental lines Q21861 and SM89010 was estimated by using four biological replicates that were randomized among the 75 doubled-haploid progeny of the QSM population see . Plants t-test with respect to QSM line between INOC and MOCK.Observation of differentially expressed genes in the parents alone sheds light on only a small fraction of the diverse genetic responses associated with defense. By contrast, the use of a segregating population provides a biallelic sampling that incorporates genotypic variability when detecting differences between treatments (INOC and MOCK). In addition, the greater number of individuals allows for a precise estimation of differential expression regardless of the allele used in our experiment t-test approaches, respectively, with an overlap of 5,325. This suggested that the difference found between INOC and MOCK either by pooling lines (ANOVA) or between paired lines (paired t-test) was consistently detected as responsive to inoculation. A significant overlap was found between genes that were differentially expressed in the progeny  and those that were differentially expressed in the parents , with the intersection consisting of 1,476 genes  \u200a=\u200a14.62). Alternately, the Rpg-TTKSK locus could impart resistance by modulating the expression of a small set of genes at 24 HAI. We identified 88 genes with Pgt race TTKSK-specific regulation at the Rpg-TTKSK locus (5H.48/49/50), with several genes known to function in PTI, ABA signaling, and reorganization of the actin cytoskeleton . This analysis revealed that probes 1\u20135 of Contig7092_s_at and probes 1\u20133 of Contig7093_at contained no SNPs between the probe source sequence (cv. Morex) and all other lines examined above. Analysis of these monomorphic probes verified the true expression level polymorphism as opposed to sequence-dependent hybridization efficiency. Note: Since the Rpg5 sequence was not identified prior to chip design, we were therefore unable to assay its expression for this analysis.We hypothesized that an eQTL hotspot would form at the skeleton . In paraSK locus ; for ConRpg-TTKSK. These loci may represent additional basal defense regulators, such as PRR-mediated recognition of PAMPs that alter the expression of genes involved in non-specific resistance Pgt race TTKSK and mock-inoculation. We used the 5,997 genes identified as differentially expressed between INOC and MOCK treatments of the QSM progeny . As shown in p<0.01; Phenotypic QTL analysis of seedling and adult progeny implicated several resistance factors distinct from  progeny  and appltrans-eQTL hotspot as a result of inoculation with Pgt race TTKSK. By comparing the positions of the most significant eQTL for each gene in the INOC and MOCK experiments, we asked whether genes regulated at this locus are specific to pathogen-induction, or alternatively, if they are regulated by different loci between MOCK and INOC. We then determined if an overlap between two loci was significant between INOC and MOCK by generating a bootstrap p-value based on the number of genes shared under a random distribution and excluding comparisons between cis-chromosomal positions. As shown in p<0.001  at 2H.16 in INOC override the regulation exerted by MOCK loci 2H.28/29, 3H.27, 6H.36/37, 6H.40, as well as at additional loci distributed across the genome. This coalescence of regulation in INOC suggests that each of the loci identified in MOCK may itself coordinate the expression of a distinct regulon. If this were true, one might expect the functional polymorphisms that underlie these eQTL to act pleiotropically on their respective targets in similar ways. To test this, we compared AEE for eQTL between INOC 2H.16 and the four MOCK loci listed in Pgt race TTKSK, over-saturation of inoculation-responsive genes at the 2H.16 locus implies that this locus largely determines the extent to which a gene is differentially expressed between INOC and MOCK. When we considered the AEE in INOC eQTL at the 2H.16 locus, we found that it was highly predictive of the direction of differential expression between INOC and MOCK, with 78 of 90 genes affirming this association  (r2\u200a=\u200a0.85 (48 genes). In contrast, genes not declared differentially expressed between treatments had a considerably weaker correlation of r2\u200a=\u200a0.51 (42 genes). These results indicate that the regulation in INOC from the 2H.16 locus is either the principal source or major component of gene expression changes due to challenge by Pgt race TTKSK.Coupled with the dependence on alternative regulation after challenge with ociation . Additio0 genes) . Selectitrans-eQTL hotspot following challenge by Pgt race TTKSK. First we examined the AEEs of these genes and observed a bias in the directionality of effects. Specifically, of the 229 genes with eQTL that have positive AEE , 162 are induced and 67 are suppressed. The opposite is seen for the 139 genes with eQTL that have negative AEE , with 39 induced and 100 suppressed. Thus, for the 368 genes regulated, the SM89010 allele at 2H.16 attenuates expression levels for 262 of them (\u223c71%).Our final analyses aimed to characterize shared aspects of the genes regulated by the 2H.16 trans-eQTL hotspot are oversaturated for differential expression in response to stem rust . In contrast, no significant GO terms were identified using SEA for eQTL with Q21861 allele contributing the greater allelic effect.Since we previously established that the genes regulated by the 2H.16 tem rust , we wishPgt race TTKSK . We also found that genes with negative allelic estimates, where the SM89010 allele results in lower expression, were moderately over-represented among the set of genes predicted to be localized to the plastid . Additional statistically significant GO terms were identified with PAGE that were associated with either up-regulation or down-regulation after inoculation with Pgt race TTKSK. GO terms associated with lower expression in INOC (down-regulation) included: biosynthetic processes (GO:0009058), nitrogen compound metabolism (GO:0006807), nucleobase, nucleoside, nucleotide and nucleic acid metabolism (GO:0006139), and cellular biosynthetic processes (GO:0044249). In contrast, protein metabolism (GO:0019538), protein modification (GO:0006464), macromolecule modification (GO:0043412), cellular protein metabolism (GO:0044267), and localization to the membrane (GO:0016020) are associated with up-regulated genes (greater expression in INOC).Although SEA directly tests for enrichment of GO terms, it does not take into account the magnitude of differential expression or allelic effects. To address this, we used a parametric analysis of gene set enrichment (PAGE) to incorporate these effects. In agreement with the results from SEA, we found that localization to the plastid was significant for genes down-regulated after challenge by trans-eQTL hotspot. This result is paradoxical, since presence of SM89010 alleles across this same locus also enhances Rpg-TTKSK-mediated resistance  that typically manipulates the expression of tens, if not hundreds, of genes. Thus, the weaker effect of trans-eQTL is compensated for by the co-localization of many genes and is identified by the occurrence of trans-eQTL hotspots. Notably, eQTL analysis of segregating populations of Arabidopsis and barley has been used to establish links between circadian rhythm and metabolism cis-eQTL The intricacies of genetic inheritance of gene expression have contributed significantly to our current understanding of gene regulation trans-eQTL hotspots at the site of major regulators has become a common theme in the control of gene expression Rpg-TTKSK locus, but several models can account for its absence. First, our selection of 24 HAI may represent a very early (or late) time point in the activation of resistance signaling, such that only the primary targets of Rpg-TTKSK-specified resistance would be differentially regulated at this time. Second, if the primary transcriptional targets of Rpg-TTKSK-mediated resistance had considerable structural variation in their promoters, then the regulatory contribution from the Rpg-TTKSK locus may be effectively masked by strong cis-eQTL effects, or be too small to be detected with this approach. In this scenario, trans-eQTL hotspots composed of secondary targets will form at the genetic positions of primary transcriptional targets of R-gene signaling, rather than the R gene itself. These two hypotheses overlap, as the selection of time points would determine whether primary, secondary, or more general responses are detected. Lastly, the primary resistance response mediated by Rpg-TTKSK may not include gene expression as a causal component in defense.The presence of trans-eQTL hotspot refocused our efforts to identify cis-eQTL that are biologically relevant at the Rpg-TTKSK locus, an approach used previously to dissect partial resistance to barley leaf rust mlo-mediated resistance in barley-powdery mildew interactions Adf3 is located within 5H.49 and is proximal to the Rpg5 resistance gene. Previously, this gene was excluded as a candidate for Rpg5, which mediates resistance to Pgs isolate 92-MN-90, because the ADF3 amino acid sequence was identical between resistant and susceptible cultivars Pgt race TTKSK, Adf3 has enhanced regulation (AEEINOC > AEEMOCK) at 5H.49, with lower expression in lines carrying Rpg-TTKSK. Therefore, resistance is associated with the suppression of Adf3 expression, a hypothesis suggested for Adf2 by Brueggeman and colleagues Adf3 was not considered a candidate for Rpg5 based on the failure to observe non-synonymous variation between Q21861 and susceptible alleles of the coding region Adf3 may be a factor contributing to Rpg-TTKSK-mediated resistance based on its strong expression polymorphism. Several hypotheses have been put forth for the biological role of ADF in plant-pathogen interactions Adf3 in plants carrying the susceptible (SM89010) allele implicates its functional role as a susceptibility factor induced by Pgt race TTKSK. Significant structural variation does occur in the promoter of Adf3Adf3 induction in compatibility. However, it has been shown that an intact host actin cytoskeleton is required for successful colonization in several plant-fungal pathosystems Adf3 may be a potential target of pathogen-derived effectors.The absence of a trans-eQTL hotspot at Rpg-TTKSK, extensive transcriptome reprogramming due to invasion by Pgt race TTKSK revealed key regulators that were altered between INOC and MOCK treatments. Most significant was the trans-eQTL hotspot at 2H.16, where saturation in both the number of eQTL and inoculation-responsive genes suggests that regulator(s) at this locus usurp the steady-state regulatory machinery to actively remodel gene expression. Dissection of this hotspot revealed a transcriptional hierarchy of several distinct loci in MOCK that converge on 2H.16 after challenge with Pgt race TTKSK, with a predictive allelic effect between INOC and MOCK conditions.Even in the absence of a R gene-mediated defense may implicate a causal relationship between gene expression and enhanced resistance. There exists some difficulty in making this connection, as the cloning of both classical and expression QTL can be confounded by the presence of tightly linked genes contributing to the phenotype. In mouse, dissection of the Qrr1 (QTL rich region on chromosome 1) region found that multiple genes likely regulate different subsets of trans-eQTL that had previously been grouped together Qrr1 region into proximal and distal portions, and found the distal region specifically regulated RNA metabolism and protein synthesis. In doing so, they were able to focus the eQTL candidate gene list that underlies a classical QTL associated with seizure susceptibility. This case study provides a model for how a single locus associated with an abundance of both QTLs and trans-eQTL was broken into two regions that are associated with entirely different pathways. Similarly, it is possible that the 2H.16 region may be comprised of several regulators. At present, our strongest evidence against this hypothesis is the predictive power in the allelic effects between the MOCK loci  and the INOC 2H.16 locus, suggesting a single regulator or family of regulators that control these regulons after challenge with Pgt race TTKSK.The co-localization of the hotspot with an enhancer of adult plant, Pgt race TTKSK, we focused on the dynamics in response to pathogen invasion. This is one approach for identifying genes that act as nodes in regulatory networks, but several other methodologies may be similarly powerful. For example, candidate genes can be identified from unrelated eQTL experiments by using additional information such as physical map position, functional annotation, expression polymorphisms, and correlation Rpg1rpr1Rpg1-mediated resistance, and physical map position to identify a sensory transduction histidine protein kinase (represented by probe set Contig13680_s_at) that was strongly down-regulated in non-inoculated rpr1 plants and physically mapped near the QPgt.StMx-2H QTLs (IF2 and PC2) trans-eQTL hotspots on chromosome 2H in our work, we linked the QSM and SxM genetic maps via the conserved TDMs used to generate both maps  and AEE of 0.67 (0.66), with greater expression contributed by SM89010 allele in INOC (MOCK). Thus, three tightly linked loci, 2H.16, 2H.21/22, and 2H.28/29 control the QTLs to Pgt race TTKSK, Pgt race MCCF, and the regulation of a steady-state trans-eQTL hotspot, respectively. It is of interest that both QTLs detected in the SxM and QSM populations against Pgt races TTKSK and MCCF, respectively, enhance the effect of their respective R genes, Rpg-TTKSK and Rpg1. As Druka and colleagues used tissue derived from germinating embryos, it is unclear whether a trans-eQTL hotspot underlies the histidine protein kinase in leaf tissue in the SxM population In our investigation of eQTL regulation after treatment with oth maps . Based otrans-eQTL hotspot at 2H.16 regulates genes that are both induced and suppressed in response to Pgt race TTKSK invasion. Overall, the allelic effects for trans-eQTL at this locus were biased for greater expression when carrying the Q21861 allele (304 Q21861 vs. 219 SM89010). This effect was mutually predictive with up-regulation in response to Pgt race TTKSK associated with the Q21861 allele. In contrast, enhancement of Rpg-TTKSK-mediated adult plant resistance was associated with the SM89010 allele. This is especially relevant, as Q21861 contributes Rpg-TTKSK and SM89010 is susceptible to Pgt race TTKSK. Taken together, these results suggest that transcriptional suppression was correlated with resistance, where enhancement would have been expected. This model of host-mediated gene suppression may be a defense mechanism against pathogen-mediated gene activation, which has been observed in several phytopathosystems as a method to distract or enhance accessibility of the host. The bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 produces the jasmonic acid-mimic coronatine that induces jasmonic acid/ethylene-associated pathways that compete with bacterial defense pathways dependent on salicylic acid signaling Xanthomonas spp. activate genes involved in host susceptibility bona fide approach for manipulating the host and enhancing virulence. Therefore this locus may provide a degree of insensitivity to effector-dependent manipulation of the host.It is interesting that the Rpg-TTKSK. Therefore the enhanced resistance conferred by 2H.16 in the presence of Rpg-TTKSK suggests that the manipulation of gene expression may only impact the interaction of barley and Pgt under the appropriate conditions. Here, regulation at 2H.16 in INOC overrides the control of several steady-state regulators in MOCK. Thus, these MOCK regulators may be prone to manipulation by Pgt, whereas 2H.16 is non-responsive. Alternatively, the 2H.16 locus may control the precise timing of gene expression, such that the full impact of these genes is maximized to strengthen Rpg-TTKSK-mediated resistance. Ultimately, increased resolution in the 2H.16 region will be required to dissect the causal polymorphisms that enhance R gene-dependent adult plant resistance and the regulator(s) that generate the trans-eQTL hotspot.It is important to recognize that genes regulated at 2H.16 in INOC were not dependent on 1 plant et al. Pgt race TTKSK isolate 04KEN156/04 was initially increased on a susceptible wheat host, collected, desiccated, and stored in tubes at \u221280\u00b0C. Nine days after sowing (PO:0007094 - first leaf unfolded), flats were inoculated with a low density of Pgt race TTKSK urediniospores (0.004 mg/plant) suspended in a lightweight mineral oil carrier using the inoculation protocols described by Sun and Steffenson The barley QSM doubled-haploid mapping population was generated from a single Q21861 \u00d7 SM89010 FPgt races TTKSK (used in seedling resistance assays) and TTKST (same virulence pattern as TTKSK with the addition of virulence for wheat stem rust resistance gene Sr24). Pgt race TTTSK (same virulence pattern as TTSKS with the addition of virulence for Sr36) may have been present at a low frequency.Field trials were carried out at the Kenya Agricultural Research Institute in Njoro, Kenya during the 2008 growing season. QSM DH lines were planted in 0.3 m rows (20\u201335 seed per row) in a completely randomized design with one replicate. Parents were included at random in the planting plan in three replicates. Infection phenotypes were scored on 7 October 2008, 17 October 2008, and 10 November 2008. The majority of lines were at the mid-dough stage of development  at the first scoring date st, 2nd, 3rd, and 4th ordered ITs, respectively. IFs were determined by averaging weights for two replicates, where full weight is given to ITs of 0, 1, 2, and 3 or partial weights for ITs of \u20180;\u2019, \u20181\u2212\u2019, \u20181+\u2019, \u20182\u2212\u2019, \u20182+\u2019, and \u20183\u2212\u2019. For partial weights, 70% is given to the IT shown  and 30% to the modified IT . In the unique case of \u20183+\u2019, a weight of 1.3 was given to IT 3. For adult plants, LES was quantified on a scale from 0.25 to 1.0 based on resistance or full susceptibility, where resistant is equal to 0.25, moderately resistant is equal to 0.5, and a fully susceptible LES score is 1.0 (www.r-project.org)  was performed with QTL Cartographer v1.17j, with a walking speed of 2 cM, window size of 10 cM, and five background markers (SRmapqtl) Prune) with reselection of background markers (SRmapqtl), where each iteration maximum LOD scores were stored and after 1,000 runs the 95th quantile (\u03b1\u200a=\u200a0.05) was selected as the EWT Eqtl.Stakman infection types (ITs) for seedling plants were normalized using a modified approach that weights the counts of ordered ITs  [30]. Wee is 1.0 . The IC ect.org) . ComposiPgt race TTKSK urediniospores (0.25 mg/plant) was used as compared to the seedling phenotypic assay. For the MOCK flat, spore-free mineral oil was used. After inoculation, both flats were placed in the same mist chamber for 16 hours in the dark, exposed to light for 5 hours, and then moved to the greenhouse for 2 hours. Five seedlings were harvested, pooled, and placed in liquid nitrogen for each line in the population within a 1.5 hour period at 24 HAI. RNA was extracted using a hot acid-phenol protocol and RNAeasy columns (Qiagen) were used for further purification of the isolated RNA. Labeling, hybridization, washing, and scanning were performed according to standard Affymetrix protocols using the Barley1 GeneChip which contains probe sets representing 22,792  genes www.biotech.iastate.edu/facilities/genechip/Genechip.htm).Two flats (each flat contained 75 doubled-haploid lines + 4 replicates of each parent \u200a=\u200a81 cones/flat) were grown in a completely randomized design at the USDA-ARS Cereal Disease Lab, University of Minnesota, St. Paul. For the INOC flat, a higher density of www.python.org). This technique identifies single feature polymorphisms (SFPs) by using individual probes on the Affymetrix Barley1 GeneChip as quantifiable measures of probe hybridization efficiency. After background correction and quantile normalization using R/Bioconductor (www.bioconductor.org), individual probe signals were separated into two distinct groups with k-means clustering. Goodness-of-fit using a Z-statistic found over 2,500 quality markers for the QSM genetic map. This analysis was performed separately with the INOC and MOCK data sets, and only those markers conserved between these data sets  that had three (of 75) or fewer data points missing were included. A scaffold of 294 markers shared with the SxM doubled-haploid mapping population was used to place the remaining 1,200 markers http://cgpdb.ucdavis.edu/XLinkage/MadMapper) At any single genetic locus, each of the 75 doubled haploid lines carries two copies of either the Q21861 or the SM89010 allele. These two genotypes can be distinguished by differential success in hybridizing RNA to Barley1 arrays, providing robust genetic markers. Transcript-derived markers were generated as described by Potokina and colleagues q-values were determined using histogram-based estimation proposed by Mosig and colleagues ANOVA was performed with SAS v9.1 . All comparisons between these data sets were generated using Python scripts. www.bioconductor.org). Composite interval mapping was performed with QTL Cartographer v1.17j, using a walking speed of 2 cM, window size of 10 cM, and five background markers Prune) with reselection of background markers (SRmapqtl) a total of 1,000 times th quantile (\u03b1 \u200a=\u200a 0.05) was selected as the individual EWT.All microarray data for eQTL analysis were normalized with the Bioconductor implementation of the MAS5.0 algorithm  that were generated with a greedy approach. This approach required a minimum number of TDMs and eQTL in INOC (73 TDM and eQTL) and MOCK (85 TDM and eQTL) to be placed within a superbin. Hence, the same bins in each experiment were collected into a single superbin. This approach is similar to that used for the identification of enes see . Two strhttp://bioinfo.cau.edu.cn/agriGO) Gene ontology enrichment analysis was carried out using agriGO v1.0\u03b2 (www.plexdb.org), accession GSE20416 at NCBI-GEO, as well as accessions GN235, GN236, GN237, GN238 at GeneNetwork (www.genenetwork.org) All MIAME-compliant GeneChip profiling data are available as accession BB64 at the PLEXdb expression resource for plants and plant pathogens (Dataset S1Excel spreadsheet of the QSM genetic map. Transcript-derived marker genetic map of the Q21861 x SM89010 doubled-haploid population. Markers have been derived from probe-level data as described in the (XLS)Click here for additional data file.Dataset S2Excel spreadsheet of the eQTL identified in the MOCK experiment. eQTL determined to be significant based on individual EWT in the MOCK experiment as described in the (XLS)Click here for additional data file.Dataset S3Excel spreadsheet of the eQTL identified in the INOC experiment. eQTL determined to be significant based on individual EWT in the INOC experiment as described in the (XLS)Click here for additional data file.Figure S1et al.st, 2nd, 3rd, and 4th ordered ITs, respectively. Infection frequencies (IF) were determined by averaging weights for two replicates, where full weight is given to ITs of 0, 1, 2, and 3 or partial weights for ITs of \u20180;\u2019, \u20181-\u2018, \u20181+\u2019, \u20182-\u2018, \u20182+\u2019, and \u20183-\u2019. For partial weights, 70% is given to the IT shown  and 30% to the modified IT . In the unique case of \u20183+\u2019, a weight of 1.3 was given to IT 3.Modified Druka (TIF)Click here for additional data file.Figure S2Ternary plot of percent effect explained by genotype (G), treatment (T), and genotype x treatment (G x T). (A) Genes shown have over 50% of their variance (from two-way ANOVA) explained by the sum of the three effects . Point colors of magenta and green show whether or not a given gene had a transcript-derived marker, respectively. The majority of genes having TDMs are those with a significant genotype effect, suggesting strong allelic polymorphisms between parents Q21861 (Q) and SM89010 (SM). The bottom panel shows representative examples for the extremes of genotype (B), treatment (C), and their interaction (D).(TIF)Click here for additional data file.Figure S3Two-point locus heat map of the QSM DH genetic map. Heat map of the non-redundant QSM DH genetic map showing linkage for all marker x marker comparisons, genetic distance between markers (right), and proportion of the contributed allele  for every marker (bottom).(TIF)Click here for additional data file.Figure S4Recombination map of the QSM DH population.(TIF)Click here for additional data file.Table S1Rpg-TTKSK) of seedlings of the QSM DH mapping population inoculated with Pgt race TTKSK.Quantitative trait loci identified in the resistant sub-population ((XLS)Click here for additional data file.Table S2rpg-TTKSK) of seedlings of the QSM DH mapping population inoculated with Pgt race TTKSK.Quantitative trait loci identified in the susceptible sub-population ((XLS)Click here for additional data file.Table S3Rpg-TTKSK locus.Genes identified with an inoculation-specific eQTL at the (XLS)Click here for additional data file.Table S4Rpg-TTKSK locus.Genes identified with eQTL in INOC and MOCK at the (XLS)Click here for additional data file.Table S5trans-eQTL hotspot.Genes identified with an inoculation-specific eQTL at the 2H (XLS)Click here for additional data file.Table S6Pgt race TTKSK.Phenotypic data for seedling and adult QSM lines inoculated with (XLS)Click here for additional data file.Table S7Coefficients of the principal components for QSM seedling phenotyping.(XLS)Click here for additional data file."}
+{"text": "Chiropractors are frequent providers of care for patients with lower back pain. Biopsychosocial approaches to managing patients are regarded as best practice and are gaining wider acceptance. Recent evidence suggests that practitioners\u2019 attitudes and beliefs may also have an important effect on patients\u2019 recovery from back pain. Past studies have pooled manual therapists from differing professions. Dissonant findings have been hypothesised as being a result of the chiropractic subpopulation within multi-practitioner participant pools who are hypothesised to focus on biomedical aspects of\u00a0treatment and minimize biopsychosocial dimensions.The aim of this study is to determine whether a study population of only chiropractors would demonstrate similar attitudes and beliefs to other manual therapists\u2019 biopsychosocial or biomedical approach to the management of their patients.A survey of chiropractors in Victoria Australia in September 2010 was undertaken utilising the Pain Attitude and Belief Scale (PABS.PT), a tool which has been developed to determine the orientation  of practitioners to the management of people with low back pain. The survey also obtained demographic data from respondents to determine whether variables such as education, gender or practice related factors influenced their orientation.The overall response rate was 29% (n\u2009=\u2009218). The majority of the sample was male (68%), with a mean age of 44 years. The 6 point Likert scale scores were 34.5 (6.3) for the biomedical factor scale and 31.4 (4.1) for the biopsychosocial scale. Internal consistency of the psychosocial subscale was poor. None of the demographic variables were found to influence the biomedical or psychosocial scales.Chiropractors in the state of Victoria were found to have similar biomedical and psychosocial orientations in their attitudes and beliefs when compared to other manual therapists\u2019 levels of previous studies from differing cultural and educational backgrounds. This study was unable to replicate any of the relationships from past studies with any of the demographic variables. The psychosocial scale internal consistency may be a significant factor in this non-finding. Future research should address the identification of more robust items of the biopsychosocial attitudes of Victorian chiropractors toward treating lower back pain. Low back pain is now the leading cause of disability globally and was estimated to be responsible for 83 million years lived with disability in 2010 [It is now well established that the problem of persistent low back pain and associated levels of disability are not fully explained by biological factors alone and that best practice care should include more than the musculoskeletal system. There is good evidence that psychological constructs such as pre-existing depression, anxiety, fear-avoidance beliefs, poor coping strategies and poor self-efficacy are significant predictors of greater functional disability and work loss . These cThe most dominant current model in understanding these is the biopsychosocial model (BPSM) of health. It describes the influence of the biological, psychological and sociological factors in the manifestation of pain and illness . It may th birthday not all manual therapists are cognisant of the impact of the psychological and sociological domains. In response to this various government and insurance agencies have attempted to implement readily applicable assessment frameworks and education programs to improve patient outcomes [Despite the BPSM recently celebrating its 25outcomes . Despiteoutcomes . SubsequOne line of exploration that researchers have turned their focus toward is that of the attitudes and beliefs of the practitioner . A systeOne instrument used to explore these attitudes is the Pain Attitudes and Beliefs Scale for Physiotherapists (PABS.PT) ,17. It wThe initial version PABS.PT was comprised of 31 items which were extracted from existing psychosocial related questionnaires by an expert physiotherapist panel . Factor Houben et al.  continueSubsequent studies have verified the two factor structure of the PABS.PT -21. ThesThis suggestion of the chiropractic population possessing different qualities to other health professionals is not without precedent. For example the STarT back screening tool had been developed to help primary care practitioners make care decisions about the likely need LBP patients have for secondary prevention based on modifiable risk factors for poor outcomes ,23. It hOther possible explanations for this professional dissonance include the presence of a significant portion who hold unorthodox views , a limitTherefore, the primary aim of this study was to further explore chiropractors\u2019 attitudes and beliefs in an effort to understand these reported differences. In particular it sought to measure and compare their attitudes and beliefs in regard to biomedical and psychosocial aspects of patient care in a sample of only chiropractors in Victoria, Australia. A secondary aim was to determine if sociodemographic characteristics may be also be associated with their beliefs and attitudes.The study consisted of a retrospective survey of chiropractors practising in the state of Victoria Australia in October 2010. Ethical approval was obtained from Human Research Ethics Committee of Macquarie University, New South Wales, Australia (HE25SEP2009-ROO143). All questionnaire responses were completed anonymously, thus written consent was not obtained from individual respondents with completion of the questionnaire indicating consent.The 19 item shortened version PABS.PT was used to evaluate the role of chiropractors\u2019 attitudes and beliefs on the development and maintenance of persistent low back pain . PreviouA recently published systematic review of the psychometric properties of the PABS.PT found positive results for its construct validity, reliability and responsiveness .A mailing list of all registered chiropractors in the state of Victoria was obtained from the national chiropractic registration board. Duplicate names, incomplete details and practitioners who were not practising in Victoria and returned mail from practitioners who were no longer practising at the designated address were excluded from the sample. This resulted in a list of 750 eligible practitioners.All 750 practitioners were mailed an invitation letter which consisted of the PABS.PT and 10 demographic questions. The survey was accompanied by a reply paid envelope to return to the authors. Alternatively, practitioners were given the opportunity to complete the survey online via an online survey tool (Survey Monkey\u2122). A reminder was forwarded to practitioners via electronic mail with the assistance of the professional associations.Data were analysed with SPSS V21. Descriptive statistics were calculated for the demographic items and Cronbach\u2019s alpha was used to examine internal consistency of the questionnaire items.Median scores on the biomedical and psychosocial factors were calculated and the sample was dichotomised using these values. Logistic regression was then used to determine whether any demographic variables  could explain the dichotomisation (i.e. whether or not the chiropractors have higher odds of the particular domain tendency).In order to compare this sample to the national population, figures were obtained from the Australian Workforce Data Analysis and Planning, Department of Health . These dThe mean and standard deviation scores for the biomedical and psychosocial scales were calculated on a \u201c1 to 6\u201d Likert scale and a \u201c0 to 5\u201d Likert scale to allow for comparison to the Magalh\u00e3es et al., ,21 studyA multiple regression analysis was used with the biomedical and psychosocial scale scores as the dependent variables and the demographic factors were entered as independent variables to test for the presence of independent predictors.The overall response rate to the survey was 29% n\u2009= 218). Demographic data are summarised in Table\u00a08. DemogrThe average 6 point Likert scale scores were 34.5 (6.3) for the biomedical factor and 31.4 (4.1) for the biopsychosocial scores. The 5 point Likert scale produced biomedical factor scores of 24.7 (6.3) and a biopsychosocial score of 22.4 (4.1) .The biomedical and psychosocial scales were inversely related as shown in several previous studies . Multiple linear regression analysis demonstrated that the chiropractic demographic variables accounted for 12% of the variance of the biomedical factor \u2009=\u20091.210, p\u2009=\u20090.298, R2\u2009=\u20090.12) and 20% of the psychosocial factor \u2009=\u20091.348, p\u2009=\u20090.226, R2\u2009=\u20090.20)  and were less represented in the younger age groups (16 -34 years) compared to the national figures (18.9% versus 35.6% respectively). Chiropractors in this study tended to have more work experience in the 10 to 20 years range (38% compared to 21%). So this sample may be over represented by \u201cmid-life\u201d chiropractors.The results of this study showed that the estimated chiropractic population\u2019s scores on the two subscales of the 19 item PABS.PT were within the ranges of previous studies. Scores were calculated on a \u201c0-5\u201d Likert scale for comparison to the Brazilian chronic pain physiotherapists in Magalh\u00e3es et al. ,21 and wMagalh\u00e3es et al. ,21 raiseIf the Magalh\u00e3es et al., study was scored on a \u201c1-6\u201d Likert scale and increased by a similar amount as those of Victorian chiropractors when re scaled, which appears highly likely as standard deviations are similar between studies, then those scores would fall within the midrange of previous studies. This suggests that practitioner responses are similar across culture and educational training.Magalh\u00e3es et al. ,21 suggeThe most significantly disparate score is that of the Irish GP\u2019s psychosocial scale, which was 16.3 (SD\u2009=\u20093.1) . The autThe biomedical orientation scale internal consistency in this study was found to replicate levels of previous studies. Other studies have reported less favourable internal consistency levels in the psychosocial scale. This study obtained the lowest Cronbach\u2019s alpha to date (0.44). The original study of Ostelo et al.  achievedWhile several demographic variables have been found to influence the biomedical orientation in other studies  this study was not able to identify any such relationships. The number of patients treated per week approached significance at the 5% level in relation to the psychosocial scale. This unexpected finding suggests that the practitioner who sees larger numbers of patients per week is more likely to believe that psychosocial factors play a role in patients\u2019 lower back pain. The poor Cronbach\u2019s alpha result for the psychosocial scale may also be a possible explanation for this trend and casts uncertainty over this finding.This study did not replicate past studies which have identified relationships with the biomedical scale and GPs and physiotherapists in early and late working life stages. This study\u2019s population was biased toward those chiropractors in \u201cmid-life\u201d and a targeted study seeking to recruit larger numbers from those age groups may be required to reveal if this pattern exists in Victorian chiropractors.The results of this study and previous studies have not found any relationship to the PABS.PT psychosocial subscale and demographic variables. The lower scores for internal consistency may be a possible explanation. Future studies exploring alternative items may overcome this concern and add to the potential utility of the PABS.PT.This study was limited by its low response rate. Comparison to the national workforce data suggests that this studies sample approximated it, aside from the younger age groups. Nonetheless non-responders may be aligned to those of high biomedical and low psychosocial patterns but only a larger sample would clarify this possibility.When compared to the national chiropractic data this sample was underrepresented by younger aged practitioners with fewer years in practice. This will be the \u201clongest practicing\u201d portion of the population who this study focused on and will subsequently have the potential to deliver suboptimal care for many years. A base-line measure would allow an insight into the changes in attitudes across their working lifespan and to determine if future generations of manual therapists are improving in their ability to deliver best practice guideline based care.This poor internal consistency of the psychosocial scale suggests that the survey items are not capturing this dimension in the chiropractic population. The development of alternative items remains an objective for future research.A strength of this study was the single professional practitioner base of participants. It enabled a comparison to previous studies comprised of various populations. Chiropractic practitioners do not appear to differ significantly from other health care providers. Thus the PABS.PT, in particular the biomedical scale may offer some utility in identifying non guideline based chiropractitioners.Practitioner attitudes and beliefs have been shown to be associated with clinical outcomes for patients with low back pain . This st"}
+{"text": "The correct title is as follows: Hypertonic saline protects brain endothelial cells against hypoxia correlated to the levels of epidermal growth factor receptor and interleukin-1\u03b2.In the article \u201cHypertonic saline protects brain endothelial cells against hypoxia correlated to the levels of estimated glomerular filtration rate and interleukin-1\u03b2\u201d,"}
+{"text": "To estimate the prevalence of World Health Organization-defined chronic suppurative otitis media (CSOM) and mild hearing impairment in a population representative sample of school-entry age children in rural Malawi. A secondary objective was to explore factors associated with CSOM in this population.We performed a community-based cross-sectional study of children aged 4\u20136 years in Chikhwawa District, Southern Malawi, utilising a village-level cluster design. Participants underwent a structured clinical assessment, including video-otoscopy and screening audiometry. Diagnoses were made remotely by two otolaryngologists who independently reviewed clinical data and images collected in the field. Hearing impairment was classified as failure to hear a pure tone of 25dB or greater at 1, 2 or 4kHz.We recruited 281 children across 10 clusters. The prevalence estimates of CSOM, unilateral hearing impairment and bilateral hearing impairment were 5.4% (95%CI 2.2\u20138.6), 24.5% (95%CI 16.3\u201330.0), and 12.5% (95%CI 6.2\u201316.9) respectively. Middle ear disease was seen in 46.9% of children with hearing impairment. A trend towards increased risk of CSOM was observed with sleeping in a house with >2 other children.We found a high burden of middle ear disease and preventable hearing impairment in our sample of school-entry age children in rural Malawi. There are important public health implications of these findings as CSOM and hearing impairment can affect educational outcomes, and may impact subsequent development. The identification and management of middle ear disease and hearing impairment represent major unmet needs in this population. Chronic suppurative otitis media (CSOM) is an infection of the middle ear cleft characterised by perforation of the tympanic membrane and persistent ottorhoea. CSOM is a substantial global health problem with an estimated incidence of about 31 million cases per year. ApproximIn sub-Saharan Africa, CSOM is a leading cause of preventable childhood hearing loss.,3. CSOM The recent development of tertiary otolaryngology and audiology services in Southern Malawi along with establishment of a national diploma in otolaryngology for healthcare workers has markedly increased the resources available to manage CSOM and hearing impairment in this setting. AdditionSurveys of CSOM in the sub-Saharan African region have reported broad variation in prevalence estimates, ranging from 1\u201310%.,11\u201320. AThe primary objective of this study was to estimate the prevalence of World Health Organization (WHO)-defined CSOM and hearing impairment in a representative sample of rural children in Southern Malawi. A secondary objective was to evaluate potential associations of CSOM in this population. The study was undertaken in communities that had participated in the Cooking and Pneumonia Study (CAPS), a cluster randomized controlled trial of two cleaner-burning biomass-fueled cookstoves (Philips HD4012LS) compared to continuation of open fire cooking on the incidence of childhood pneumonia. Since emWe conducted a community-based cross-sectional study of children between 4 and 6 years old living in Chikhwawa district in Southern Malawi, and utilised a village level cluster design. The study was based at the Malawi-Liverpool-Wellcome Trust (MLW) clinical research program Chikhwawa field site.2 with a population of 245,000, of which 45.9% are under 15 years old.[Chikhwawa district  covers an area of 4755Kmears old. The distears old. In 2010,ears old. Infrastrears old.The sampling frame listed all villages that utilised Chikhwawa District Hospital for primary healthcare needs. This was created by MLW field workers with the District Medical Officer as part of CAPS. We utilised a two-stage sampling strategy. At the first stage, 10 villages were selected from the sampling frame by simple random sampling without replacement. The number of children aged 4\u20136 years in each of these villages was established through door to door census conducted for the current study. Cluster sizes were determined by dividing the required sample size by the total number of eligible children in each village. At stage two, we subsampled observational units from each cluster using simple random sampling without replacement. All children identified in the initial survey were assigned a random number. Children with the highest number were invited to participate sequentially until the desired sample size was achieved in each cluster.Children were eligible for inclusion if aged 4 to 6 years and resident in a selected village at the time of the study. The age inclusion criteria were chosen to estimate the prevalence of disease at school-entry age. Children were excluded if they had died or moved since the initial census, or if they did not provide assent to participation. Written informed consent was obtained from the guardian of all participants by a field worker fluent in the local language, Chichewa.We calculated that approximately 270 participants would be sufficient to estimate the prevalence of CSOM with a precision of +/-3% at a 5% alpha level. A design effect of 1.2 and a prevalence of 6% were assumed after a review of prevalence surveys in comparable settings and ages.,11\u201320A structured interview was performed using a standardised questionnaire . DemograTM screening audiometer , with supra-aural Sennheiser HD280 headphones , calibrated to ANSI S3.6 standard.[A hearing assessment was performed using a HearScreenstandard. Each parTests were conducted in the quietest available room in each community. Ambient noise levels were monitored and tests repeated if maximum permissible noise levels were exceeded . ParticipTM MEDL4E otoscope  following study-specific training. TM otoscope. Where appropriate, cerumen management was performed under direct vision using a BionixTM lighted ear curette . Cerumen removal was performed to tolerance and halted in the event of discomfort or withdrawal of assent.Tympanic membrane examination was performed by a Clinical Health Officer using a Dino-LiteAll participants with ear pathology received appropriate medical treatment at diagnosis. All children with CSOM were given assistance to attend the tertiary otolaryngology centre at Queen Elizabeth Central Hospital, Blantyre, Malawi for specialist assessment. Guardians were compensated for associated financial and opportunity costs.Anonymised data were viewed remotely by two otolaryngologists  with 20 years\u2019 cumulative experience. Assessors commented on image quality and assigned a diagnosis to each ear using a structured image interpretation tool. Assessors had access to tympanic membrane images, screening audiometry results, and clinical findings. Diagnoses were assigned as per the WHO classification of ear and hearing disorders and are summarised in Each participant was assigned a single middle ear diagnosis. A normal diagnosis was assigned if participants had healthy tympanic membranes bilaterally. Where participants had a differing diagnosis in each ear, the most clinically serious diagnosis was taken: CSOM, followed by dry perforation, serous otitis media, acute otitis media, foreign body and cerumen impaction.Prevalence estimates were calculated as a weighted average of the cluster level means and reported alongside 95% confidence intervals. Weighting was determined by the sampling fraction with additional post-hoc weighting at an individual level to account for sex. Due to the narrow age inclusion criteria, analyses were not weighted for age. Confidence intervals were constructed assuming a normal distribution with standard errors calculated from a bootstrapping without replacement approach, modified for two stage sampling. 100,000 Associations of CSOM were evaluated using univariate analyses with no formal statistical testing. Relative risk estimates for categorical variables were calculated as the ratio of the sample means between each group. A binomial generalised linear model with a log link function was fitted to continuous variables without post-hoc weighting. The relative risk is reported as the exponential of the estimated coefficient. Confidence intervals for relative risk estimates were calculated assuming a log-normal distribution with standard errors estimated from the bootstrap samples above. A constant of a half was added to each cell entry when calculating relative risk for each bootstrap replicate to avoid 0 and infinity estimates. PatientsEthical approval was granted by the science and ethics review committees of the Liverpool School of Tropical Medicine and the Malawi College of Medicine. The protocol was prospectively registered with ClinTrials.gov (Protocol ID NCT02779907). The full anonymized dataset are available in th May and 23rd June 2016 (We recruited 281 children aged 4 to 6 years from 10 villages between 11une 2016 . Of 569 The median age of children included in the study was 5.2 years (IQR 4.6\u20135.8) with 42.0, 43.4 and 14.6% aged 4, 5 and 6 respectively. There were slightly more male (54.1%) than female participants. The age and gender distribution of our sample was comparable to 2008 census data with theIn total, 562 ears were assessed in 281 participants. Image quality was rated as excellent in 15.3% (86/562), good in 72.4% (407/562) and inadequate in 3.2% (18/562). Cerumen impaction prevented visualisation of the tympanic membrane in 9.1% (51/562).CSOM was diagnosed in 15/281 (5.3%) of participants, with 4/15 (26.7%) having bilateral disease. In all cases the diagnosis was confirmed by a third otolaryngologist on referral to the otolaryngology centre. No participant displayed clinical features of mastoiditis or cholesteatoma. The weighted prevalence of CSOM was estimated at 5.4%  1.49).Audiometry data were complete for all but 2/281 patients. Background ambient noise was monitored throughout the study and found to be an average of 45.3 dB. Median screening audiometry time was 123 seconds (IQR 73\u2013171). Hearing impairment at 25 dB or greater hearing level in at least one ear was observed in 64/279 participants, with a weighted prevalence estimate of 24.5% . Bilateral hearing impairment was observed in 32/279 participants, with a weighted prevalence of 12.5% . Of those with hearing impairment, 46.9% (30/64) had an identifiable and potentially reversible aetiology . The comUnivariate analysis for potential associations of CSOM was limited by the low number of CSOM cases observed. There was a trend in increasing risk of CSOM with increasing age and sleeping in a house with >2 other children . There wThe prevalence of CSOM in our study population is estimated at 5.4% 95% CI, 2.2\u20138.6), placing this rural Malawian population in the highest WHO category for disease burden..2\u20138.6, p,18,20,33We report a prevalence of unilateral hearing impairment at 25dB of 24.5% (95% CI 15.0\u201333.9) and bilateral hearing impairment of 12.5% (95% CI 6.2\u201316.9). In this study we provide hearing impairment estimates from screening audiometry. In the validation study, HearScreen was compared to diagnostic audiometry using a pure tone average of >25 dB at 0.5, 1,2 and 4 kHz. HearScreen and was shown to have a sensitivity of 75% and a specificity of 98.5%. We utiliOur hearing impairment estimate is relatively high when compared to a recent systematic review of hearing impairment in Africa. This revOf participants with hearing impairment, half had evidence of middle ear disease. Identification and management of CSOM and bilateral hearing impairment at 25 dB is particularly important in this age group, as both have been associated with decreased academic attainment in primary school children across a range of settings.,34\u201336 ThThroughout sub-Saharan Africa, primary healthcare outside large cities is often delivered by practitioners with limited training and equipment to diagnose and manage ear and hearing disorders. In this context, provision of otolaryngology services to rural communities presents a substantial challenge. In this study, we utilised a telehealth approach, with a protocolised ear assessment performed by a non-specialist Clinical Officer and evaluated remotely by an Otolaryngologist. This approach links centralised specialist otolaryngology services with rural communities, where the burden of middle ear disease is highest. A telehealth approach, piloted for both otoscopy and screening audiometry in rural South Africa, has proven promising in terms of clinical validity and acceptability.,28,37 HoTM MEDL4E otoscopes used in this study represent an affordable option which may be suited to use in low-resource settings. We found the otoscopes to be reliable and produced diagnostic quality images. However, due to high magnification levels, multiple photographs were required to image the whole tympanic membrane when utilising paediatric specula. This may be problematic where assent is withdrawn and may have contributed to poor image quality recorded in a minority of cases.The Dino-LiteThis study has limitations. We did not perform tympanometry to confirm diagnoses of acute and serous otitis media, and pneumatic otoscopy was considered unfeasible due to the asynchronous telehealth approach. These factors are likely to lead to a decreased diagnostic accuracy for acute and serous otitis media and the true prevalence may be higher than measured. AdditionThe confidence intervals around our prevalence estimate for CSOM are slightly broader than those specified in the sample size. We had planned to use probability proportional to size sampling to select the village clusters, however recent flooding and food insecurity in the area resulted in substantial population movements, invalidating previously available census data on village sizes. To ensure a probability sample, we selected the village clusters by simple random sampling, which resulted in unequal cluster sizes and likely increased the sampling design effect and uncertainty around our prevalence estimate. Additionally, we sampled more 4 and 5 year-olds than 6 year-olds. Children commence schooling aged 6 and it may be these children were more likely to be a source of non-response. Due to the narrow age-inclusion criteria, this is not thought to substantially affect the prevalence estimate.The study was not powered to formally assess potential associations of CSOM, and analysis was limited by the low number of CSOM cases observed. Crowding and increasing age appeared to display a positive effect size. There was no difference observed in CSOM prevalence in CAPS intervention versus control households. Possible explanations for this include inadequate observed case numbers, inadequate emissions reductions from the cookstoves in the field, inadequaIn conclusion, we found a high burden of CSOM and other potentially treatable ear diseases and preventable hearing impairment in our sample of school-entry age children in rural Malawi. Since CSOM can adversely affect academic performance and development there are substantial public health implications of these findings which, we suggest, are generalizable to surrounding areas with similar geography and development indicators. The identification and management of middle ear disease and hearing impairment represent major unmet needs in this vulnerable population.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "We have reported immunotargeting of \u03b16\u03b24 for radionuclide-based and near-infrared fluorescence imaging in a pancreatic cancer model. In this study, we prepared yttrium-90 labeled anti-\u03b16\u03b24 antibody (90Y-ITGA6B4) and evaluated its radioimmunotherapeutic efficacy against pancreatic cancer xenografts in nude mice. Mice bearing xenograft tumors were randomly divided into 5 groups: (1) single administration of 90Y-ITGA6B4 (3.7MBq), (2) double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq \u00d7 2), (3) single administration of unlabeled ITGA6B4, (4) double administrations of unlabeled ITGA6B4 with once-weekly schedule and (5) the untreated control. Biweekly tumor volume measurements and immunohistochemical analyses of tumors at 2 days post-administration were performed to monitor the response to treatments. To assess the toxicity, body weight was measured biweekly. Additionally, at 27 days post-administration, blood samples were collected through cardiac puncture, and hematological parameters, hepatic and renal functions were analyzed. Both 90Y-ITGA6B4 treatment groups showed reduction in tumor volumes (P < 0.04), decreased cell proliferation marker Ki-67-positive cells and increased DNA damage marker p-H2AX-positive cells, compared with the other groups. Mice treated with double administrations of 90Y-ITGA6B4, exhibited myelosuppression. There were no significant differences in hepatic and renal functions between the 2 treatment groups and the other groups. Our results suggest that 90Y-ITGA6B4 is a promising radioimmunotherapeutic agent against \u03b16\u03b24 overexpressing tumors. In the future studies, dose adjustment for fractionated RIT should be considered carefully in order to get the optimal effect while avoiding myelotoxicity.The contribution of integrin \u03b1 Pancreatic cancer is a malignant disease with poor prognosis. According to the Globocan 2012 data, the estimated numbers of new cases and deaths caused by pancreatic cancer worldwide in 2012 were about 338000 and 331000, respectively , white blood cell  and platelet counts [mean (43.7 \u00b1 25.2) \u00d7 104/\u03bcl (57.8% of the untreated control)]  \u00d7 105/\u03bcl (88.5% of the untreated control)]. Additional biochemical tests showed minimal side effect of RIT on liver and renal functions. The values of serum GOT , GPT (glutamic pyruvate transaminase), ALP  values and serum CRE (creatine) and BUN (blood urea nitrogen) were not increased  accumulates in tumor. Although the software and hardware improvements are necessary, the 90Y-ITGA6B4 PET images were successfully acquired, validating the localization of the antibody in target tumor, through the CT image co-registration  -trans--cyclohexane-1,2-diamine-N,N,N\u2032,N\u2032,N\u2032-pentaacetic acid (CHX-A\u2032\u2032-DTPA)  were mixed in a molar ratio of 1:2.5 and incubated overnight at 37\u00b0C. The conjugation ratio of DTPA and antibody was estimated to be 1.6, calculated from the ratio of 90Y-DTPA-antibody to 90Y-DTPA, determined by isoelectric focusing. Unconjugated DTPA was removed using a Sephadex G-50 column . Afterward, DTPA-conjugated antibody (83 \u03bcg in 0.1 M sodium acetate buffer [pH 6.0]) was incubated with the mixture of 90Y-chloride  and 1 M sodium acetate buffer (pH 6.0) for 30 min at room temperature (RT). The radiolabeled antibody was purified using a Sephadex G-50 column (730\u00d7 g for 2 min). The radiochemical purity determined by TLC was > 99%. The radiochemical yield was approximately 85 to 90%, and the specific activity was approximately 758 to 802 kBq/\u03bcg.Human anti-\u03b1reported . Briefly90Y-ITGA6B4 (3.7MBq), group (2) received double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq \u00d7 2), group (3) received a single administration of unlabeled ITGA6B4, group (4) received double administrations of unlabeled ITGA6B4 with the once-weekly schedule, and group (5) received no injections. Each quantity of the injected antibody was adjusted to 20 \u03bcg, by the addition of the intact antibody. Tumor- and tissue-absorbed doses for 90Y-labeled antibody were estimated from the biodistribution data of 111In-labeled antibody )\u00d7(width [mm])2/2. Relative tumor volume was calculated as the volume at the indicated day divided by the volume at 1 day before start of the treatment.To monitor the tumor response, tumor volumes were measured twice a week throughout the experiment, using caliper, and approximated using the equation: volume  were extirpated, fixed in 4% paraformaldehyde, and embedded in paraffin. Untreated tumor was used as control. Ki-67 staining of 4 \u03bcm thick sections was performed using an anti-human Ki-67 polyclonal antibody , as previously described [4 expression in tumor cells, rabbit anti-human \u03b24 integrin (H-101) polyclonal antibody  was used as primary antibody. HRP-linked polymer anti-rabbit (Dako Denmark), was used as secondary antibody. To detect the apoptotic tumor cells, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining with an ApopTag Peroxidase In Stiu Apoptosis Detection Kits  was performed. Each slide was observed by using microscope Olympus BX43 system .At 2 days after the treatment with escribed . Phospho18F-FDG (2.0 MBq). At 50 min following the injection, static PET data acquisition was conducted for 10 min, using a small-animal PET system , under 1.5% isoflurane anesthesia. Images were reconstructed using a maximum a posteriori (MAP) method with attenuation correction using Inveon Acquisition Workplace software . Using ASIPro VM software , regions of interest (ROIs) were manually drawn on 3 slices in coronal, transverse, and sagittal planes of each tumor, and the standardized uptake value (SUVmax) was measured in each ROI, for the quantitative analysis.FDG-PET scans were conducted before, and at 6 and 13 days after antibody administration. Tumor bearing mice from each group (n=3-4) were intravenously injected with To assess the toxicity, body weight of each mouse was measured every 3-4 days. At 27 days after the first administration, all mice were euthanized. Their blood samples were collected through cardiac puncture, and the hematological parameters and biochemical parameters relevant to hepatic and renal functions were analyzed. We used an automated hematology analyzer, Celltac Alpha , for hematological and automated clinical chemistry analyzer, DRI-CHEM 7000V , for biochemical tests.t-test . P-values < 0.05 were considered significant.Significant differences between the groups were determined by Student's"}
+{"text": "In the last decade, the attention for health literacy has increased in the European Union. This is due to three main reasons. First, reviews have shown that inadequate health literacy is associated with worse health outcomes, higher health care use and expenditure. Second, in all European countries the population is aging and the number of chronically ill people is rising. Improving health literacy in this group can offer greater opportunities to take an active part in society, be independent and improve quality of life. Third, since most research on health literacy has been conducted outside Europe and relatively little is known about the development of health literacy interventions and its effects on outcome measures in European countries. The aim of this systematic review was to assess the evidence on the effectiveness of health literacy interventions in the European Union published between 1995 and 2018.Searches have been performed in Medline, PubMed, EMBASE, CINAHL, Cochrane library, PsychINFO, ERIC, Web of Science and SCOPUS for publications on health literacy intervention studies in European Union countries. Studies were included if the research was conducted in one or more Member States of the European Union, the publication described an intervention study, the intervention was aimed at health literacy, the publication described an outcome measure related to health literacy and the publication was written in English, French or German.A total of 23 studies were included. Three types of interventions were identified; aimed at improving health literacy, tailored to different health literacy levels and aimed at improving health outcomes in general that differentiated in effects for people with different health literacy levels. Most interventions identified in the review focus on the functional level of health literacy or numeracy. The strength of evidence from the European health literacy intervention studies was low and there was a huge heterogeneity in study design, measurement tools and outcomes measured.Promising interventions were tailored to the needs of patients, addressing functional, interactive and critical skills and use not difficult animated spoken text. Future research should focus on the development and assessment of such interventions and use stronger designs.The online version of this article (10.1186/s12889-018-6331-7) contains supplementary material, which is available to authorized users. Health literacy is a topic of growing importance in European public health research. In general, health literacy is \u2018linked to literacy and encompasses people\u2019s knowledge, motivation and competences to access, understand, appraise, and apply health information in order to make judgments and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life during the life course.\u2019 This is the definition of health literacy as it was developed in the European Health Literacy Project (HLS-EU) , 2. ThisApart from this one, there are many different definitions and conceptualisations of health literacy . Narrow Especially in the last decade, the attention for health literacy has increased in the European Union (EU). This is due to three main reasons. First, studies mainly from the United States of America (USA) have shown that inadequate health literacy is associated with worse health outcomes, poor preventive care behaviours, higher health care service use and expenditures. In addition these studies showed that health literacy influences the effects social determinants of health have on health status and as such is an important determinant of health inequalities \u20138. ThereTo determine the efficacy of health literacy interventions in the EU context, a similar systematic review as the review conducted by Berkman et al.  and Dennhttps://ec.europa.eu/health/sites/health/files/health_policies/docs/2015_health_literacy_en.pdf.This systematic review was conducted in accordance with the PRISMA  guidelines . This reStudies were identified by searching Medline, PubMed, EMBASE, CINAHL, the Cochrane library, PsychINFO, ERIC, Web of Science and SCOPUS. English, French and German language publications on health literacy intervention studies in EU countries. According to our knowledge, no research on health literacy has been done before 1995, therefore studies from January 1995 to Augusts 2018 were included. Recent reviews on health literacy that developed search strategies based on a list of key words and text words for use in the different databases were used to construct our own search strategy. For the search strategy, the reviews of Berkman et al.  and DennAll studies that describe an intervention study with one of the following designs were included: randomized controlled trials, quasi randomized controlled trials, controlled before and after studies or interrupted time series. Studies with no original data, studies with only case report and studies with only ecological data were excluded.Studies involving people living in one or more member states of the EU were included. The Member States of the EU are Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden and the United Kingdom.Studies with an intervention that focused on health literacy were included. Interventions at population level, as well as interventions on specific populations and individual level were searched for and included. Studies on the basic experimental science of reading ability were excluded as were studies examining normal reading development in children and studies about dyslexia. Contrary to the strategy of Berkman et al. , the seaStudies that described an outcome measure related to health literacy were included. These outcome measure included among others: knowledge, skills, attitudes, self-efficacy, stages of change, motivation and patient activation, behaviour change, health care access, service use, health status, costs of care.The study selection consisted of two phases: first the selection on title and/or abstract and second the selection of the remaining articles based on full text. The search results were screened by two researchers each in two independent phases ; BV, BS (2015\u20132018)). Consensus meetings were held with the researchers of both phases to resolve disagreements. A 20% sample of the excluded scientific publications was screened by a third researcher (JR 1995\u20132018). Studies were included if they met the inclusion criteria.global rating is calculated using information across all six domains : strong (no weak ratings), moderate (one weak rating), or weak (two or more weak ratings).The abstracts were systematically screened on the basis of our in- and exclusion criteria. In case a publication did not meet a criterion, the publication was excluded and the next publication was screened. Of all the studies, fulfilling the inclusion criteria, full texts were read. For the inclusion of full texts the same in- and exclusion criteria were used. To assess the quality of the studies the \u201cQuality Assessment Tool For Quantitative Studies\u201d developed by the Effective Public Health Practice Project (EPHPP)  was usedOur literature search yielded 6206 publications between 1995 and mid-2018. Of these publications 6042 (97%) were excluded based on title and abstract because they did not fulfil one or more of the inclusion criteria: 3950 (65%) were excluded because not describing an intervention, 1037 (17%) did not meet the first criterion (being conducted in one or more of the European Member States), 1024 (17%) of the European interventions were excluded because they were not focusing on health literacy and 26 (1%) of the studies were excluded because there was no health literacy outcome measure  of data; therefore a qualitative analysis was performed. The 23 included intervention studies and their characteristics are summarized in Table Studies varied considerably in their measurement of health literacy. Commonly used instruments in the USA to assess health literacy such as the Raped Estimate of Adult Literacy in Medicine (REALM) , 19, then\u2009=\u20099). The web-based interventions were conducted during the most recent years, most of them (n\u2009=\u20097) in 2015, 2016 and 2017. In only one study it was explicitly mentioned that the patients were involved in the development of the intervention on a module designed for the development of a decision aid about MS-immunotherapy [There was also a huge variation in the type of interventions given: group interventions, individual interventions, web-based interventions, one component interventions (e.g. an information leaflet) and multi-component interventions including chat-groups, lectures, training sessions, a help-desk, computer programs and leaflets among others. Most interventions were web-based interventions (otherapy .n\u2009=\u20099) used the same group that was pretested and post-tested immediately after the intervention (Cohort study). The study by White et al. [n\u2009=\u200915) the quality was rated as weak  design , 27, 34 e et al.  used an k  the health literacy level of individuals.Interventions that were specifically tailored to different health literacy levels.General interventions that aimed at improving health outcomes, which described the specific effects for patients with different health literacy or numeracy levels.The types of interventions in the 23 studies could be categorized as follows;A group training of 2\u2009\u00d7\u20092,5\u2009days in evidence based-medicine for patients, patient counsellors, consumer representatives and healthcare professionals resulted in a significant increase in health related knowledge and in the level of critical health literacy of the participants . In the An intervention that was developed to improve self-care among diabetes patients was evaluated after two years. The patients had received tailored tele-carer education as well as support to change specific lifestyle behaviours . The evaFive interventions specifically focused on the improvement of numeracy skills, i.e. the ability to understand numerical risk information , 35\u201337. Three studies , 27, 28 Three studies focused on the way of presenting information to persons with different health literacy levels , 25, 34.In general, patients with low health literacy benefit less from general interventions compared to patients with higher levels of literacy, e.g. with respect to understanding medication labels , 35 and Four studies reported on outcomes relevant for the daily management of chronic illness or health in general such as knowledge, empowerment, ability to self-manage, decision-making skills, ability to taken an active role in treatment. Increased levels of health literacy were associated with higher levels of empowerment, better decision-making skills, and a more active role in treatment \u201332. The In this systematic review the evidence on the effectiveness of health literacy interventions in the EU published from 1995 until mid-2018 was assessed. There were not a sufficient number of studies with similar outcome measures or similar interventions to consider quantitative analysis  of data; therefore a qualitative analysis was performed. The evidence collected gives insight into the gaps in research in the context of the European Union, compared to the evidence presented in the already published reviews outside Europe, and provide recommendations for research. To our knowledge, this is the first systematic review on health literacy interventions in the EU context. The results of this review are compared to the results of reviews in the USA context.In total, 23 intervention studies were identified. The interventions described in these studies either (a) aimed at improving (aspects of) health literacy, (b) were specifically tailored to different health literacy levels or (c) were general interventions that aimed at improving health outcomes, which described the specific effects for patients with different health literacy or numeracy levels. As was found in other review studies , 14, 40 The studies varied with respect to their study design, measurement instruments and outcomes. Health literacy was also operationalized and measured differently, thus impeding comparability of the results. Most studies did not give information whether their study results were stratified across health literacy levels. This was also concluded in the review of D \u2018Eath et al. . As a reBecause of the low quality of the studies no firm conclusions can be drawn with respect to the effective components of health literacy interventions. It seems that the type of intervention  is not of major importance. However, three factors are likely to be distinctive of promising interventions: (1) they tailor their activities to the needs of the  participants, (2) they also address interactive and/or critical skills (instead of knowledge only) and (3) they present the information in an appropriate way, i.e. not difficult and using animated spoken text. Studies that also focus on interactive and/or critical skills led to improvements in outcomes such as motivation, knowledge, empowerment and self-confidence. These findings are congruent with those from the review by Berkman et al. . InterveDespite the small number of studies, findings from the EU are in line with the results from other international reviews , 14, 40.There are definitely considerable gaps in the research evidence concerning which interventions are most effective in improving health literacy or health literacy related outcomes in Europe. In order to be able to draw firm conclusions, there should be more agreement among researchers about the definition of health literacy, and more systematic use of validated measurement tools in interventions as a \u201cgolden standard\u201d. In the past years several studies on the development, translation and validation of (both subjective and objective) health literacy measurement instruments have been done. As a consequence, the assessment of health literacy varies depending on the setting and scope of the health literacy definition. The results of future intervention research then become more comparable and generalizable, leading to a more rapid insight in what constitutes effective health literacy interventions in the EU context.New developed interventions should be tailored to the needs of the patients; address functional, critical and interactive skills and the way of presenting should be not difficult animated spoken text. Web-based interventions might be suitable for patients that have digital skills, but also blended interventions (combining face-to-face with online activities) and other types of interventions might integrate these three factors in their design. Future research should focus on the assessment of such interventions and use stronger designs e.g. in well-reported, large-sampled randomized controlled trials.Additional file 1:Final search strategy (DOCX 13 kb)"}
+{"text": "Low health literacy has been associated with poor cancer screening uptake, difficulty in making treatment choices and reduced quality of life following a cancer diagnosis, yet it is unclear whether and how health literacy influences the pathway to diagnosis for patients with cancer symptoms. This systematic review aimed to evaluate the influence of health literacy on the timely diagnosis of symptomatic cancer. Literature was searched between January 1990 and May 2017 using MEDLINE, Embase, Scopus, ASSIA, CINAHL and PsycINFO. Only three papers met the inclusion criteria. These reported two qualitative studies and one quantitative, with adult patients diagnosed with gastrointestinal , cervical and breast cancer. The definition and assessment of health literacy varied between the studies, as did the descriptions of the pathway to diagnosis. Due to the methodological weaknesses identified, the conclusions are limited; however, the studies did highlight important considerations in the definition and measurement of health literacy. Further research is required that clearly defines health literacy and follows the principles of the Aarhus Statement to assess the influence of health literacy on the pathway to cancer diagnosis. The protocol for this review was registered with PROSPERO (CRD42016048917). The search was limited to 1990 onwards as health literacy is a relatively new field, with the number of studies expanding following the publication in 1991 of the first widely used health literacy instrument, the Rapid Estimate of Adult Literacy in Medicine (REALM)  with a primary diagnosis of any cancer and explored the influence of health literacy  in relation to the time to diagnosis of symptomatic cancer. This included studies evaluating the total time to diagnosis, from symptom onset to diagnosis, or focusing on one or more intervals along the pathway: appraisal, help\u2010seeking or diagnostic intervals. Systematic reviews, reviews, editorials and letters were excluded, along with studies reporting time to diagnosis without health literacy, or vice versa, or studies focusing on recurrent cancers, cancer incidence, survival and mortality, risk factors, genetics, screening and prevention, or assessing the validity of referral decisions.2.3Following removal of duplicate references, EH screened the titles and abstracts against the inclusion and exclusion criteria, with a random sample  assessed by FMW and JB to confirm agreement. The full text was obtained for all studies identified as potentially relevant to the review. Three reviewers  screened all the full\u2010text articles to identify studies for inclusion in the review.2.4Data were extracted by EH from each of the included studies: study type, recruitment setting, data collection details, participant characteristics, patient pathway/interval data as defined within the study, health literacy data including the definition and health literacy instrument used (if any), and the findings in relation to time to diagnosis. The extracted data were reviewed by FMW and JB to confirm completeness.2.5Quality of the studies was assessed by EH and reviewed by FMW and JB, using the Joanna Briggs Institute Critical Appraisal Tools , an international prospective register of systematic reviews. The review is reported based on the guidelines proposed by the PRISMA Statement: Preferred Reporting Items for Systematic Reviews and Meta\u2010Analyses , cervical or breast cancer Table . Two stu3.3The studies were assessed based on methodological quality and conceptual clarity in relation to definitions of \u201ctime to diagnosis\u201d and \u201chealth literacy.\u201d3.3.1Table 3.3.2All the studies aimed to explore the diagnostic pathway from the patients\u2019 perspective, from symptom onset to diagnosis or start of treatment. Table SD unknown), while symptom onset to treatment onset  was 8.82\u00a0months . The two qualitative studies did not seek to quantify the time from symptom onset to diagnosis, although patients\u2019 narratives were used to demonstrate how the time to diagnosis may be influenced  was 5.26\u00a0months  , with eight (22%) women scoring 54 or less and demonstrating low health literacy. The authors stated that no statistically significant correlations were found between the REALM\u2010SF scores and the patient interval (time from symptom onset to first presentation) across the whole cohort; however, they did not provide data to substantiate this. A sub\u2010group analysis of the eight women with low health literacy found that four had a patient interval of 6\u201312\u00a0months; although, patient intervals were not reported for the 29 women who scored >54 on the REALM\u2010SF. The survey also explored symptom experience, knowledge and help\u2010seeking behaviours, yet presented these as descriptive statistics without any analysis of how these factors may correlate with health literacy or the patient interval.In contrast to the qualitative studies, the survey study conducted by Tecu and Potter quantified the time to diagnosis and explored health literacy using the REALM\u2010SF. The mean REALM\u2010SF score was 60.08 (4This systematic review sought to explore how health literacy can influence the patient's pathway to diagnosis with cancer, as health literacy may affect a patient's ability to access and understand cancer symptom information, appraise the information in relation to bodily changes and navigate the healthcare system to access the specialist care required to obtain a diagnosis. Although the search strategy was broad and undertaken across multiple databases, only three studies, all methodologically poor, met the inclusion criteria. There is an important gap in the literature when considering the role of health literacy in timely diagnosis of cancer.The included studies used three very different approaches to assess health literacy, which reflects the current challenge of health literacy research in respect to definitions and measurement. Sorensen et al.  conducte4.1Two of the 23 studies excluded from the review following full\u2010text assessment used validated measurement tools to assess health literacy in relation to stage at diagnosis (Bennett et al., 4.2In searching a wide range of databases with a broad search strategy, we are confident that we identified all articles that aimed to explore health literacy on the time to diagnosis of cancer. However, definitions of health literacy vary considerably, as seen in the study by McEwan et al. where aspects of health literacy were explored without defining them as \u201chealth literacy.\u201d In this review, we specifically searched for \u201chealth literacy\u201d or variations of the term and therefore other studies exploring time to diagnosis of cancer, particularly using qualitative methodology, could have investigated areas that are linked to health literacy yet not recognised them within this context and therefore these papers would not have been identified within the review.A further review investigating qualitative time to diagnosis research and evaluating the results in respect to a recent multi\u2010dimensional definition of health literacy may now be needed.5Due to the few studies identified from the systematic search, and their methodological weakness and relatively poor quality, it was not possible to fully evaluate the influence of health literacy on the timely diagnosis of symptomatic cancer. However, the studies provide a starting point for research within this area and identify important aspects that need to be addressed in future research. When exploring diagnostic routes for cancer, researchers should be guided by the Aarhus Statement and underpin their research with a conceptual framework and clear definitions. Where health literacy is explored, researchers should also be aware of the range of health literacy definitions and assessment tools currently in use, and how these could relate to their research. Again, they would be advised to choose a definition best suited to their research area and to reflect on this and the aim of the research when choosing an appropriate instrument or method for exploring health literacy.Reducing the patient interval is important for earlier diagnosis of cancer and it is possible that health literacy may influence the pathway, which could have important implications for developing targeted awareness campaigns for recognition of cancer symptoms and to prompt timely help\u2010seeking, as well as informing GP\u2010patient communication strategies. Research exploring the time to diagnosis should also consider the relation of health literacy to other factors affecting the pathway. In conclusion, further research is required that clearly defines health literacy and adheres to the principles of the Aarhus Statement to assess the influence of health literacy on the timely diagnosis of symptomatic cancer.None.All authors contributed to the design of the study and analysis of data, and were involved in drafting and revising the manuscript."}
+{"text": "The quality of the present day fluoroscopic images is sufficiently high for use as exposure images depending on the environment where the fluoroscopic images are recorded. In some facilities which use fluoroscopic images as exposure images they are recorded with a radiological x\u2010ray diagnostic device equipped with a fluoroscopic storage function. There are, however, cases where fluoroscopic images cannot be used as exposure images because the quality of the fluoroscopic image cannot be assured in the environment where the fluoroscopic images are recorded. This poses problems when stored fluoroscopic images are used in place of exposure images without any clearly established standard. In the present study, we establish that stored fluoroscopic images can be used as exposure images by using gray values obtained from profile curves. This study finds that replacement of stored fluoroscopic images with exposure images requires 20.1 or higher gray scale value differences between the background and signal, using a 20\u00a0cm thick acrylic phantom (here an adult abdomen as representing the human body) as the specific geometry. This suggests the conclusion that the gray value can be considered a useful index when using stored fluoroscopic images as exposure images. Fluoroscopic time (exposure), particularly when dealing with complicated diseases is prolonged,The reason for the lengthening of the fluoroscopy time is that it takes time to orient the guidewire to the intended direction when inserting the guidewire used for intravascular treatment.Images are also used in documenting that appropriate treatment has been performed. However, the quality of the present day fluoroscopic images is sufficiently high for use as exposure images depending on the environment where the fluoroscopic images are recorded,22.ADigital images are collected as numerical data that need to be converted to brightness values to be observed as images. The brightness is expressed as the gray level. With x\u2010ray images that have 8 bits per pixel of information, values from 0 to 256 in gray scale are represented by gradations from dark (black) to light (white). The representation with numerical values using this gradation is termed the gray value.This study uses the gray value as a value to determine balloon expansion in coronary artery treatment, as depicted in fluoroscopic and exposure images obtained with a cardiovascular x\u2010ray diagnostic device.2.BFor the cardiovascular x\u2010ray diagnosis device, we used the Allura10/10 manufactured by Philips, Innova 2100IQ manufactured by GE, and the Trinias B8 MiX package manufactured by Shimazu Co., Ltd., Hamamatsu, Japan. For the video view, we used the kada\u2010View manufactured by Photron M&E Solutions Inc., Tokyo, Japan, and ImageJ was used to analyze the images. We used a 20\u00a0cm thick acrylic phantom as the subject, and the area dose meter installed in the manufacturers\u2019 equipment as the dosimeter. For visual and physical evaluations, we used the balloon for coronary artery treatment commonly used in clinical settings: 2.5/15\u00a0mm, 3.0/10\u00a0mm, 3.5/10\u00a0mm, and 4.0/10\u00a0mm  manufactured by Kaneka Corporation, and Iovelin 350  as the contrast agent.2.CUsing each of the tested devices, we measured the x\u2010ray doses of fluoroscopic and exposure images at the irradiation reference point of patients. Figure\u00a02.DThe concentration of 350\u00a0mgI/mL contrast agent enclosed in a balloon for clinical coronary arteries, 4.0, 3.5, 3.0, 2.5, and 2.0\u00a0mm in diameter, was diluted twofold, and the balloon was dilated to the nominal size. The geometry of Fig.\u00a02.EBased on the images used for visual evaluation of balloons at different imaging angles, we calculated profile curves using ImageJ. To unify the measurement points, we calculated the profile curves at position (a), the midpoint of the balloon size in the length direction as shown in Fig\u00a033.ATable\u00a03.BFigure\u00a03.CFigure\u00a044.AIn the imaging condition with device A, the tube current was limited to 200\u00a0mA, and the tube current during fluoroscopy was 4.3\u00a0mA. The dose is determined by the mA value (tube current multiplied by irradiation time), and the manufacturer of device A may have set the low current value considering reducing the patient exposure. Further, the manufacturer may have tried to ensure the image quality by using digital filters as shown in Table\u00a04.B.In the visual evaluation, all the exposure images at the imaging angle AP scored 8 points with all devices. The dose rates of the exposure images showed about 1.3 times of difference between the lowest (device A) and the highest (device B) devices. Also, in device A, the visual evaluation scores with balloons with sizes other than 5.0\u00a0mm tended to decrease at the RAO/CAU and RAO/CRA angles. However, with devices B and C, even when the imaging angles were changed, the score showed 8 points like in AP. This is because there is a difference in the method of setting the image quality and the x\u2010ray output dose by the various facilities: device A performs visual evaluations at the AP angle which has the lowest load of object thickness, while devices B and C perform this at the angle which passes through the \u201cthickest\u201d part of the phantom (has the highest load of the object thickness) leading to the suggestion that the dose setting of the AP of devices B and C is unnecessarily high. Next, in the visual evaluations of stored fluoroscopic images, balloons with smaller diameters resulted in all devices receiving low evaluation scores. For the fluoroscopic dose rate, when assuming device A, which has the lowest dose setting, as the standard, the device B setting is 1.6 times and for device C it is about 2.2 times higher than that of device A. Usually, when the dose rate is high, the image quality improves, but the evaluation of the balloons with small diameters in devices B and C with the higher dose rate was lower than in device A. This is because device A controls the image quality by controlling the x\u2010ray dose. However, in devices B and C the signals of the balloons are flattened because these devices cut soft x\u2010rays using an additional filter, and this may lower the visual evaluation due to poor control of the image quality with the digital filter as shown in Table\u00a04.CIn the physical evaluation of balloons with different sizes at different imaging angles, the AP was the highest score  among all the balloon sizes, and the physical evaluation became poorer as the imaging angle became larger. This may be because the tube voltage increases and the image blurs due to the larger imaging angles than that of AP, eliminating the difference between the contrasts recorded with the contrast agent in the balloon and the gray value of the background. Further, it may also be because, as the balloon diameter decreases, the amount of contrast agent in the balloon decreases, and as a result the differences in the background gray value disappear when the contrast decreases.Figure\u00a05This study finds that replacement of stored fluoroscopic images with exposure images requires 20.1 or higher gray value differences between the background and signal, using an acrylic phantom of 20\u00a0cm thickness (representing the abdomen of a human adult) a specific geometry. This suggests the conclusion that the gray value can be considered a useful index when using stored fluoroscopic images as exposure images.The authors declare no conflicts of interest associated with this manuscript."}
+{"text": "Musa spp.) are the most important fruit crops worldwide due to their high nutrition value. Fusarium wilt of banana, caused by fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc 4), is considered as the most destructive disease in the world and results in extensive damage leading to productivity loss. The widespread use of plant resistance inducers (PRIs), such as benzothiadiazole (BTH), is a novel strategy to stimulate defense responses in banana plants to protect against pathogens infection. The recent focus on the crop defense against fungal infections has led to a renewed interest on understanding the molecular mechanisms of specific PRIs-mediated resistance. This transcriptome study aimed to identify genes that are associated with BTH-induced resistance. Patterns of gene expression in the leaves and roots of BTH-sprayed banana plants were studied using RNA-Seq.Bananas  contains supplementary material, which is available to authorized users. The inability of higher plants to escape from microbial pathogen attacking has led to the development of a diverse set of constitutive and/or inducible defence mechanisms to resist and coexist with their pathogens . Ongoing2+, reactive oxygen species (ROS) and multiple protein kinases. These signaling components modulate downstream regulatory protein activities, such as transcription factors (TFs), which lead to massive transcriptional reprogramming and result in accumulation of various enzymes, pathogenesis-related (PR) proteins and pathogen infection-responsive metabolites [2+ concentration in plant cells are believed to be one of the early intracellular reactions following pathogen perception, which are sensed by Ca2+-binding proteins and kinases, such as calcium dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs) [Arabidopsis, CDPKs have proved to be required for FLS2-dependent immunity [Arabidopsis [ERFs) and WRKYs can be activated by MAPKs, and play broad and pivotal roles in regulating defenses [WRKY genes that are responsive to Sclerotinia. sclerotiorum in Brassica. napus have been reported [Both PTI- and ETI-associated immune responses act through a common set of signaling components, including Caabolites . The cha (MAPKs) . In Arabimmunity . Importabidopsis . Moreovedefenses , 8. Genoreported . ROS serreported . A rapidreported . Accordireported , indicatFusarium pathogen infection, indicating the importanceof SA in plant defence [Arabidopsis, tomato, wheat and cucumber [NPR1A and NPR1B were strongly induced by Foc infection in banana [Fusarium oxysporum f.sp. conglutinans (Focn) or Fusarium oxysporum f.sp. raphani (Forp) inoculation in Arabidopsis [In the last decades, phytohormones such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and auxin, have been extensively studied and demonstrated to play conserved and divergent roles downstream of PTI or ETI activation in defense responses , 13. Acc defence . Exogenocucumber \u201321. The n banana . Consistn banana . Mutatiobidopsis , 24. Accbidopsis . In contbidopsis , 27. Recbidopsis , 29.Fusarium oxysporum f. sp. cubense tropical race 4 (Foc 4), is a soil-borne lethal disease of Cavendish bananas in the world [Fusarium wilt, which is mainly caused by the fungal pathogen he world , 30, 31.he world . Typicalhe world . Previouhe world . A greathe world . BTH hashe world , 34, 35,he world , 36\u201339, Musa spp. AAA group) was used in the study. Two-month-old seedlings were grown in plastic pots filled with sterilized soil at 26\u2009\u00b1\u20092\u00a0\u00b0C with a 16\u00a0h/8\u00a0h light/dark period and with 80\u201390% relative humidity in greenhouse of Chinese Academy of Tropical Agricultural Sciences, Haikou of China (N20\u00b002\u2032and E110\u00b011\u2032). The BTH treatment was performed by spraying the leaves of the banana plant with the 50\u00a0mg/L, 100\u00a0mg/L BTH. Plants sprayed with distilled water were used as the negative controls. For inoculation, the Foc 4 was used to inoculate roots as previously described [Cavendish banana . A total of 105 gigabase raw sequence reads of 18 libraries was obtained initially on a HiSeq 2500 instrument that generated paired-end reads with 150 nucleotides in length. We discarded reads with adapters, those with more than 5% unknown nucleotides, and those of low quality \u2009\u2264\u200919). The average proportion of clean reads in each sample was 88.5\u201390.2%. The filtered clean reads from each sample were aligned to the reference banana genome with mapping rate 73\u201378%, and were assembled into non-redundant unigenes using Cufflinks v2 method . The FPK\u2212\u20095. As described previously [P-value in GO and KEGG analyses. We used an FDR of <\u20090.05 as thresholds to define significantly enriched GO terms and KEGG pathways.To obtain functional annotation and identify the putative biological pathways of DEGs, we annotated the DEGs using the NR protein database (NCBI), GO and KEGG databases with similarity set at >\u200930% and an E-value \u22641eeviously , The GO eviously . KOBAS seviously . The FDR-\u0394\u0394Ct. The banana MaACTIN gene was used as a constitutive reference. The qPCR data were presented as means \u00b1 SD. Asterisks indicate significant differences compared with the control by Student\u2019s t-test.Total RNA was isolated from the leaf and roots of banana plants sprayed with 100\u00a0mg/L BTH, as described above. First-strand cDNA synthesis was performed using PrimeScript\u2122 RT reagent Kit (Takara) according to the manufacturer\u2019s instructions, using 2\u00a0\u03bcg total RNA and oligo(dT) primers. qRT-PCR was performed using SYBR\u00ae qPCR Mix  according to the manufacturer\u2019s protocol. The cycling conditions were as follows: 5\u00a0min denaturation at 95\u00a0\u00b0C, followed by 40\u00a0cycles at 95\u00a0\u00b0C for 15\u00a0s, and 60\u00a0\u00b0C for 40\u00a0s. Melting curve analysis was performed over the range of 65 to 98\u00a0\u00b0C. The primers used in this study were listed in Additional\u00a0file\u00a0BTH has already been documented to be an identical efficient activator of disease resistance in several plant species , 34, 35.Musa spp. genome and assembled into 38,995 transcripts . By analyzing the expression patterns of these genes in leaves and roots for each time point, we observed a large number of genes that exhibited highly dynamic changes after 1 day BTH treatment in both leaves and roots Fig.\u00a0. FurtherTo functionally classify those BTH-responsive DEGs, gene ontology (GO) enrichment analysis of all the DEGs was utilized, and a total of 298 GO terms were significantly enriched  database to discover those genes involved in metabolic or signal transduction pathways. We identified 15 and 25 pathways that were significantly enriched in leaves and roots, respectively, which were in agreement with the GO terms results , 57% (16 genes) of WRKY genes showed altered expression after biotic stress induced by a common fungal pathogen (Coniothyrium diplodiella) infection [Ralstonia solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways in tobacco [ERF genes in transgenic Arabidopsis or tobacco plants induces expression of several PR and hormone-responsive genes, resulting in enhanced resistance to pathogen [Transcription factors (TFs) play major roles in plant development and defense response through the temporary and spatial inducing or repressing the transcription of their target genes . In thisnfection . In pepp tobacco . The ERFpathogen , 57. TheSlCycB2, play a critical role in reproductive organ development, trichome initiation and Prodenia litura defense [KEGG analysis revealed the induction of 34 DEGs encoding for cell cycle-related proteins and enzymes in roots at the early time point, while no significant enrichment was observed in leaves confirming the tissue-dependent involvement of cell cycle pathway in the BTH response. In Fig.\u00a0 defense . The incYUCCAs, such as Ma8G00130, Ma3G08830 and Ma11G12150, were up-regulated by BTH in roots. Similarly, Two PIN proteins (Ma7G17140 and Ma8G29780), which mainly function as auxin efflux carriers, were also triggered by BTH in roots. However, the majority of these auxin-related DEGs (80.9%) were found to be down-regulated or unchanged during the two stages in leaves. Oppositely, genes involved in ET biosynthetic and signaling transduction processes were predominantly increased in leaves, and only a few of these genes were affected at both time points in roots  were increased significantly after BTH treatment. Moreover, one ethylene receptor  and two EIN3-binding F-box proteins , which play a significant role in perceiving and transducing the ET signals, were also induced specifically in leaves but not in roots in response to BTH-spraying  proteins have been widely documented to play specific roles in plant-pathogen interaction through recognizing the effector molecules . In the flavonol synthase caffeic acid (FLS), caffeic acid 3-O-methyltransferase (COMT), caffeoyl-CoA O-methyltransferase (CCOMT), caffeoylshikimate esterase (CSE), isoflavone reductase (IFR) and leucoanthocyanidin dioxygenase (LDOX)  were induced by BTH in roots  plays an essential role in plant defense via catalyzing the production of S-adenosyl-L-methionine, which supplies as a methyl-group donor in the biosynthesis of numerous secondary metabolites. SAMs were observed to be down-regulated in leaves at 1 dpt, but were restored at 3dpt. Whereas, at 1 dpt, SAMs were mainly up-regulated by BTH in roots , GLUTAREDOXIN (GRX), L-ascorbate oxidase (ASOD), L-ascorbate peroxidase (APX) and glutathione s-transferase (GST) genes were differently modulated by BTH in the two tissues. Approximately 21.2% of up-regulated DEGs in roots exhibited a different expression pattern in leaves, where they were mainly suppressed by BTH. For example, the TRX genes Ma10G21720 and Ma3G25160 were up-regulated at 1 dpt and 3 dpt in roots (Fold change\u2009=\u20095.7 to 145.2), but were down-regulated in leaves. In roots, BTH primarily induced the majority of these DEGs at 1 dpt. However, a different trend was observed in leaves, as the numbers of up-regulated and down-regulated DEGs were similar , polygalacturonase, pectinesterase, cellulose synthase, galacturonosyl transferase and expansin involved in cell wall modification or maintenance were significantly modulated by BTH , and only a few of these genes were affected at 3 dpt. In contrast, the numbers of up-regulated genes at 1 dpt and 3 dpt in leaves were similar , MaARF (Ma4G22520), MaERF (Ma10G04450), MaMCM8 (Ma11G08430), PR proteins (Ma9G16540) and RGA1 (Ma6G19890). As shown in Fig.\u00a0Arabidopsis have often been reported to be associated with increased accumulation of transcripts encoding LRR-RLK proteins. Brassinos teroid insensitive 1-associated receptor kinase 1 (BAK1) constituted one of the best studied plant LRR-RLK, and was shown to function as pattern recognition receptors mediating the recognition of microbial surface structures [R genes represent important intracellular receptors that play specific roles in pathogens\u2019 resistance [VaRGA1 gene enhances disease resistance and abiotic stresses tolerance in Nicotiana benthamian [MaLRR-RLK, MaARF, MaMCM8 and MaRGA1  and ARFs were induced in root especially at the relatively early time points analyzed. Additionally, the expression levels of ACC synthases (ACSs), ethylene receptors (ERSs), EIN3-binding F-box protein (EBFs) and ERFs were significantly increased in leaves. An interesting feature about the activation by BTH was the differential tissue-specific response. Briefly, DEGs associated with ET signaling pathway were primarily modulated by BTH in leaves, whereas DEGs participate in auxin biosynthesis, transport and response were mainly induced in a root-specific manner. Previously studies on transcriptomic analysis of Foc 4 infected banana roots have revealed the differential expression of JA- and ET-related genes, but SA and auxin marker genes were unaffected [Plant hormones such as SA, JA, auxin and ET, are well-known to play critical roles in plant-fungal interactions . In genenization . Transcraffected , 37, indaffected , 32, 38.thaumatin-like proteins (TLPs) genes and lipid transfer proteins (LTPs) were intensely induced at early stage of treatment. TLPs affect the fungus growth with \u03b2-glucanase and xylanase inhibitor activities, and have been reported against a variety of filamentous fungal pathogen [TLP genes in transgenic plants have shown enhanced resistance and protection against different fungal pathogens [Arabidopsis thaliana and in Saccharomyces cerevisiae [In response to pathogen infection, plants develop systemic acquired resistance (SAR), a state of preparedness that provides elevated resistance during subsequent infections . Besidespathogen . Overexpathogens . Lipid trevisiae . Additioendoglucanase, chitinase, glucan endo-1,3-beta-glucosidase, polygalacturonase, pectinesterase, cellulose synthase, XTHs, galacturonosyl transferase and expansins were found to increase in BTH-treated banana plants. It should be mentioned that chitinases and glucanases represent two large apoplastic protein families which are known to limit fungal growth via the degradation of the glucans from fungal cell walls [Verticillium longisporum infection of Arabidopsis leads to a specific increase of several extracellular proteins which mainly function in defense and cell wall metabolism [The plant cell wall provides a structural framework to support plant growth and acts as a pathological and environmental barrier that defends against pathogens. Typical components of the cell wall include pectins, cellulose, hemicelluloses, lignins, proteins and water . In respll walls . Previoutabolism . The agrtabolism . These rtabolism . TherefoZmWAK, which encodes a maize wall-associated kinase gene and confers quantitative resistance to head smut, was isolated by map-based cloning [Arabidopsis lectin S-domain-1 receptor-like kinase (LORE) acts as a receptor in sensing the lipid A moiety of bacterial LPSs (lipopolysaccharides), and contributes to plant immunity in response to bacteria [Arabidopsis LysM-RLKs have been shown to be essential for chitin-induced defenses in plants [RLKs play essential roles in plant immunity and development . Plants  cloning . Lectin- cloning . The Arabacteria . LysM don plants . LRR-RLKn plants . Therefo2+ transduction to activate downstream responsive genes, including TFs, PR genes, disease resistance and SAR-related genes. Furthermore, BTH altered the concentration of Ca2+ and ROS in the cytoplasm and elevated a series of ROS homeostasis related genes. The significantly altered TFs, such as bHLHs, ARFs and ZFs activate the expression of cell wall remodeling proteins, which may further affect cell wall organization and form a physical barrier that results in decreased Foc 4 infection.Taken together, comparative analyses of transcriptomic data suggest that a different initial impact of BTH on shoots and roots Fig.\u00a0. More spThe current study delineates BTH-mediated endogenous biotic defense mechanisms adapted by banana plants under Foc 4 infection. A large number of DEGs associated with signal transduction, transcription activation/repression, disease resistant and ROS/hormone signaling pathways were identified in leaves and roots of BTH-sprayed plants. More importantly, the unique positive impacts of BTH on the cell cycle process and cell wall organization in roots haves also been observed. This ultimately may help to modify the structure of plant cell wall, which attributes a key role to protect plant roots from Foc 4 infection. Taking such insights into account, The DEGs identified in the present study could provide a resource for further developing resistant varieties against Foc 4 infection.Additional file 1:Table S7. Gene-specific primers used for qRT-PCR. (XLSX 10 kb)Additional file 2:Table S1. The information of 3996 identified banana novel protein-coding transcripts in the study. (XLSX 4797 kb)Additional file 3:Table S2a. Expression information of DEGs in leaves at 1\u00a0day after BTH treatment. Table S2b. Expression information of DEGs in leaves at 3\u00a0day after BTH treatment. Table S2c. Expression information of DEGs in roots at 1\u00a0day after BTH treatment. Table S2d. Expression information of DEGs in roots at 3\u00a0day after BTH treatment. (XLSX 2340 kb)Additional file 4:Table S3. GO terms of biological process enriched in banana leaves and roots. (XLSX 29 kb)Additional file 5:Table S4. The significantly enriched pathways of DEGs in banana leaves and roots. (XLSX 11 kb)Additional file 6:Table S5. Differentially expressed genes involved in protein kinases and transcription factors. (XLSX 225 kb)Additional file 7:Table S6. Expression information of DEGs in Figs."}
+{"text": "Clupea harengus), a vital ecosystem component and target of the largest Northwest Atlantic pelagic fishery, undergo seasonal spawning migrations that result in elusive sympatric population structure. Herring spawn mostly in fall or spring, and genomic differentiation was recently detected between these groups. Here we used a subset of this differentiation, 66 single nucleotide polymorphisms (SNPs) to analyze the temporal dynamics of this local adaptation and the applicability of SNP subsets in stock assessment. We showed remarkable temporal stability of genomic differentiation corresponding to spawning season, between samples taken a decade apart (2005 N\u00a0=\u00a090 vs. 2014 N\u00a0=\u00a071) in the Gulf of St. Lawrence, and new evidence of limited interbreeding between spawning components. We also examined an understudied and overexploited herring population in Bras d'Or lake (N\u00a0=\u00a097); using highly reduced SNP panels (NSNPs\u00a0>\u00a06), we verified little\u2010known sympatric spawning populations within this unique inland sea. These results describe consistent local adaptation, arising from asynchronous reproduction in a migratory and dynamic marine species. Our research demonstrates the efficiency and precision of SNP\u2010based assessments of sympatric subpopulations; and indeed, this temporally stable local adaptation underlines the importance of such fine\u2010scale management practices.Atlantic herring ( Recently, adaptive SNP\u2010based approaches have shown increased power over neutral markers regarding population structuring  is an ideal species for studying spatial and temporal population structuring and local adaptation in the sea. It is one of the most abundant and widely distributed fish in the Northwest Atlantic  temporal stability of genomic differentiation between seasonal spawning components in the GSL and examined evidence for spawning\u2010component interbreeding. We screened 66 SNPs, representing 10 genomic scaffolds found by Lamichhaney et al.  to disti22.1A total of 276 adult herring were collected during spring and fall spawning seasons from two locations in the GSL in 2005 and 2014, and in the spring season from BDO in 2016 and 2017  for genotyping in the 66 SNP panel using the Agena MassARRAY system.Total genomic DNA was extracted following a standard phenol\u2010chloroform protocol. DNA quality was assessed in 0.8% agarose gel electrophoresis (0.5\u00d7 TBE buffer) using a 1\u00a0Kb molecular weight ladder. DNA quantity was measured using the Quant\u2010iT PicoGreen dsDNA assay (Thermo Fisher Scientific) with a Roche LightCycler 480 Instrument . Given that DNA of archived samples may exhibit different levels of degradation, we evaluated whether these samples were suitable for the Polymerase Chain Reaction (PCR), and thus, for SNP genotyping. For this, in 8 randomly chosen individuals with fragmented DNA, we amplified by PCR a total of 66 SNP loci reported in Lamichhaney et al.  as stron2.3PLINK  was nested within spawning season (FST). The proportion of heterozygosity (PHt) was calculated for all individuals using GENHET  : Jombart, ). This pR2. SNPs with R2\u00a0>\u00a00.5 were clustered, and only the top ranked SNP per cluster was used. (c) Panel of only transatlantic loci: To maximize geographic applicability, only SNPs that were found in both the Northwest and NE were used. These three SNP sets were then further reduced using the best ranking method as assessed above. Optimal panels  were found for the three screened subsets of loci, as well as all 64 unscreened loci.Secondly, three further SNP thinning methods were used, each matching a specific objective: (a) Panel of loci on separate scaffolds: only the top ranked SNP in each scaffold was chosen. (b) Panel of loci with low redundancy: To minimize redundant information, loci underwent complete linkage clustering, based on 1\u00a0\u2013\u00a02.5BDO herring has been categorized as predominantly spring spawning, though fall spawning may occur  while actively spawning in the spring (N\u00a0=\u00a061) or fall (N\u00a0=\u00a087). The remaining N\u00a0=\u00a097 individuals were collected from BDO in the spring but comprised both spawning (N\u00a0=\u00a012) and nonspawning individuals (N\u00a0=\u00a085) , while both FSC and FCT were insignificant   correction  based on 64 SNPs clustered individuals by spawning season regardless of collection year Figure . A globay et al.  ; from this reduced SNP set only 10 SNPs were needed for 100% cross\u2010validated accuracy via DAPC  transatlantic SNPs , which is seasonally upregulated during spawning in goldfish  is the largest inland sea in North America, unique in both ecology and physical geology  and spatially (Lamichhaney et al., The authors declare no competing financial interests.D.E.R. and A.P.F.P. designed the study; Q.K., J.K. A.P.F.P and J. L. M. performed tissue collection, laboratory work and data analysis; Q.K. wrote a first draft of the paper and all authors contributed to the writing of the final document. All authors approved the manuscript before submission.The raw genotyped SNP data used in this study will be made available in a public repository upon acceptance.\u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "The present study aimed to evaluate the effects of other common oils on CCl4 induced LF. Healthy male ICR mice were administered with CCl4 intraperitoneally at 2.5 ml/kg twice a week for total 3 weeks. Mice were pre-treated with olive oil, soybean oil, corn oil or lard oil. After treatment, histopathological changes were observed using Masson trichrome staining, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), hydroxyproline (HYP) and triglyceride (TG) were measured by commercial kits. The expression of LF related genes was detected by quantitative real-time PCR. We found that soybean oil or olive oil significantly reduced ALT and AST levels in serum, and MDA, HYP and TG levels in the liver, compared with corn oil or lard oil. Moreover, Masson trichrome staining and real-time PCR showed that the mice treated with CCl4 dissolved in soybean oil or olive oil had less fibrosis and apoptosis in the liver comparted to the mice treated with CCl4 dissolved in corn oil or lard oil. In conclusion, soybean oil but not corn or lard oil exerts protective effects against CCl4 induced LF in mice, possibly due to its antioxidant activity.Olive oil could attenuate carbon tetrachloride (CCl Howeverix (ECM) . LF coulix (ECM) . CurrentReactive oxygen species (ROS) released by injured HSCs play an important role in oxidative damage during chronic liver injury and LF . Matrix in vivo [4-induced LF [4 induced LF.Olive oil, soybean oil and corn oil contain a large amount of unsaturated fatty acids, while lard oil is enriched in saturated fatty acids. Several studies have suggested that saturated fatty acids are more cytotoxic than unsaturated fatty acids in vivo . Olive oduced LF . Soybeanduced LF . Howeverad libitum. A total of 108 mice were allowed to acclimatize for 3 days and then randomly divided into eight groups: (1) olive oil control group (olive oil CCl4\u2212); (2) soybean oil control group (soybean oil CCl4\u2212); (3) corn oil control group (corn oil CCl4\u2212); (4) lard oil control group (lard oil CCl4\u2212); (5) olive oil CCl4 group (olive oil CCl4+); (6) soybean oil CCl4 group (soybean oil CCl4+); (7) corn oil CCl4 group (corn oil CCl4+); (8) lard oil CCl4 group (lard oil CCl4+). Mice from olive oil CCl4+, soybean oil CCl4+, corn oil CCl4+ and lard oil CCl4+ groups were injected intraperitoneally (i.p.) with 20% CCl4 dissolved in olive oil , soybean oil , corn oil  and lard oil , respectively. Mice from olive oil CCl4\u2212, soybean oil CCl4\u2212, corn oil CCl4\u2212 and lard oil CCl4\u2212 groups were injected i.p. with olive oil, soybean oil, corn oil and lard oil, respectively. All mice received a dose of 2.5 ml CCl4 solution or oil per kilogram of body weight twice a week for 3 weeks. The mice were euthanized, and their blood and livers were collected immediately and stored.All animal care and experimental procedures were performed at Hainan Medical University and approved by Animal Care and Use Committee of Hainan Medical University. Healthy male ICR mice (20\u201322 g) were purchased from the experimental Animal Center of Harbin Medical University and maintained at 22 \u00b1 2\u00b0C with the humility of 45 \u00b1 10% and regular 12:12 h light\u2013dark cycle. All animals were given water and food The blood collected from right heart ventricle was stored at room temperature for 4 h and centrifuged at 3000 rmp at 4\u00b0C for 15 min. Serum was separated, and ALT and AST were detected using ELISA kits  following the manufacturer\u2019s protocols.Part of the liver tissues were fixed in 4% paraformaldehyde for 48 h at room temperature, paraffin-embedded and sectioned for staining with Masson\u2019s trichrome. The morphology of liver tissues was observed under microscope and the images were analzyed using Image Pro Plus 5.1 .Part of the liver tissues were washed with cold sterile phosphate-buffered saline (PBS), dried and weighted precisely, and hydroxyproline (HYP), malondialdehyde (MDA) and triglyceride (TG) were measured using assay kits  following the manufacturer\u2019s protocols .\u00ae Premix kit  according to the manufacturer\u2019s instructions. PCR conditions were: 95\u00b0C for 30 s, then 40 cycles of 95\u00b0C for 5 s, 60\u00b0C for 30 s The data were presented as the fold change in mRNA expression levels and normalized to reference gene GAPDH.Total RNA was isolated from the liver tissues using Trizol reagent , followed by reverse transcription to cDNA using a PrimeScriptTM RT kit . Real-time PCR was performed using primers  listed in post hoc test. The P<0.05 was considered significant difference.All data are presented as the mean \u00b1 S.E.M. and analyzed by GraphPad Prism 7 . The significant differences among groups were determined by a one-way ANOVA followed by Dunnett\u2019s 4 had increased levels of ALT and AST compared with control group, indicating that CCl4 successfully induced liver injury. However, administration with olive oil or soybean oil significantly decreased ALT and AST in CCl4 groups compared with corn oil or lard oil CCl4 group. There was no significant difference between olive oil and soybean oil CCl4 groups.As most commonly used biochemical markers, ALT and AST reflect liver injury during the progression of LF. Therefore, ALT and AST were detected in serum of control and experimental mice. As shown in 4\u2212 groups showed normal structure with little fibre portal expansion, whereas the livers in CCl4+ groups exhibited massive fatty changes and pericentral fibrosis with extensive blue-stained fiber. The fatty changes and fiber formation in olive oil or soybean oil CCl4 group were similar but much weaker compared with corn oil or lard oil CCl4 group. Moreover, the contents of HYP, a specific amino acid for collagen, decreased in olive oil or soybean oil CCl4 group compared with corn oil or lard oil CCl4 group (To observe pathological changes in the livers, we used Masson\u2019s trichrome staining. As shown in l4 group B. Consisl4 group C.4 treated mice, suggesting that olive oil or soybean oil may reduce HSCs apoptosis to alleviate LF.TNF-\u03b1 and FasL regulate HSCs apoptosis and aggravate LF . Therefo4 treatment clearly decreased the expressions of TIMP-1 and TIMP-2, and MMP-2 and MMP-13 compared with those from corn oil or lard oil with CCl4 treatment, suggesting that olive oil or soybean oil may alleviate LF by reducing ECM.TIMPs and MMPs play an important role in the degradation of ECM. Therefore, we determined the effects of four oils on the expression of TIMPs and MMPs. As shown in 4-treated mice (TGF-\u03b21, PDGF and PDGFR are related with the proliferation of HSCs. Therefore, we determined the effects of four oils on their expression. Compared with corn oil or lard oil, olive oil or soybean oil significantly reduced the expression of TGF-\u03b21, PDGF and PDGFR in CClted mice , suggest4+ groups increased markedly compared with CCl4\u2212 groups, but decreased significantly in olive oil or soybean oil CCl4 group compared with corn oil or lard oil CCl4 group (4 groups.To detect lipid peroxidation and deposition in the livers, we measured hepatic MDA and TG levels. MDA and TG levels in CCll4 group . Moreove4-induced LF. Specifically, olive oil and soybean oil significantly alleviated CCl4-induced LF as evidenced by reduced serum ALT and AST as hepatic injury indicators; reduced LF pathology accompanied by less HYP in ECM; reduced expression of TNF-\u03b1, FasL, \u03b1-SMA, TGF-\u03b21, PDGF and PDGFR in the liver; and reduced oxidative stress in the liver with less hepatic MAD and TG.In the present study, we demonstrated that olive oil and soybean oil had similar effects on CCl4 group compared with corn oil or lard oil CCl4 group. Furthermore, mRNA expression of Col1\u03b11 and \u03b1-SMA reduced in olive oil or soybean oil CCl4-treated mice. Our results suggest that the antifibrotic effects of olive oil or soybean oil are partly due to the inhibition of collagen production.HYP only exists in collagen and its contents can directly reflect the levels of collagen . In addiHSCs constantly secrete \u03b11-collagen, TGF-\u03b21, PDGF and PDGFR, which in turn stimulate the activation and proliferation of HSCs to promote LF . Moreove4-treated mice compared with corn oil or lard oil CCl4-treated mice. It has been reported that TIMP-1 and -2, and MMP-2 expression significantly increased during LF both in human and in animal models [MMPs and TIMPs play an important role in maintaining the dynamic equilibrium of the production and degradation of ECM, and are involved in LF . We founl models . In addil models . MMP-13 l models . In our 4 is known to induce oxidative damage during chronic liver injury and LF [4-treated mice compared with corn oil or lard oil. Therefore, we could infer that soybean oil, similar as olive oil, might alleviate LF by reducing lipid peroxidation. Furthermore, olive oil or soybean oil led to significant decrease in TG in CCl4-treated mice compared with corn oil or lard oil, maybe due to high contents of phenolic compounds and unsaturated fatty acids in soybean oil and olive oil [4 group. Collectively, these results suggest that olive oil and soybean oil might alleviate LF by reducing lipid deposition compared with corn oil and lard oil, because the total fatty acid composition ratio of the four oils are different. Recent studies showed olive oil could activate Nfr2 pathway to induce cellular antioxidant response and inhibit PERK pathway to prevent endoplasmic reticulum stress, autophagy, and lipogenic response, and essential oils provided hepatoprotection by accelerating acetaminophen harmless metabolism [CCly and LF . In our live oil . It has live oil . Furtherlive oil , consisttabolism . Further4-induced LF in mice, which is related to the anti-oxidative effects of olive oil and soybean oil. To our knowledge, the present study is the first comprehensive analysis of beneficial effects of olive oil or soybean oil in comparison with corn oil or lard oil on CCl4-induced LF. Our findings provide a better understanding of the mechanisms underlying the different effects of dietary lipids on LF.In conclusion, olive oil and soybean oil show hepatoprotective effects against CCl"}
+{"text": "Essential oils (EOs) are known to possess a number of beneficial properties. Their antimicrobial, anti-inflammatory, antioxidant, antidiabetic, and cancer-preventing activities have been extensively reported. Due to their wide use as food preservers and additives, as well as their use in agriculture, perfumes, and make-up products, these complex mixtures of volatile compounds have gained importance from a commercial point of view, not only in the pharmaceutical industry, but also in agronomic, food, cosmetic, and perfume industries. An analysis of the recent scientific literature allowed us to highlight the presence of an increasing number of studies on the potential antiarthritic properties of EOs and their main constituents, which seems to suggest a new interesting potential therapeutic application. The aim of this review is to examine the current knowledge on the beneficial effects of essential oils in the treatment of arthritic diseases, providing an overview of the reports on the in vivo and in vitro effects of EOs. Furthermore, this review critically examines the recent findings on the potential roles of the main components of EOs in the exerted beneficial effects. Obtained negative results are also reported. Arthritis is one of the most common chronic health problems and a major cause of disability . The terArthritis includes more than 100 different disorders, with osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis, gout, and fibromyalgia being the most common types .Osteoarthritis is the most frequent form of arthritis . This deThe pharmacological management of both these major forms of arthritis is mostly symptomatic and is aimed at reducing pain and inflammation. This includes the use of non-steroidal anti-inflammatory drugs (NSAIDs) and oral glucocorticoids. Current treatment modalities for RA are based on the use of disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, hydroxychloroquine, and sulfasalazine. However, these drugs can cause serious side effects, including gastrointestinal complaints, pneumonitis, and liver function abnormalities. More recently, biologic drugs  have gained importance in the treatment of OA and RA. These agents are able to reduce inflammation and joint destruction, however besides the higher cost of such new agents, which limits their use, both their long-term side effects and benefits have not yet been fully understood. These drugs are not always effective and severe side effects may occur, such as serious infections and increased risk of cancer. Immunosuppressive and cytotoxic drugs  may be used as well, but these drugs also cause a number of toxic side effects ,9.As a consequence, the use of botanicals and nutritional supplements has gained importance, and the number of studies on the potential health benefits of plant extracts in the treatment of arthritis is increasing .The antiarthritic potential of plant species and extracts has been extensively reviewed in the last decade, and a number of papers dealing with such botanicals have been published ,18,19,20In contrast, the present review aims to specifically offer an overview of the plant essential oils (EOs) with antiarthritic potential.Essential oils are complex mixtures containing low molecular weight compounds, mainly monoterpenes, sesquiterpenes, and oxygenated derivatives, extracted from aromatic plants . A numbeThe analysis of the most recent literature allowed us to identify an increasing number of studies focusing on the in vitro and in vivo antiarthritic properties of essential oils. Overall, these findings give interesting perspectives on the therapeutic use of EOs and their chemical constituents.This search, conducted over a period of 5 months, included references published up until July 2020. The academic search engines Google Scholar, Pubmed, Microsoft Academic, and Scopus were utilized. The keywords \u201cessential oils\u201d, \u201carthritis\u201d, \u201cantiarthritic\u201d, \u201canti-rheumatoid arthritic effect\u201d, \u201cterpenes\u201d, and \u201cvolatile oil\u201d were searched in the databases. These descriptors were combined by using Boolean operators, e.g., \u201cand\u201d, \u201c+\u201d, allowing for more targeted results. Studies focusing on the antiarthritic potential of essential oils in their whole forms or their single pure active components were included in the research, and both in vitro and in vivo studies were taken into account.The purpose of this review is to provide an overview of the studies dealing with the potential beneficial effects of EOs and their phytochemical constituents on arthritic conditions. A total of 31 EOs from different plant species have been reported in the literature to have in vitro or in vivo effectiveness. The investigated EOs that have been proved to have antiarthritic potential belong to a number of plant families , mainly Aquilaria agallocha Roxb. (Thymelaeaceae), commonly known as \u201cagarwood\u201d, grows wild in the Himalayan region and East India, and it is cultivated in India, Bangladesh, Indonesia, and Sri Lanka [ri Lanka . Agarwoori Lanka ,27.A. agallocha Roxb. heartwood essential oil (EO) [A. agallocha EO inhibited the increase in paw volume, with maximum inhibition values equal to 19.78% and 27.88%, respectively.Rahman and colleagues reported the anti-arthtritic potential of oil (EO) . In vitrCedrus deodara (Roxb.) Loud. (Pinaceae), commonly named deodar, is a large evergreen tree native to the western Himalayas, traditionally used in Ayurvedic medicine for the treatment of inflammatory diseases, such as arthritis and bronchitis [onchitis ,30.C. deodara wood EO in adjuvant arthritic male albino rats [C. deodara wood EO inhibited the acute phase of adjuvant-induced response (first 7 days following the injection of FCA). Moreover, the EO showed analgesic activity against acetic-acid-induced writhing and hot plate reaction in mice.Shinde and coworkers investigated the anti-inflammatory activity of ino rats . The oilChamaecyparis obtusa (Siebold and Zucc.) Endl. (Cupressaceae) on pain-related behavior and pro-inflammatory cytokines in rats with carrageenan-induced arthritis [C. obtusa EO inhibited pain-related behavior and the expression of pro-inflammatory cytokines TNF-\u03b1, IL-1\u03b2, IL-6, and COX-2 in inflamed knee joints in rats.Suh and colleagues tested the effects of EO from the leaves of rthritis . The obtCyperus species (Cyperaceae) from India, C. esculenthus L. and C. rotondus L. [C. rotondus) and 76.58% (C. esculenthus).Biradar and coworkers investigated the in vivo antiarthritic potential of the EOs from two ondus L. . SamplesGaultheria fragrantissima Wall. is an evergreen shrub belonging to the Ericaceae family that is widely distributed in the Himalayan region and northeastern India. Its EO has been reported to have antioxidant, antibacterial, insecticidal, and nematicidal activities [tivities .G. fragrantissima [Uriah and colleagues evaluated the in vitro biological potential of the EO obtained from the leaves of ntissima . The antLagerstroemia speciosa (L.) Pers. is a common ornamental tree belonging to the Lythraceae family. The leaves are known to contain triterpenes, tannins, and flavones [L. speciosa has been studied for its beneficial effects towards glucose and lipid metabolism [flavones . L. spectabolism . The in tabolism . SamplesLitsea cubeba (Lour.) Pers. (Lauraceae) is a shrub or tree distributed in southeastern Asia. It is well known for the EO that can be extracted from the different parts of the plant, with various compositions and yields. The fruit EO is a major source for citral, which is widely used as an aroma additive in food and cosmetics [osmetics .L. cubeba EO was tested by Zhao and coworkers [The anti-arthritis potential of oworkers . LimonenOcimum americanum L. (Lamiaceae) is an annual plant native to Africa, also named American basil, which is commonly used for its flavor properties [O. americanum L. fresh leaves EO was tested in vivo in mice in an experimental model of zymosan-induced arthritis [O. americanum EO significantly inhibited leukocyte migration to the articular cavity in the knee joint of zymosan-induced arthritic Balb/c mice. Moreover, at 150 mg/kg the EO was able to reduce paw edema in mice, suggesting a suppression of the release of inflammatory mediators such as PGE2 and COX-2.operties ,42. The rthritis . The EO Rhododendron tomentosum Harmaja (Ericaceae) is a small evergreen shrub that is widely distributed in northern and central Europe, North America, and northern Asia. This species has been widely used in folk medicine against arthrosis, rheumatism, pain, wounds, fever, and cough, even if the internal use of its extracts has become rare due to it containing the toxic sesquiterpenoid ledol [R. tomentosum on CD4+ and CD8+ limphocytes, which are involved in the pathogenesis of rheumatoid arthritis. The pro-apoptotic effects on synovia-infiltrating limphocytes and monocytes or macrophages, which impact rheumatoid-arthritis-affected joint synovium, were also evaluated. In both experiments promising results were obtained.id ledol . The antid ledol . The autStrobilanthus ixiocephala Benth. flowering tops [S. ixiocephala EO was effective in Freund\u2019s adjuvant-induced arthritis in rats. At the oral dose of 1 mL/Kg, it inhibited the rat paw edema by 35.56% after 21 days of treatment. The EO was able to suppress the increased lymphocyte count in arthritic rats and the migration of leucocytes into the inflamed area [Agarwal and coworkers reported the in vivo anti-inflammatory and antiarthritic activities of the EO from ing tops . This sming tops . The plamed area .Zingiber officinale Roscoe, Zingiberaceae) is a well-known and widely utilized spice plant. This species has been traditionally used for centuries in Chinese, Ayurvedic, and Tibb-Unani medicines for the treatment of different diseases, including rheumatoid arthritis [Ginger  Willd., Alpinia oxyphylla Miq., Amomum kravanh Pierre ex Gagnep., and Kaempferia galanga L. [K. galanga being the most effective one.Zhang and coworkers recently evaluated the antiarthritic potential of Zingiberaceae plant EOs, including langa L. . These sCalophyllum inophyllum L. (Clusiaceae), Citrus aurantium L. (Rutaceae), Eucalyptus globulus Labill. (Myrtaceae), Eugenia caryophyllata L. (Myrtaceae), Foeniculum vulgare L. (Apiaceae), Helichrysum angustifolium DC. (Asteraceae), Juniperus virginiana L. (Cupressaceae), Lavandula angustifolia Mill. (Lamiaceae), Myristica fragrans Houtt. (Myristicaceae), Ocimum basilicum L. (Lamiaceae), Pinus sylvestris L. (Pinaceae), Piper nigrum L. (Piperaceae), Rosmarinus officinalis L. (Lamiaceae), Salvia sclarea L. (Lamiaceae), Salvia officinalis L. (Lamiaceae), and Zingiber officinale Roscoe [M. tuberculosis (Mtb). It was observed that arthritic rats treated with the EO mixture developed less severe clinical arthritis compared to the control group. Moreover, the treatment was able to significantly reduce the levels of TNF-\u03b1 and IL-1\u03b2, as well as the activity of matrix metalloproteinases (MMPs) in the synovial fluid (SF) and synovium-infiltrating cell (SIC) lysate.Komeh-Nkrumah and coworkers tested the antiarthritic potential of an ointment containing essential oils from 16 species: e Roscoe . EOs werStudies on the antiarthritic activity of EOs reported in this review are summarized in Essential oils are complex mixtures containing low molecular weight components, whose extraction is above all carried out by steam distillation. The main chemical constituents in EOs include terpenoids and phenylpropanoids. Additionally, several aromatic and aliphatic compounds are also present. Monoterpenes, sesquiterpenes, and their oxygenated derivatives are the major groups of EOs chemical compounds .Paeonia emodi Wall., and the sesquiterpene torilin, which were both demonstrated to modulate the levels of pro-inflammatory cytokines TNF-\u03b1, IL-1\u03b2, and IL-6 in vivo.The antiarthritic potential of terpenes, the main components in EOs, has been reviewed recently by Carvalho and colleagues . An in vEven more recently, other interesting terpenes, such as nerolidol, were described for their beneficial effects on arthritic conditions. The present review will focus on these further terpenes and other kinds of EOs components, which were investigated through both in vivo and in vitro assays. Seven EOs chemical constituents have been recently described for their potential antiarthritic activity .\u03b2-caryophyllene  is a bicVijayalaxmi and colleagues verified the effectiveness of this compound on FCA-induced arthritic rats . Paw volMore recently, El-Sheikh and coworkers assessed the ability of \u03b2-caryophyllene to increase the efficacy of methotrexate and leflunomide and to improve their side effects . Experimtrans-cinnamic aldehyde) is known to be the principal component of cinnamon flavor and a potent antimicrobial compound. It is also detectable in other EOs and is commonly used as a natural food flavorant [Cinnamaldehyde -stimulated macrophages .Cheng and coworkers reported the interesting antiarthritic effects of cinnamaldehyde . At concThe antiarthritic activity was also evaluated in a rat model of arthritis by Mateen and colleagues . CinnamaThe monoterpene oxide eucalyptol , the principal component of the essential oils from Eucalyptus leaves , is wellYin and coworkers recently verified the anti-inflammatory and analgesic potential of this compound on a mouse model of gout arthritis . The disMyristica fragrans Houtt. [Ocimum basilicum L. [Eugenia caryophyllata Thunb.) is the principal source of this compound, representing 45\u201390% of the total EOs. A wide spectrum of biological properties has been reported for this molecule, including antioxidant, antimicrobial, anti-inflammatory, analgesic, and anticancer activities [Eugenol is present in numerous aromatic plants, such as s Houtt.  and Ocimlicum L. . The clotivities .The efficacy of this allylbenzene in arthritic rats was verified by Grespan and colleagues . ArthritGinkgo biloba L. [Piper claussenianum (Miq.) C. DC. [Momordica charantia L. [Baccharis dracunculifolia DC. [Nerolidol is a naturally occurring sesquiterpene alcohol used in cosmetics and as a food-flavoring agent ,78. It hiloba L. , Piper c) C. DC. , Momordiantia L. , and Bacolia DC. . DiffereNano-encapsulated nerolidol was recently studied for its effects on zymosan-induced arthritic mice . An in vSalvia sclarea L.) [Cleome spinosa Jacq. [Cistus creticus L. [Sclareol s a labdane diterpene ditertiary alcohol of high value in the fragrance industry, as this molecule is a good starting material for the semisynthesis of different commercial substances due to its labdane carbon skeleton and the two hydroxyl groups . This tearea L.) , but it sa Jacq.  and Cistticus L. . Sclareoticus L. .Zhong and colleagues assessed the anti-osteoarthritic properties of sclareol in IL-1\u03b2-induced rabbit chondrocytes and in a rabbit model of osteoarthritis induced by ACLT . SclareoTsai and coworkers assessed the potential therapeutic effects of sclareol on RA using the human synovial cell line SW982 and the collagen-induced arthritis (CIA) mouse model . The autNigella sativa L. seeds EO [The monoterpene thymoquinone is the major constituent of seeds EO . Antioxiseeds EO .Tekeoglu and coworkers investigated the anti-inflammatory effects of this monoterpene on arthritis in rat model . ArthritThe in vitro protein denaturation method using bovine serum albumin as a protein model has often been used to assess the antiarthritic potential of some of the EOs reported here, while their anti-inflammatory potential has generally been assessed using a rat ear edema model.C. obtusa was able to inhibit the expression of IL-1\u03b2, TNF-\u03b1, and IL-6 in the inflamed synovial membrane, as well as IL-1\u03b2 and IL-6 in an inflamed meniscus [Litsea cubeba, whose EO was able to decrease the TNF-\u03b1, IL-1\u03b2, IL-6, IL-8, and IL-17A levels and to increase IL-10 in type II collagen-induced arthritic rats [K. galanga and some other species belonging to the Zingiberaceae family also demonstrated impacts on the expression of COX-2, TNF-\u03b1, IL-6, and IL-1 in Freund\u2019s adjuvant-induced arthritic rats [E. caryophyllata, C. inophyllum, C. aurantium, and E. globulus, as described by Komeh-Nkrumah and coworkers [The mechanism of action underlying the antiarthritic activity of some of the investigated species reported above was more deeply investigated. In some cases, the different expression levels of pro-inflammatory cytokines and COX-2 in inflamed synovial membranes among control and treated groups were verified. As a results of their investigations, Suh et al. demonstrated that meniscus . Such eftic rats . K. galatic rats . The levoworkers .O. americanum EO (150 mg/kg) for seven days in arthritic mice was able to inhibit leukocyte migration in the synovial membrane and to attenuate cartilage destruction. Moreover, the treatment significantly reduced IFN-\u03b3 levels in synovial tissue.Furthermore, Yamada and coworkers  demonstrR. tomentosum EO was demonstrated to have antiproliferative and pro-apoptotic activity toward CD4 and CD8 T cells. The same effects were also observed toward synovia-infiltrating monocytes and macrophages [rophages .The mechanisms of action of some EOs\u2019 pure components were also investigated. Cinnamaldehyde, for example, has been demonstrated to significantly suppress IL-1\u03b2-induced inflammatory cytokine production in human synoviocyte cell line MH7A via the suppression of JAK/STAT pathways  and to iM. spicata L. EO and its main constituents, such as menthol, carvone, and limonene, demonstrated analgesic effects in animal models, while spearmint oil was able to reduce pain in osteoarthritis patients [Cymbopogon citratus (DC.) Stapf.) [Zingiber officinale Roscoe) [EOs and their chemical components could also be useful in the treatment of arthritis-related pain. Various monoterpenes, sesquiterpenes, and other essential oil constituents have been investigated for their potential antinociceptive activity and have demonstrated interesting analgesic-like activity  and diffpatients . Recentlpatients , a monot Stapf.)  and ging Roscoe) ,96. It w Roscoe) .Curcuma longa L. (Zingiberaceae) is known to contain two main classes of secondary metabolites, phenolic curcuminoids, and essential oils, including the major components \u03b1-turmerone and \u03b2-turmerone. Despite the essential oil from Curcuma longa L. administered via intraperitoneal injection demonstrating a strong antiarthritic effect in female rats with SCW-induced arthritis, significant morbidity and mortality have been observed, which do not support the use of this EO against arthritis [rthritis . On the rthritis , and goorthritis .The low toxicity and reduced genotoxicity are some of the advantages associated with the use of EOs . HoweverMoreover, some old essential oils and oxidized terpenoids have demonstrated skin-sensitizing activity causing contact dermatitis. A study of the toxicity profile of EOs should be carried out, even if this kind of investigation is not simple due to the variability of EO compositions, which in turn may be affected by a number of factors ,100.The application of EOs and their constituents in the treatment of arthritis appears to be an interesting new perspective on their potential health benefits. A total of 31 plant EOs and some chemical compounds commonly distributed in different EOs have been reported in the literature to have in vitro or in vivo antiarthritic potential. Many other EOs and components could still be potentially identified.Overall, the analysis of the existent scientific literature focusing on the antiarthritic potential of EOs reveals that the information obtained by in vitro and in vivo studies still needs to be confirmed through clinical investigations."}
+{"text": "Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China, in December 2019, Coronavirus - 19 (COVID-19) has become a global pandemic with multiple neurological complications. In December 2020, two vaccines have been approved in the United States for the prevention of COVID-19. We report a case of Guillain-Barre Syndrome (GBS) after receiving the first dose of Pfizer - COVID-19 vaccine. The first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China, in December 2019 . In DeceAn 82-year-old highly functional female at baseline without significant comorbidities presented to the emergency department with generalized body aches, paresthesia, and difficulty walking. She received her first dose of the Pfizer COVID vaccine two weeks prior to presentation\u00a0(see Figures GBS\u00a0is an inflammatory polyradiculoneuropathy associated with numerous viral infections . ApproxiSince the pandemic outbreak and constantly increasing numbers of COVID-19 respiratory illness caused by SARS-CoV-2, worldwide mass vaccination campaigns have taken place in order to control the pandemic ,7. UnderWe report the first case of COVID-19 post vaccine associated GBS. In this pandemic and with ongoing worldwide mass vaccination campaign, it is critically important for clinicians to rapidly recognize neurological complications or other side effects associated with COVID-19 vaccination. We would like to highlight that the risk of neurological complications or any other adverse effect associated with COVID-19 vaccination is low and the benefits of the vaccination outweigh any potential risks or side effects, both at the individual and society levels. We encourage and support the recommendations of the CDC and WHO guidelines for COVID-19 vaccination."}
+{"text": "IL6) -174G/C and -572G/C are related to bone deterioration. However, no study of the interaction between diet and IL6 polymorphisms has been conducted among Asians. Thus, the objective of this study was to determine whether IL6 gene polymorphisms modified the association between dietary acidity and the rate of bone resorption. Methods: This cross-sectional study recruited 203 postmenopausal women (age ranged from 51 to 85 years old) in community settings. The dietary intakes of the participants were assessed using a validated interviewer-administered semi-quantitative food frequency questionnaire (FFQ), while dietary acid load (DAL) was estimated using net endogenous acid production (NEAP). Agena\u00ae MassARRAY genotyping analysis and serum collagen type 1 cross-linked C-telopeptide (CTX1) were used to identify the IL6 genotype and as a bone resorption marker, respectively. The interactions between diet and single-nucleotide polymorphisms (SNPs) were assessed using linear regressions. Results: A total of 203 healthy postmenopausal women aged between 51 and 85 years participated in this study. The mean BMI of the participants was 24.3 kg/m2. In IL6 -174 G/C, all the participants carried the GG genotype, while the C allele was absent. Approximately 40% of the participants had a high dietary acid load. Dietary acid load  and the IL6 -572 CC genotype group  were positively associated with a higher bone resorption. However, there was no moderating effect of the IL6 genetic polymorphism on the relationship between and acid ash diet and bone resorption markers among the postmenopausal women (p = 0.79). Conclusion: High consumption of an acid ash diet and the IL6 -572 C allele seem to attribute to high bone resorption among postmenopausal women. However, our finding does not support the interaction effect of dietary acidity and IL6 (-174G/C and -572G/C) polymorphisms on the rate of bone resorption. Taken together, these results have given scientific research other candidate genes to focus on which may interact with DAL on bone resorption, to enhance planning for preventing or delaying the onset of osteoporosis among postmenopausal women.Background: Evidence is growing that a high-acid diet might accelerate the rate of bone loss, and gene polymorphisms such as Interleukin 6 ( A bourgeoning body of research has focused on osteoporotic fractures in light of their great impact on public health . OsteopoDespite being the gold standard for the quantitative assessment of bone and an important predictor of fracture risk , bone miIL6) single-nucleotide polymorphism (SNP) is one of the susceptible genes that is commonly used to detect bone deterioration [IL6 is a key moderator of inflammation which has been found to play an important role in the pathogenesis of atherosclerosis, while IL6 gene -174G/C (rs 1800795) and -572G/C (rs 1800796) polymorphisms were related to bone metabolism and BMD among postmenopausal women [IL6 -572G/C polymorphism as being common in the Asian population [IL6 -174 G/C polymorphism studies were among Europeans, with a scarcity of data from Asians. Sun et al. [IL6 -174 G/C polymorphism too.Attributable to the complex predominance of osteoclastic activity, genetic polymorphism factors contributed up to 80% in determining BMD . Interleioration . IL6 is al women . Severalpulation ,17,18. Apulation  reportedn et al.  showed tDietary factors are other independent factors that may affect body acidity and the rate of bone loss. Generally, a highly acidic diet is recognized as a diet that is high in animal proteins and may generate endogenous acids. Although body acidity is generally tightly regulated, the habitual intake of a high-acid ash diet without adequate compensation by alkaline ions such as potassium and magnesium from fruit and vegetables may lead to a lower blood pH , which mIL6 (-174G/C and -572G/C) genetic polymorphisms with the presence of C alleles is associated with a higher risk of bone resorption, the potential association between dietary acidity and bone health remains inconclusive. Therefore, we hypothesized that the complex interaction between IL6 (-174G/C and -572G/C) genetic polymorphisms and dietary acidity may be implicated in lower bone mass. To date, this is the first study to examine how genetic factors may modify the association between diet and bone resorption among postmenopausal women. We aimed to explore whether IL6 SNPs modulate the effect of dietary acidity on bone resorption among postmenopausal women.On the other hand, while there is growing evidence that 2 was 0.15, which gave a study power of 95%. The written informed consent of participants were obtained prior to study enrollment. The study protocol was approved by the Ethics Committee for Research Involving Human Subjects (project reference number FPSK (FR16) P019).A total of 203 postmenopausal women with ages ranging from 51 to 85 years old was randomly recruited from National Council of Senior Citizens Organizations Malaysia (NACSCOM). This was an analytical cross-sectional study conducted on a representative population . The exclusion criteria were the presence of any systemic diseases  and on medication  that could affect bone health within the past one year. 2 (m2), and was classified according to the WHO (2000) [The sociodemographic factors of participants were obtained using a pre-tested structured questionnaire. Anthropometric measurements including height, weight, and waist circumference were ascertained using standardized techniques ,29. A unO (2000) . The perO (2000) . The adeO (2000) .The habitual food intake of participants over the past one month was assessed using a validated 165-item semi-quantitative food frequency questionnaire (FFQ) , which wA total of 10 mL of fasting blood was collected between 9:00 and 10:30 h by certified phlebotomists and was then sent to a certified commercial laboratory for biochemical analysis. The fasting blood glucose was estimated by the Hexokinase method performed by the Olympus AU analyzer. The serum levels of 25(OH) vitamin D were determined using the Siemens ADVIA Centaur Vitamin D Total assay , with the analytical measuring range of 4.2 to 150 ng/mL 10.5 to 375 nmol/L). This assay has been standardized to the University of Ghent ID-LC/MS/MS reference measurement procedure and has achieved the Centers for Disease Control Vitamin D Standardization Certification program . The vit5 nmol/L.\u00ae MassARRAY platform. After the SNP detection process, the Typer Analyzer was used to analyze the output data from the Agena\u00ae MassARRAY platform.Genomic DNA was extracted from non-coagulated whole blood samples  using a commercially available DNA extraction kit  according to the manufacturer\u2019s protocol. The quality of the extracted DNA was evaluated by means of electrophoresis, and the concentration of the extracted DNA was estimated using the spectrophotometer. For the SNP control, built-in controls were used to easily quantify amplifiable copies of DNA. The panel includes 5 DNA copy number control assays  to quantify from as little as 500 to 18,000 amplifiable copies (~1 ng to 60 ng) of DNA. Each genotyping was performed with the AgenaThe Statistical Package for Social Sciences for Windows version 22.0  was used to perform the statistical analysis. The data normality was checked by a normal P-P plot of the standardized residuals. First, the descriptive data were expressed as mean \u00b1 SD or frequency and percentage. In light of the absence of established cut offs for NEAP and CTX1, the mean values of the participants were arbitrarily used to identify the overall dietary acid load and rate of bone resorption of the participants. Second, bivariate analyses of Pearson\u2019s correlation were used to determine the relationships between CTX1 with NEAP, sociodemographic background (age and education level), anthropometry parameters (waist circumference and height), and biochemical indices (serum vitamin D and fasting blood glucose). Next, the Hardy\u2013Weinberg equilibrium for genotypic distribution was examined by the Hardy\u2013Weinberg equilibrium exact test. For the IL6 gene -572 G/C polymorphism, with a small number of GG homozygous (5 participants), GG and CG genotypes were collapsed into one single group. Comparisons of the study variables by the IL6 gene -572 G/C polymorphism were examined by the t test (continuous variables) or chi-square test .A hierarchical multiple linear regression model was used to determine the interaction effect between an acid ash diet and SNPs, and also to determine the contribution of adjusted variables and variables of interest (acid ash diet and IL6 SNPs) to bone resorption. Hierarchical regression is more flexible, as it allows the researcher to specify the order of entry of the independent variables in the regression equation . Among t2, with the majority of them normal or overweight, while approximately 5% were underweight. Less than two thirds of the participants met the recommended duration of physical activity. The mean score of NEAP was 72.8 \u00b1 28.7, with approximately 45% having a high dietary acid load. The prevalence of vitamin D deficiency and inadequacy was extremely high (more than 80%). The mean serum CTX1 of the participants was 45% \u00b1 0.2 ng/mL, and approximately 43% had an elevated rate of bone resorption. The IL6 -174G/C polymorphism was absent in this study population, with all the participants carrying the GG wild type. As the CC and CG genotypes were absent, the comparison and linear regression analysis were therefore not pursued for this SNP. The genotype frequencies for rs 1,800,796 were 2.5%, 41.4%, and 56.1% for GG, CG, and CC, respectively.As shown in r = \u22120.19, p = 0.007), while the NEAP, height, serum 25(OH) vitamin D, educational level, fasting blood glucose, and waist circumference of the participants were not associated with the CTX1 , biochemical measures  vitamin D), and CTX1 .2 = 0.066). In model 2, the NEAP score  and IL6 CC genotype group  showed positive associations with the CTX1 rate. The inclusion of the NEAP and IL6 -572G/C increased the explained variance in CTX1 by 3.8%. However, inclusion of the interaction term (NEAP*IL6) in model 3 did not reflect any changes in R Square, and hence did not support the interaction term (NEAP*IL6) associated with CTX1   .IL6 is a cytokine that involved inflammation and infection responses and was reported to play a role in the pathogenesis of osteoporosis [IL6 -174 G/C GG genotype. Our findings are consistent with other Asian studies [IL6 polymorphism at \u2212174 is rare and unlikely to contribute significantly to disease susceptibility in the Malaysian Chinese population.The present study was the first study on dietary acidity among Malaysians. The higher NEAP score as compared to studies in Hong Kong among an older Chinese population  and Caucoporosis ,49. In t studies ,53,54,55 studies . Similar studies ,52 and JIL6 -572G/C with age, BMI, and serum 25(OH)D among Japanese postmenopausal women.We did not find any significant association between -572G/C genotypes and sociodemographics, anthropometrics, lifestyle factors, or biochemical measures. The results are in line with a Caucasian study conducted in Brazil, indicating that the -572G/C genetic polymorphism was not associated with the fasting blood glucose . MoreoveThe present study shows that a higher acid ash diet as determined by NEAP score may accentuate the bone resorption marker. Prior studies have suggested a few mechanisms to interpret the influence of diet-induced acidosis on bone metabolisms ,60,61. FIL6 -174G/C and -572G/C SNPs were considered as candidate genes for bone resorption because of their potential involvement in regulating the proliferation, differentiation, and maturation of osteoblasts in bone turnover pathways [IL6 and accelerate the bone resorption process. The findings of this study indicated that the participants with the CC genotype have a significant positive relationship with bone resorption. To the best of our knowledge, there is no other study that investigates the association between IL6 \u2013572G/C SNP and bone resorption, despite earlier studies by Lee et al. [In this study, the pathways . Postmenpathways . Estrogee et al.  and Chune et al.  reportinIL6 genetic polymorphism and diet interaction data on bone resorption. The present study shows that higher dietary acidity, as determined by NEAP score and IL6 polymorphism at the -572 CC genotype, may accentuate the rate of bone resorption. Studies on the effects of dietary acidity on bone have yielded mixed and inconsistent findings. While some studies suggested that high dietary acidity are detrimental to bone [IL6 SNPs, but to no avail. Although the interaction between IL6 -572G/C and dietary acidity was not statistically significant in the analysis, the replication of this interaction in another large population study is highly warranted.In this study, we provide the first  to bone ,45,70, o to bone ,71,72. WIL6 SNP and CTX1. Future studies should also include the investigation of long-term changes in IL6 and the quantification of bone mineral. Secondly, the acid ash diet was estimated from dietary intake. The absorption of individual ions from food is variable and quite complex. It is highly dependent on an individual\u2019s gastrointestinal absorption of the nutrients from food and also on kidney function to filter the net acid excretion. Thus, measurement bias in determining the acid ash diet\u2019s effect on bone health was one of the major limitations that could not be avoided. In addition, although we have included the confounders in the analysis, other potential confounding factors such as gene\u2013gene interactions due to other potential candidate genes (vitamin D receptor [ER\u03b2) [TGF-\u03b21) [An important strength in our study was that it was population-based. Bone health is highly dependent on heritability, and a population-based study may minimize the bias of the result. There are several limitations in this study. Firstly, it was a cross-sectional study, and hence we were unable to determine the causality between the consumption of acid ash diet/receptor , estrogeor [ER\u03b2) , transfo[TGF-\u03b21) , C-react[TGF-\u03b21)  were not[TGF-\u03b21) ,78,79\u2014foIL6 polymorphism at -174 G/C is rare in the Chinese population and does not contribute significantly to CTX1. Besides this, our results also do not support the interaction effect of dietary acidity and IL6-572 G/C with CTX1. These data add to the limited literature regarding the interaction between DAL and the IL6 polymorphism associated with bone health, especially in Asian populations. It is suggested that future studies should include other bone biomarkers for bone resorption and formation, and should be accompanied by more time points of assessment for the better understanding of the role of genetic polymorphism and dietary acidity in bone health.In summary, our study confirms that the"}
+{"text": "The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they have similar or antagonistic function depending on genes. H2A.Z.1 and H2A.Z.2 can replace each other at Transcription Start Sites, providing a molecular explanation for this interplay. Mass spectrometry analysis showed that H2A.Z.1 and H2A.Z.2 have specific interactors, which can mediate their functional antagonism. Our data indicate that the balance between H2A.Z.1 and H2A.Z.2 at promoters is critically important to regulate specific gene expression, providing an additional layer of complexity to the control of gene expression by histone variants. The H2A.Z histone variant is one of the two histone variants conserved from yeast to human. It is enriched at the \u22121 and +1 nucleosomes surrounding the nucleosome-depleted region of active promoters . It can H2AFZ and H2AFV, leading to the production of three proteins, one produced from the H2AFZ gene, called H2A.Z.1, and two splicing variants produced from H2AFV, called H2A.Z.2 and H2A.Z.2.2 , indicating that H2A.Z isoforms regulate overlapping sets of genes. However, they also have independent functions, since 2814 and 741 genes are regulated specifically by H2A.Z.1 and H2A.Z.2 respectively. The lists of genes up-regulated or down-regulated upon H2A.Z.1 or H2A.Z.2 depletion in WI38 cells are shown in We next analysed whether H2A.Z.1 and H2A.Z.2 regulate the same set of genes. We found that among the 3573 genes regulated by H2A.Z.1, 759 are also regulated by H2A.Z.2 , whereas the expected overlap for random lists of gene of the same size would be 255. Actually, more than half of the genes regulated by H2A.Z.2 are also regulated by H2A.Z.1. This intersection is highly significant  deregulated upon either H2A.Z isoform depletion. This showed a striking similarity with RNA-Seq results, validating the analysis . MoreoveImportantly, very similar results were observed in RNA-Seq data obtained following depletion of H2A.Z.1 or H2A.Z.2 in tumoral U2OS cells. We found more genes regulated by H2A.Z.1 (5196), less by H2A.Z.2 (673) than in WI38 cells, with roughly an equivalent number of activated and repressed genes . The lisFinally, we crossed the results obtained in U2OS cells with those obtained in WI38 cells . DespiteCDKN1A/p21, GAPDH genes were very similar and showed accumulation of H2A.Z.1 and H2A.Z.2 signal mostly at the TSS (http://www.enhanceratlas.org/). We next computed the ratio of H2A.Z.1 to H2A.Z.2 ChIP-Seq signals at these enhancers as well as at all TSS or on control genomic regions. As previously found by We next tested whether genes specifically regulated by a given isoform were characterized by a specific feature with respect to the presence of this isoform at their promoters. Thanks to the U2OS cells lines with tagged endogenous isoforms, we performed H2A.Z.1 and H2A.Z.2 ChIP-Seq experiments. ChIP-Seq profiles on the  the TSS . We then the TSS , as alre the TSS . Importa the TSS . Althoug the TSS . Similar the TSS . To testWe next asked whether the depletion of both isoforms together in WI38 non-transformed cells could lead to cumulative effects, as could be guessed if the total amount of H2A.Z was functionally important. As mentioned, only upon transfection of both siRNAs could we achieve efficient inhibition of total H2A.Z expression see . We thusNote however that cumulative effects can be observed on genes which are deregulated only upon the combined depletion of the isoforms, as expected, as well as on some genes which are similarly regulated by H2A.Z.1 and H2A.Z.2 .Again, very similar results were observed in U2OS cells, in which the effects of depleting one isoform were attenuated upon depletion of the other , with thAltogether, these data indicate a conserved complex interplay between H2A.Z.1 and H2A.Z.2 in specific gene regulation, with two types of H2A.Z-regulated genes: genes that they regulate similarly or on which they can compensate each other and genes that they differentially regulate and on which there is a rather general antagonism between the two isoforms.We next investigated the molecular mechanism underlying this complex interplay. H2A.Z is proposed to regulate specific gene expression by binding around gene Transcription Start Sites (TSSs) . One posGAPDH and CDKN1A/p21 promoters in U2OS cells expressing endogenous H2A.Z with a 3xFlag-2xStrep tag. Moreover, at the genome-wide level, we observed a strong correlation between the levels of H2A.Z.1 and H2A.Z.2 binding to gene promoters   . Likewisctively) .Thus, taken together, these data indicate that PHF14 and SIRT1 are major mediators of H2A.Z.1- and H2A.Z.2-specific gene regulation, since i) depletion of H2A.Z isoforms specifically affects their localisation ii) they affect transcription of a significant proportion of H2A.Z-regulated genes in a manner similar to the H2A.Z isoforms. Given that these effects can be positive or negative, our data further underline the function of PHF14 and SIRT1 in mediating the context-dependent regulation of specific gene transcription by H2A.Z isoforms.Strikingly, when we analysed the PLAT mRNA, we found that co-depletion of SIRT1 along with H2A.Z.1 or PHF14 also abolishes the effects of H2A.Z.1 and PHF14 depletion . This shRRM2 and AKAP12 promoters (GAPDH and \u03b2-actin) and on the H2A.Z.1-regulated PLAT promoter Thus, altogether these data indicate that PHF14 favours histone acetylation, at least at the RRM2 and AKAP12 promoters.We next investigated the mechanism involved in this antagonism. Given that SIRT1 is a protein deacetylase, we tested whether PHF14 could favour protein acetylation. Indeed, PHF14 and associated proteins were already proposed to function by counteracting the function of the repressive LSD1-CoREST complex, which contains histone deacetylases along the H3K4 demethylase . Thereforomoters . Note thHere, we performed an integrated study of the role of H2A.Z isoforms H2A.Z.1 and H2A.Z.2 on gene expression in non-transformed cells.We first confirmed that H2A.Z is a major regulator of specific gene expression. If we take into accounts all genes differentially regulated upon depletion of H2A.Z.1, H2A.Z.2 or both together, we found that 5005 and 6317 genes are deregulated in WI38 and U2OS cells, which represent roughly 1/4th of all expressed genes. Interestingly, the number of genes activated by either H2A.Z isoform is roughly equivalent to the number of genes they repress, in agreement with previous studies underlining a positive and negative role for H2A.Z in gene expression .We also found that there are many genes at which H2A.Z isoforms play similar roles including some on which they can compensate each other. This compensation probably explains why a strong phenotype is observed upon depletion of both H2A.Z isoforms in mouse intestinal stem cells whereas depletion of individual isoforms had no or weak effects . At thesWe further found that both isoforms regulate different sets of genes, as previously shown , again cAltogether, these data underline that H2A.Z isoforms H2A.Z.1 and H2A.Z.2 can regulate gene expression similarly and antagonistically depending on the gene, uncovering a complex interplay.We also provide major insights into the mechanism of this interplay. By ChIP-Seq analysis, we found H2A.Z isoforms are present on most promoters of expressed genes or genes prone to be expressed, as already shown for total H2A.Z . ClearlyThese data also suggest that the two isoforms compete each other for the binding to gene promoters. As a consequence, at a promoter specifically regulated by a given isoform, the presence of the other isoform would counteract the presence and the function of the regulatory isoform. Such a model would explain the rather general antagonism we observe on genes specifically regulated by an isoform. It underlines the functional importance of the relative levels between the two isoforms.Thus, the same mechanism, that\u00a0is the ability to be incorporated at the same promoters, could explain both the compensation between H2A.Z isoforms at some promoters and their antagonism at others. It also fits with the fact that they can both interact with incorporation machineries (p400- and SRCAP complexes).We next investigated how H2A.Z isoforms could differentially regulate gene expression. We identified proteins interacting preferentially with one isoform or the other, that\u00a0is PHF14 with H2A.Z.1 and SIRT1 with H2A.Z.2. To our knowledge, this is the first demonstration that endogenous H2A.Z.1 and H2A.Z.2 can differentially bind to proteins. Note however that it was previously found using overexpressed proteins that H2A.Z.1 nucleosomes were slightly more efficient than H2A.Z.2 nucleosomes in interacting with BRD2 as well as with PHF14, HMG20A and TCF20 .A functional interaction between H2A.Z and SIRT1 in the nucleus was also already described . The autFunctional analysis of PHF14 and SIRT1 indicates that they can mediate, at least in part, the specific effects of H2A.Z.1 and H2A.Z.2 on gene expression. In particular, we provide evidence that PHF14 and SIRT1 can mediate the functional antagonism between both H2A.Z isoforms. These two proteins would function as readers of the presence of a specific H2A.Z isoform at gene promoters. Upon depletion of one isoform, the other isoform takes over, resulting in changes in the amount of promoter-bound PHF14 and/or SIRT1. These changes may in turn affect gene expression in a context-dependent manner. Importantly, we found that PHF14 favours acetylation of histones, at least on H3K9 residues at its target promoters. Thus, this may provide a mechanism by which PHF14 and SIRT1 mediate opposite effects on gene expression: when H2A.Z.1 is depleted, there is less recruitment of PHF14 and more recruitment of SIRT1, shifting the balance towards local deacetylation, whereas when H2A.Z.2 is depleted, the balance is shifted towards local acetylation. Such changes in acetylation of histones or non-histone proteins could mediate the context-dependent regulation of gene expression. However, the fact that there are many genes regulated by H2A.Z isoforms H2A.Z.1 and H2A.Z.2 but not by PHF14 and SIRT1 suggests that other mechanisms mediate H2A.Z gene expression regulation, such as changes in nucleosome stability, as previously proposed, or interaction with other specific effectors.H2AFZ promoter is a direct target of the Wnt signalling pathway  is located in the N-terminal tail basic patch important for H2A.Z function and target of multiple post-translational modifications such as acetylation, methylation or ubiquitylation. Furthermore, T14 of H2A.Z.1 could itself be phosphorylated which would likely affect adjacent lysine modifications (K13/15). Acetylation of H2A.Z has indeed been linked to transcriptional activation by many studies . It is tInterestingly, a recent study showed that a S38T\u00a0substitution in H2A.Z.1,\u00a0mimicking T38\u00a0found in H2A.Z.2, rescues\u00a0the SRCAP-dependent Floating-Harbor Syndrome . UnderstLung Fibroblastic cells WI38 hTERT RAF1-ER, which are immortalized by hTERT expression and contain an inducible RAF1 oncogene fused to estrogen receptor (ER) , were grOsteosarcoma U2OS cells were obtained from the ATCC and were grown in DMEM media with Glutamax (1 g/L glucose), supplemented with 10% of FBS, sodium pyruvate and penicillin-streptomycin.2 in RPMI medium supplemented with 10% newborn calf serum (Wisent) and GlutaMAX. When cultivated in spinner flasks, 25 mM HEPES-NaOH (pH 7.4) was added. Cells were transfected using the Amaxa 4D-Nucleofector (Lonza) per the manufacturer\u2019s recommendations.K562 cells were obtained from the ATCC and maintained at 37\u00b0C under 5% COAll cell lines were regularly checked for the absence of mycoplasma contamination.siRNA transfection was performed using the Dharmafect four reagent (Dharmacon) according to the manufacturer\u2019s instructions. 24 hr before transfection, 1.4 million of Wi-38 cells, and 850,000 of U2OS cells were plated in 10 cm dish. 100 nM of siRNA were used, and an equal volume of the culture medium was added 24 hr after transfection. 48 hr later, cells were harvested. The list of siRNAs used is indicated in H2AFZ and H2AFV were selected using a web-based tool  and assembled using the Zero Blunt TOPO cloning kit (Invitrogen). The donor plasmid for H2AFV was obtained by cloning. Briefly, a PCR fragment of 1.7 kb genomic DNA (1008 bp before stop codon and 690 bp after stop codon) was integrated in Zero Blunt TOPO plasmid following manufacturer\u2019s instructions. Subsequently, the PAM motif corresponding to the gRNA was mutated and the 3xFlag-2xStrep sequence -driven human-codon optimized Cas9 nuclease (Addgene #41815) was used. gRNA sequences targeting sequence  was inteH2AFZ by homology directed repair. One million K562 cells were transfected using 2 \u03bcg gRNA plasmid, 2 \u03bcg Cas9 vector, and 4 \u03bcg donor (plus 600 ng i53 plasmid for H2AFZ). Limiting dilution cloning was performed 3 days post-transfection, and targeted clones were identified via out-out PCR as before . Primers were then chosen according to the website proposition. To clone guides, a phosphorylation step consisting on incubation of 1 \u03bcl of each oligo (100 \u03bcM) at 37\u00b0C for 30 min in the presence of the PNK enzyme (Promega) was performed. Then, samples were heated for 5 min at 95\u00b0C, and cooled over night at room temperature. The annealed phosphorylated product was then digested-ligated in a one step reaction into the ATP1A1 G3 dual sgRNA plasmid in the presence of bbs1 restriction enzyme (NEB) and T7 ligase (NEB), and the reaction was subjected to the following PCR cycle . The product of the ligation was exonuclease-digested using exonuclease V RecBCD for 30 min at 37\u00b0C. Competent cells were then transformed with the final product, and positive clones were selected by bbs1 digestion. 200,000 U2OS cells plated in 6-well plates were transfected with this plasmid, a double stranded 3xFlag-2xStrept tagged PHF14 DNA donor  or donor plasmids for H2AFZ and H2AFV described above and Ouabaine-resistance donor  and guide to a final DNA concentration of 500 ng, using jetPEI polyplus reagent according to manufacturer\u2019s instructions. 48 hr later, cells were trypsinised and transferred into 10 cm dish, and ouabaine (sigma) was added to a final concentration of 0.5 \u03bcM, for one week. Clones were then recovered and screened by PCR for the presence of Flag-tag. Positive clones were verified by sequencing. Primers for each guide are detailed in RNA guides for targetting Total Cell extracts were prepared and analysed by standard Western blotting protocol, using antibodies against total H2AZ , GAPDH , PHF14 , SIRT1 , Flag , Pol I , H3 , pan Acetyl-H3 , Acetyl-H3K9 , Flag-HRP , Brd8 (Bethyl A300-219A), DMAP1 ,\u00a0ACTR6(ARP6) ,\u00a0p400 , SRCAP (gift from J. Chrivia),\u00a0BAF53a ,\u00a0EPC1 .Purification of endogenously tagged H2AZ.1 and H2AZ.2 was performed basically as described . Typical[pH 7.9], 10% glycerol, 300 mM KCl, 0.1% Tween 20, 1 mM DTT, 1 mM PMSF, 2 \u03bcg/mL Leupeptin, 5 \u03bcg Aprotinin, 2 \u03bcg/mL Pepstatin, 10 mM Na-butyrate, 10 mM \u03b2-glycerophosphate, 100 \u03bcM Na-orthovanadate, 5 mM N-Ethylmaleimide, 2 mM Ortho-Phenanthroline) followed by 40 CV of buffer #2 . Complexes were eluted in two fractions with 2.5 CV of buffer #2 supplemented with 200 ug/ml 3xFLAG peptide (Sigma) for 1 hr at 4\u00b0C. Next, fractions were mixed with 125 ul Strep-Tactin Sepharose (IBA) affinity matrix for 2 hr at 4\u00b0C, and the beads were washed with 20 CV of buffer #2. Complexes were eluted in two fractions with 2 CV of buffer #2 supplemented with 4 mM D-biotin, flash frozen in liquid nitrogen, and stored at \u221280\u00b0C. Typically, 15 ul of the first elution  was loaded on NuPAGE 4\u201312% Bis-Tris gels (Invitrogen) and analyzed by silver staining.The analyses were performed at the proteomic platform of the Quebec Genomics Center.18  stationary phase. The peptides were eluted from the column by a gradient generated by an Agilent 1200 HPLC system  equipped with a nano electrospray ion source coupled to a 5600+ Triple TOF mass spectrometer . A 65 min linear gradient of a 5\u201335% mixture of acetonitrile, 0.1% formic acid injected at 300 nL/min was used to elute peptides. Data dependent acquisition mode was used in Analyst version 1.7 (Sciex) to acquire mass spectra. Full scan mass spectrum (400 to 1250 m/z) were acquired and followed by collision-induced dissociation of the twenty most intense ions. A period of 20 s and a tolerance of 100 ppm were set for dynamic exclusion.The peptides were directly loaded at 300 nL/min onto a New Objective PicoFrit column  packed with Jupiter 5 \u03bcm CHomo sapiens database  assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance and a parent ion tolerance of 0.1 Da. Oxidation of methionine and deamidation of asparagine and glutamine were specified as variable modifications and carbamidomethylation as fixed modification. Two missed cleavages were allowed. Scaffold (version 4.0.1), Proteome Software Inc, Portland, OR) was used to validate MS/MS based peptide and protein identifications. Proteins/peptides FDR rate was set to 1% or less based on decoy database searching. The Protein Prophet algorithm assigned protein probabilities. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.Protein Pilot version 5.0 (Sciex) was used to generate MS/MS peak lists. Mascot  was used to analyze MGF sample files. Mascot was set up to search the UniprotKB www.crapome.org; https://prohits-viz.lunenfeld.ca/;Data were further analyzed using the CRAPome online tool (Cell pellets (5 million cells) were resuspended in 250\u2013300 \u03bcl of lysis buffer  and incubated at 4\u00b0C for 5 min. 10 \u03bcl were kept to prepare whole cell extracts. 50 \u03bcl/ml of NP40 were then added and samples were incubated for additional 10 min at 4\u00b0C and then centrifuged at 3000 g for 5 min. The supernatant was collected and represented the cytoplasmic fraction. The remaining pellet was resuspended in 35 \u03bcl of nuclear buffer  and incubated 30 min at 4\u00b0C. Samples were centrifuged at full speed for 5 min. The supernatant was removed and a second extraction was performed on the remaining pellet with a nuclear buffer high in salt . After 30 min of incubation at 4\u00b0C, samples were centrifuged at full speed. The supernatant was collected and represented the soluble nuclear fraction. The pellet corresponding to the chromatin was resuspended in boiling buffer  and sonicated 5 times at 25% amplitude for 10 s. Whole cell extracts were also resuspended in boiling buffer and sonicated. All the buffers were supplemented with EDTA-free protease inhibitor cocktail (Roche).7 cells/ml in buffer A  and incubated at 4\u00b0C for 8 min. Small volume was kept to prepare whole cell extracts. The samples were then centrifugated at 1300 g for 5 min. The supernatant was collected and represented the cytoplasmic fraction. The nuclei pellet was washed once in buffer A and lysed for 30 min on ice in buffer B . Samples were centrifuged at full speed for 5 min. The supernatant was collected and represented the soluble nuclear fraction. The pellet corresponding to the insoluble chromatin was washed once in buffer B. The chromatin was then extracted with buffer A and sonicated 3 times at 25% amplitude for 10 s. Whole cell extracts were sonicated with the same protocole. All frations were clarified by full speed centrifugation. The buffers were supplemented with EDTA-free protease inhibitor cocktail (Roche).Cells were trypsinated, counted, and resuspended at a concentration of 4 \u00d7 10Total RNA was prepared using the MasterPure RNA Purification Kit (Epicentre) supplemented with Baseline-ZERO DNase, according to the manufacturer\u2019s instructions. For random-primed RT-qPCR, 200 ng of RNA were used for each reverse transcription reaction. The reverse-transcription was performed using random primers and superscript III reverse transcriptase (Invitrogen) at 50\u00b0C according to manufacturer\u2019s protocol. qPCR analysis was performed on CFX96 devices (BioRad) using the SYBR Premix Ex Taq II (Takara), according to the manufacturer\u2019s instructions. All samples was analysed in duplicates. All data was normalized relative to GAPDH mRNA levels. The list of primers can be found in Drosophila melanogaster chromatin , and 1 ug of an antibody recognizing H2Av, aDrosophilaspecific histone variant, , were incubated overnight with 4 \u03bcg of antibody at 4\u00b0C. A mock sample without antibody was processed similarly. Then, 20 \u03bcl of blocked A/G beads were added for 2 hr at 4\u00b0C to recover immune complexes. Beads were washed once in dialysis buffer , four times in wash buffer  and twice in TE buffer . The bead/chromatin complexes were resuspended in 200 \u03bcl of TE buffer and incubated 30 min at 37\u00b0C with 10 \u03bcg of RNase A (Abcam), as well as input DNA. Formaldehyde crosslink was reversed in the presence of 0.2% SDS at 70\u00b0C overnight with shaking. After 2 hr of proteinase K (0.2 mg/ml) treatment at 45\u00b0C, immunoprecipitated and input DNA were purified on columns using Illustra GFX kit . All buffers for ChIP experiment were supplemented with EDTA-free protease inhibitor cocktail (Roche) and filtered 0.2 \u03bcM. Results were analysed by qPCR. The list of primers used can be found in 15 million cells transfected with siRNA were crosslinked for 15 min using 1% formaldehyde directly in the culture medium. 0.125 M of Glycine were then added for 5 min. After two washes with PBS, cells were scraped and frozen at \u221280\u00b0C. Cells were lysed with 3 ml of a lysis buffer  and homogenized 40 times with a dounce . After centrifugation, nuclear pellets were resuspended in 1.5 ml of nuclear lysis buffer , and sonicated 10 times for 10 s , to obtain DNA fragments of about 500 bp. DNA concentration was determined using a Nanodrop and samples were adjusted to the same concentration of chromatin. Samples were diluted at least one time in dilution buffer  and precleared for 2 hr with 250 \u03bcl of previously blocked protein-A and protein-G beads (Sigma P-7786 and P-3296 respectively). Blocking was achieved by incubating the beads with 0.5 mg/ml of Ultrapure BSA for 3 hr at 4\u00b0C. ChIP reaction was performed in 1 ml final volume. 100 \u03bcl of chromatin were kept for inputs. 50 \u03bcg of pre-cleared samples per ChIP supplemented with 10 ng of 8 million cells were lysed with 600 \u03bcl the lysis buffer . Samples were incubated 15 min on ice and vortexed each 5 min. After 15 min of centrifugation at full speed, supernatant was recovered and diluted 3 times with dilution buffer . NP40 final concentration was adjusted to 0.5%, and lysates were quantified. 1 mg was used per immunoprecipitation reaction. Preclearing was done by incubating lysates 2 hr at 4\u00b0C with 15 \u03bcl of A/G beads (Sigma). 4 \u03bcg of Flag antibody were then added ON at 4\u00b0C. 15 \u03bcl of A/G beads were added for 2 hr at 4\u00b0C, and immuno-complexes were washed 4 times with wash buffer . Results were analysed by Western blot.In WI38 cells, to identify the interplay between H2A.Z. isoforms, two samples of siCtrl, siH2A.Z.2, and siH2A.Z.1 + siH2A.Z.2 and one sample of siH2A.Z. one were transfected in parallel. The other siH2A.Z.1 sample came from In U2OS cells, two samples of siCtrl, two sample of si Ctrl#, siH2A.Z.1, siH2A.Z.2 and siH2A.Z.1+siH2A.Z.2 were transfected in parallel.We used strand-specific RNA-Seq method, relying on UTP incorporation in the second cDNA strand. For each sample, 5\u201310 \u03bcg of total RNA, extracted as described above in (RNA extraction and Reverse transcription), was submitted to EMBL-GeneCore, Heidelberg, Germany. Paired-end sequencing was performed by Illumina\u2019s NextSeq 500 technology. Two replicates of each sample were sequenced.For the ChIP-seq in U2OS cells expressing Flag-H2AZ.1 and Flag-H2AZ.2, 100 \u03bcg of chromatin supplemented with 10 ng of spike in chromatin (active motif) were used per ChIP experiment. For each reaction, 4 ug of Flag M2 antibody (sigma) and 1 ug of spike in antibody (active motif), were used. About 10 ng of immunoprecipated DNA  were obtained at each time, and samples were submitted to EMBL-GeneCore Heidelberg for sequencing, that was performed by Illumina\u2019s NextSeq 500 technology.ChIP-seq in K562 cells expressing Flag-H2A.Z.1 and Flag-H2A.Z.2 were performed and analysed as previously described .RNA-Seq samples were sequenced using Illumina NextSeq 500 sequencer, paired-end, 80 bp reads, at EMBL Genomics core facilities . The quality of each raw sequencing file (fastq) was verified with FastQC . Files wSeveral differential analysis  were done with DESeq2 Bioconductor R package, Version 1.22.1  with defLists of gene of interest were crossed to identify common genes, and results were represented using Venn Diagrams with R and VennDiagram package. For each crossing, the Chi-square test (chisq.test function in R) was applied to the associated contengency table. The test evaluate if the 2 lists of genes are independent that\u00a0is whether there is a significant association between the categories of the two variables. Chi-square p-values were then corrected for multiple testing (one test per crossing) with the Benjamini\u2013Hochberg method.Box-plots representing the RPKM value were generated with R-base based on the mean of replicates from Tables S1-8. The center line represents the median, box ends represent respectively the first and third quartiles, whiskers represent the minimum and maximum values without outliers. Outliers were defined as below 1stQuartile \u2212 1.5\u00a0\u00d7\u00a0InterquartileRange and above 3rdQuartile + 1,5\u00a0\u00d7\u00a0InterquartileRange. Nonparametric Mann\u2013Whitney\u2013Wilcoxon test (wilcoxon.test function in R) was applied to test distribution differences between two populations.ChIP-Seq samples were sequenced using Illumina NextSeq 500 sequencer, single-end, 80 bp reads, at EMBL Genomics core facilities .The quality of each raw sequencing file was verified with FastQC. Files were aligned to the reference human genome (hg38) in single-end mode with  and procChiP-Seq mean coverage per base was computed for each annotated gene, in a window of +/-\u00a02 kb around Transcription Start Site (TSS). For each gene, the log2 mean value in these windows was computed and plotted using R-base, in a dot-plot representing H2AZ1 versus H2AZ2. Lastly, the log2 ratio of the mean value in these windows for H2AZ1 divided by the mean value in these windows for H2AZ2 was computed for 5 list of genes selected through the RNA-Seq differential analysis: genes up-regulated from siH2AZ1 versus siCtle, genes down-regulated from siH2AZ1 versus siCtle, genes up-regulated from siH2AZ2 versus siCtle, and un-regulated genes in both analysis. Results of these calculations were shown in a boxplot. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper begins to resolve the previously enigmatic roles in gene regulation of two closely related isoforms of the variant histone H2A.Z. By engineering cells to allow the targeted depletion of one or both of these isoforms, the authors show, surprisingly, that these two nearly identical proteins work antagonistically, and make progress towards understanding the molecular basis of this interaction. The antagonism of two forms of the same histone variant/reader combination represents a novel mechanism for modulating gene activity, and should be of broad interest.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Michael Eisen as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article \"Integrated analysis of H2A.Z isoforms function reveals a complex interplay in gene regulation\" 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.The manuscript deals with the functional analysis of the two histone variant H2A.Z isoforms. These isoforms are highly similar with only three amino acid differences, but have been recently shown to play distinct roles in tumor development, gene regulation and DNA repair. The authors have used siRNAs against edited epitope-tagged versions of the isoforms to disentangle their individual roles in gene regulation. H2A.Z occupies nucleosomes that flank active promoters and enhancers and has long been enigmatic in its effects on gene regulation, showing up-regulation at some genes and down-regulation at others. Here this finding is confirmed by isoform depletions in two different human cell lines, but the unexpected result is that depletion of both isoforms results in effects opposite to expectation, namely depletion of both isoforms restores a more wild-type-like situation.eLife policy not to request such experiments, we are leaving the inclusion of additional data to the authors' discretion.Overall, the reviewers judged that the authors made important observations that are of interest for the histone variant and chromatin research field. There was some discussion of whether additional replicates and therefore more robust statistical analysis is required. Recognizing that doing such experiments might take more than 2 months and would therefore be inconsistent with The reviewers made specific comments and questions listed below that we request be addressed in the revision.Essential revisions:In Figures 2 and 6 Venn diagrams are shown to indicate the number of genes showing effects caused by knockdown of either isoform and the overlap indicates the number of genes affected by both, with p-values based on Chi-square tests. However, astronomical p-values are the norm for such comparisons involving changes in thousands of genes, and the p-values are potentially sensitive to minor technical biases. Also p-values compare observations to random expectation, which is not the same as asking how different the results are from what no treatment would show. However, each experiment included a control siRNA, and so the degree to which technical variation might have inflated these p-values can be estimated by comparing each to its siRNA control as was done in the histograms and box-plots.It is not always clear how many replicates are being analyzed, or what kind of statistics are being used to report on the data . In some places it appears that error bars are being used for two samples.Figure 1B: What is the band observed at around 20 kDa? Could this be modified, possibly ubiquitinated or sumoylated, endogenous protein? In this case, is such a shift also observed with the Flag-tagged histones? A larger view of the blot would be helpful, as well as blots with a Flag antibody.Why do the authors use K562 cells instead of the in the study well-characterized U2OS cells for protein-pull-down assays? Do K562 cells show a similar effect in deregulated genes upon H2A.Z.1 or H2A.Z.2 depletion?What is the experimental difference compared to previously performed mass-spectrometry-based identifications of H2A.Z interactors? The authors should indicate in the Results section whether they used nuclear extracts, chromatin-fractions, mononucleosomes, whole cell extracts or others. Why were the authors not able to identify direct and strong H2A.Z interacting proteins such as e.g. BRD2 , PWWP2A , but were able to identify the p400 chaperone complex that interacts mostly with free nuclear H2A.Z proteins? Did the authors use chromatin-free nuclear extracts?Figure 5\u2014figure supplement 1C: The figure legend is not sufficient to describe the experimental data in a way that it is understandable. What does NT mean? Not transfected, no tag? It would be good to show where the primers bind to explain the size differences between H2AFZ and H2AFV PCR products. Ideally, the authors would include western blot analysis with H2A.Z antibody for these cells as done for U2OS cells in Figure 1.Figure 5\u2014figure supplement 1E: The legend describes 5 classes, shown are 4 groups labelled with G1-G4. Are these groups labelled according to gene expression levels: G1 is lowest and G4 highest? More information is required to understand the figure.Figure 6: the authors should indicate how many times these experiments were repeated. In A) H2A.Z western blots should be shown to document knock-down efficiency. Also, H2A.Z.1 seems to lead to an increase in Flag-PHF14 while H2A.Z.2 depletion to a decrease. The authors should quantify this result and, if reproduced, check whether PHF14 RNA transcripts are also affected. Hence, more experiments are required to strengthen the statement: \"Thus H2A.Z.1 favours the interaction of PHF14 with chromatin\", especially since there is no difference observed in PHF14 levels in the chromatin fraction upon H2A.Z.1 depletion. Similar for SIRT1 .Figure 6C and Figure 1D: when comparing the effects of PHF14 or SIRT1 depletion on the expression of those six gene with the effects observed with H2A.Z.1 or H2A.Z.2 depletion, I do not agree with the conclusions made by the authors in the subsection \u201cPHF14 and SIRT1 are major H2A.Z1 and H2A.Z.2 effectors\u201d. The authors should use the same axis values to better compare the results. It appears that only the PLAT and AKAP12 genes shows a similar trend, all other genes look different. ZDHHC20 shows only minimal increase when PHF14 is depleted (4-fold in siZ1 conditions), RRM2 is not much lower in siSIRT1 conditions, but half the amount in siZ2, COLEC12 and ADAMTS1 do not show any change, but 3/4-fold increases in siZ2 treatments. Also, while there is some overlap of deregulated genes in Figure 6D, E, the statement \"Thus, taken together, these data indicate that PHF14 and SIRT1 are major mediators of H2A.Z.1- and H2A.Z.2-specific gene regulation\u2026\" is an over-interpretation of the data. The conclusion should be toned down or other data should be included that strengthen this statement.Figure 7: Additional replication would be ideal for 7B and additional targets, including ones not dependent on isoform, for 7C.The authors state: \"The conclusion from these experiments is that PHF14 and SIRT1 could have antagonistic effect on local protein acetylation balance, providing a molecular mechanism by which PHF14 and the SIRT1 deacetylase can mediate H2A.Z.1 and H2A.Z.2 antagonism\". This is a too strong conclusion, as no experiments have been performed to show that H3K9ac changes upon PHF14 depletion are due to SIRT1 activity at those sites. Many other proteins can contribute to the observed effects. In order to make such a statement, the authors need to perform PHF14 and SIRT1 ChIP-seq experiments to show their localization at H2A.Z sites and how their localization changes when H2A.Z.1, H2A.Z.2, PHF14 or SIRT1 are depleted. Hence, I would also remove the model in Figure 7D.Discussion: This is not the first study showing that H2A.Z.1 and H2A.Z.2 bind differentially to proteins. Draker et al. already showed a preference of BRD2 binding to H2A.Z.1, an observation also confirmed by P\u00fcnzeler et al. Further, in P\u00fcnzeler et al., PHF14, HMG20A, TCF20 and RAI1 were seen to be enriched on H2A.Z.2 nucleosomes when compared to H2A.Z.1 nucleosomes, as was also seen for other interacting proteins. The authors should rephrase their conclusions. Essential revisions:In Figures 2 and 6 Venn diagrams are shown to indicate the number of genes showing effects caused by knockdown of either isoform and the overlap indicates the number of genes affected by both, with p-values based on Chi-square tests. However, astronomical p-values are the norm for such comparisons involving changes in thousands of genes, and the p-values are potentially sensitive to minor technical biases. Also p-values compare observations to random expectation, which is not the same as asking how different the results are from what no treatment would show. However, each experiment included a control siRNA, and so the degree to which technical variation might have inflated these p-values can be estimated by comparing each to its siRNA control as was done in the histograms and box-plots.First, we want to point out that, in RNA Seq experiments following each protein depletion, all samples were performed together, and two control samples were always included. Differentially expressed genes were then identified compared to these two control samples , while the alternative hypothesis H1 claims that some association does exist. In all the dependencies we tested , the tests indicated that we can reject the H0 hypothesis.However, we do agree with the reviewer that the Chi-square test gives astronomical p values when analyzing large populations. As the reviewer says, it compares to random expectations. In order to clarify how different the result is from random, we now indicate in all Venn diagrams of the revised manuscript the expected number of common genes in the case of random intersection, providing the quantitative data requested by the reviewer.It is not always clear how many replicates are being analyzed, or what kind of statistics are being used to report on the data . In some places it appears that error bars are being used for two samples.We changed the manuscript to carefully state this information in figure legends. Note that in the original manuscript (as well as in data added for revision), all histograms presenting qPCR data were calculated on three entirely independent experiments. The only exception were histograms comparing RT-qPCR with RNA Seq data , given that in this figure, we analyzed by RT-qPCR the two experiments used for RNA-Seq. We now show the RT-qPCR results from the two samples side-by-side.The only other experiment where error bars were calculated using two replicates was the quantification of the western blot shown in Figure 1C. We now present a representative experiment out of two.Figure 1B: What is the band observed at around 20 kDa? Could this be modified, possibly ubiquitinated or sumoylated, endogenous protein? In this case, is such a shift also observed with the Flag-tagged histones? A larger view of the blot would be helpful, as well as blots with a Flag antibody.As requested by the reviewer, we performed a Flag western blot both in U2OS and K562 cells. These data indicate that in these two cell lines this band is present, confirming that it is indeed produced from endogenous H2A.Z, and that it is observed with the two isoforms. It most likely corresponds to ubiquitinated H2A.Z, in a ratio that has been reported before in the literature. This Flag western blot in U2OS cells is shown in the revised manuscript in Figure 1\u2014figure supplement 1 and the Flag western blot in K562 cells is shown in Figure 5\u2014figure supplement 1. The text of the manuscript has been changed accordingly.Why do the authors use K562 cells instead of the in the study well-characterized U2OS cells for protein-pull-down assays? Do K562 cells show a similar effect in deregulated genes upon H2A.Z.1 or H2A.Z.2 depletion?We use K562 cells because they are nonadherent cells which are easier to produce in large amounts for endogenous nuclear proteins purifications and mass spectrometry analysis of interacting proteins. This is now stated in the manuscript.In the time allowed during revision, we confirmed the preferred interactions between H2A.Z isoforms with SIRT1 or PHF14 in U2OS cells by western blotting after affinity purification. These data have been included in the revised manuscript in Figure 5\u2014figure supplement 2.What is the experimental difference compared to previously performed mass-spectrometry-based identifications of H2A.Z interactors? The authors should indicate in the Results section whether they used nuclear extracts, chromatin-fractions, mononucleosomes, whole cell extracts or others. Why were the authors not able to identify direct and strong H2A.Z interacting proteins such as e.g. BRD2 , PWWP2A , but were able to identify the p400 chaperone complex that interacts mostly with free nuclear H2A.Z proteins? Did the authors use chromatin-free nuclear extracts?The reviewer is right in thinking that we were using chromatin-free nuclear extracts . This was actually indicated in the Materials and methods section, but we agree with the reviewer that it is important. Indeed, it explains why we detect the p400, SRCAP and chaperone complexes but not BRD2 and PWWP2A proteins which were identified in over-expressed H2A.Z nucleosome pull-downs from digested chromatin fractions. Accordingly our mass spec data indicates a majority of H2A.Z-H2B dimers compared to octamers. In the revised manuscript, we modified the Results section to indicate the different purification approach compared to what has been reported in other studies. At the same time our different approach provides a new distinct interactome dataset, in a completely physiological setting since we purified endogenous H2A.Z isoforms.Figure 5\u2014figure supplement 1C: The figure legend is not sufficient to describe the experimental data in a way that it is understandable. What does NT mean? Not transfected, no tag? It would be good to show where the primers bind to explain the size differences between H2AFZ and H2AFV PCR products. Ideally, the authors would include western blot analysis with H2A.Z antibody for these cells as done for U2OS cells in Figure 1.We changed the figure to specify what NT is and included western blot data as requested by the reviewer . Primers were already used in Figure 1\u2014figure supplement 1, which is now referred to in the legend.Figure 5\u2014figure supplement 1E: The legend describes 5 classes, shown are 4 groups labelled with G1-G4. Are these groups labelled according to gene expression levels: G1 is lowest and G4 highest? More information is required to understand the figure.We apologize for this mistake. We corrected it in Figure 5\u2014figure supplement 1 from revised manuscript.Figure 6: the authors should indicate how many times these experiments were repeated. In A) H2A.Z western blots should be shown to document knock-down efficiency.This information has now been included in the revised manuscript. H2A.Z western blot is shown in Figure 6\u2014figure supplement 1.Also, H2A.Z.1 seems to lead to an increase in Flag-PHF14 while H2A.Z.2 depletion to a decrease. The authors should quantify this result and, if reproduced, check whether PHF14 RNA transcripts are also affected. Hence, more experiments are required to strengthen the statement: \"Thus H2A.Z.1 favours the interaction of PHF14 with chromatin\", especially since there is no difference observed in PHF14 levels in the chromatin fraction upon H2A.Z.1 depletion. Similar for SIRT1 .No effect could be seen by RT-qPCR. This is now stated in the manuscript. The results are reproducible and we now show two independent experiments . In addition, we now show in Figure 6\u2014figure supplement 2 a quantification showing that the repartition of PHF14 between chromatin and soluble nuclear fraction changes upon H2A.Z1 depletion, therefore suggesting that the presence of H2A.Z.1 favours chromatin localisation of PHF14. We analysed and present similarly SIRT1 data. However, we agree with the reviewer that more experiments would be needed to strengthen this point, and we toned down our conclusions when describing these data.Figure 6C and Figure 1D: when comparing the effects of PHF14 or SIRT1 depletion on the expression of those six gene with the effects observed with H2A.Z.1 or H2A.Z.2 depletion, I do not agree with the conclusions made by the authors in the subsection \u201cPHF14 and SIRT1 are major H2A.Z1 and H2A.Z.2 effectors\u201d. The authors should use the same axis values to better compare the results. It appears that only the PLAT and AKAP12 genes shows a similar trend, all other genes look different. ZDHHC20 shows only minimal increase when PHF14 is depleted (4-fold in siZ1 conditions), RRM2 is not much lower in siSIRT1 conditions, but half the amount in siZ2, COLEC12 and ADAMTS1 do not show any change, but 3/4-fold increases in siZ2 treatments. Also, while there is some overlap of deregulated genes in Figure 6D, E, the statement \"Thus, taken together, these data indicate that PHF14 and SIRT1 are major mediators of H2A.Z.1- and H2A.Z.2-specific gene regulation\u2026\" is an over-interpretation of the data. The conclusion should be toned down or other data should be included that strengthen this statement.We agree with this reviewer that phenocopying was an over-interpretation. As suggested by this reviewer, we now use the same axes in Figure 6C and 1D  and have now toned down our conclusion. Indeed, as indicated above, H2A.Z1 probably regulates genes independently of PHF14, and vice-versa.Figure 7: Additional replication would be ideal for 7B and additional targets, including ones not dependent on isoform, for 7C.We performed the requested experiments and included them in the manuscript .The authors state: \"The conclusion from these experiments is that PHF14 and SIRT1 could have antagonistic effect on local protein acetylation balance, providing a molecular mechanism by which PHF14 and the SIRT1 deacetylase can mediate H2A.Z.1 and H2A.Z.2 antagonism\". This is a too strong conclusion, as no experiments have been performed to show that H3K9ac changes upon PHF14 depletion are due to SIRT1 activity at those sites. Many other proteins can contribute to the observed effects. In order to make such a statement, the authors need to perform PHF14 and SIRT1 ChIP-seq experiments to show their localization at H2A.Z sites and how their localization changes when H2A.Z.1, H2A.Z.2, PHF14 or SIRT1 are depleted. Hence, I would also remove the model in Figure 7D.We agree with the reviewer that many more experiments would be needed to formally demonstrate the original Figure 7D model, that we actually presented as a working model. Therefore, according to the reviewer suggestion, we removed the model from Figure 7D and toned down our conclusions.Discussion: This is not the first study showing that H2A.Z.1 and H2A.Z.2 bind differentially to proteins. Draker et al. already showed a preference of BRD2 binding to H2A.Z.1, an observation also confirmed by P\u00fcnzeler et al. Further, in P\u00fcnzeler et al., PHF14, HMG20A, TCF20 and RAI1 were seen to be enriched on H2A.Z.2 nucleosomes when compared to H2A.Z.1 nucleosomes, as was also seen for other interacting proteins. The authors should rephrase their conclusions.We agree that our phrasing was misleading . Our data are indeed the first to be performed using endogenous proteins, which is now clearly stated in the Discussion section of the revised manuscript. According to this reviewer's suggestions, we now detail Draker et al. and P\u00fcnzeler et al. results when discussing our interaction results."}
+{"text": "Feijoa sellowiana on stem cells.Feijoa is widely used in medicine due to their anti-inflammatory, antioxidant, antimicrobial and antitumor properties. The current investigation studied the proliferative and regenerative effect of acetonic extract of in vitro model and characterized morphologically, by flowcytometry, and differentiation properties. The toxicity of the extract on hBMSCs was determined by MTT assay. The viability and growth kinetics of hBMSCs treated to Feijoa was determined. Real time PCR was used for changes in expression of proliferative and apoptotic genes on day 7th. Acetone extract of Feijoa was prepared using percolator and rotary machines. Human bone marrow stem cells (hBMSCs) were used as experimental rd followed by a non-significant slow decreasing trend until day 7th. Population doubling time (PDT) showed a decline until day 3rd followed by an increase until day 7th. A significant rise in expression of Bax and decline in Bcl-2 expression were noted on day 7th.MTT assay demondtrated that Feijoa at doses less than 200 ng/ml did not show any cytotoxic effect on hBMSCs and increased the cell proliferation until day 3rd. Therefore, when faster proliferation during a shorter time period is targeted, Feijoa can be safely added to the culture media in the first three days.The modulatory activity of Feijoa may be responsible for its increasing effect on cell proliferation till day 3 Feijoa sellowiana (O. Berg) Burret) is an evergreen tree belonging to Myrtaceae family, with gray branches, elliptical buds, white and red flowers, and sweet-smelling leaves and is indigenous to western highlands of Paraguay, southern Brazil, Uruguay and northern Argentina.1-6 This plant entered the Mediterranean region in the late nineteenth century and also entered into Islamic Republic of Iran in 1973 through the Republic of Azerbaijan. This shrub is just grown on the northern strip of Iran.7 Feijoa has several biological features such as antibacterial,8 analgesic and anti-inflammatory,9-11 antioxidant and anticancer12,13 effects. Feijoa  denoting to the medicinal property of Feijoa. MDMA is a ring-substituted amphetamine derivative that was synthesized in 1912 by Merck chemical company and has attracted a great deal of attention in recent years. It has widespread abuse as a recreational drug by the young generation.19 This study was undertaken to evaluate the proliferative and regenerative effect of acetonic extract of Feijoa sellowiana on human bone marrow derived stem cells (hBMSCs).Investigation on human myeloid leukemia cells showed that flavonoids in Feijoa induce apoptosis through caspase activation, overexpression of p16, p21 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), increase in histone and non-histone acetylation and inhibition in histone deacetylase (HDAC) pathways.Feijoa sellowiana [(O. Berg) Burret] leaves were collected from Ramsar Research Center, northern Iran in spring of 2017. To remove the epiphytic hosts, which are normally present on the surface, the leaves were treated with 0.8% Triton X-100. Distilled water was used for extensive washing and then the leaves were dried on filter papers in a dark and dry place for two weeks and were later changed into powder. Totally, 100 g of the powder was placed in a percolator machine for 72 hours in 1000 mL of acetone , while the acetone solvent was rotated at 45\u00b0C and 50 rpm in a rotary machine  to be completely evaporated for the solvent.20Feijoa . Bone marrow (BM) samples were provided from the Bone Marrow Transplantation Center of Nemazi Hospital, affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. A written signed consent form was provided from the subjects by Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. BM was diluted with an equal volume of Dulbecco\u2019s Modified Eagle Medium  and was carefully layered over an equal volume of Ficoll-Paque product  as described before.2 incubator at 37\u00b0C with saturated humidity . The medium was changed every 3 days until 80% confluence and sub-cultured using 0.25% trypsin  till passage 3rd. It was centrifuged at 1500 rpm and 20\u00b0C for 30 min to separate a second layer. The interface between the plasma and the Ficoll-Paque layer consisted of mononuclear cells (MNCs) were seeded in 75 cm flasks containing 88% alpha minimal essential medium , 10% fetal bovine serum , 1% penicillin and streptomycin , and 1% L-glutamine . hBMSCs were transferred in 5% COIn vitro differentiation to osteogenic lineage was assessed by seeding 5\u00d7104 hBMSCs in a 12-well plate containing osteogenic medium of DMEM, 10% FBS, 200 \u03bcM L-ascorbic acid, 10 mM glycerol phosphate and 100 nM dexamethasone, while the media was changed every three days until 3 weeks. Alizarin red  staining that bound to calcium mineralized deposits revealing a red color when osteogenic differentiation was induced.The adhered cells were assessed morphologically to be spindle shape. In vitro differentiation to adipogenic lineage was investigated by seeding 5\u00d7104 hBMSCs in a 12-well plate containing adipogenic medium of DMEM, 10% FBS, 1 \u03bcmol/L dexamethasone, 200 \u03bcg/mL insulin, 0.5 mmol/L isobutylmethylxanthine, and 60 \u03bcmol/L indomethacin  for 21 days, while the medium was replaced every 3 days. Then, the cells were stained with Oil red O  to evaluate presence of red color droplets, when adipogenic differentiation was induced. Flowcytometry of hBMSCs was conducted for expression of mesenchymal markers (CD73 and CD90) and hematopoietic markers   in passage 3rd.4 hBMSCs were seeded in a 96-well plate . After 24 hours, the medium was changed and different doses of Feijoa  were added and transferred to 5% CO2 incubator for one day at 37\u00b0C and saturated humidity. Twenty micro-liter of MTT was added for 4 hours for each 200 \u03bcL of the culture medium. It was later centrifuged for 5 minutes, the supernatant was removed and 200 \u03bcL of DMSO  was added to the remaining. Optical absorption at 570 nm was read by an ELISA plate reader . Based on the highest percentage of cell viability that 200 \u03bcL/mL of Feijoa induced in hBMSCs until 7 days, this concentration was selected as optimum dose in all experiments  was added to the cell suspension and counted each day using a Neubauer hemocytometer slide and a phase contrast microscope until 7 days. The cell viability was calculated at optical density of 570 nm on a microplate reader  (n=4) using the following formula, while Ac and Ab were considered as the absorbance in the control and blank wells. %Survival rate=A sample\u2013(Ab\u00d7Ac\u2013Ab)\u00d7100.22hBMSCs from passage 36 cells/mL until use.22The population doubling time (PDT) was measured and the growth curve was plotted using the following the formula of PDT=T ln2=ln(Xe/Xb); while T is the incubation time in hours, Xb is the cell number at the beginning of the incubation time, and Xe is considered as the cell number at the end of the incubation time. The mean number of cells at each time point was plotted by GraphPad Prism . Cells were cryopreserved in 10% (V/V) dimethyl sulphoxide , 50% (V/V) fetal bovine serum and 40% DMEM, at a density of 1\u00d71022Total cellular RNA was isolated from hBMSCs using RNA extraction kit . Applying a nanodrop\u2122 spectrophotometer , the quantity and quality of RNA were assessed by determining the ratio of optical density at 260/280 nm. They were stored at 80\u02daC until cDNA synthesis. The cDNA was synthesized by 1,000 ng total RNA in a first strand cDNA synthesis reaction using RevertAid\u2122 First Strand cDNA Synthesis kit .Quantitative real time PCR (qPCR) was conducted by ABI Biosystems StepOne and the RealQ Plus 2x Master Mix Green . In each reaction, 200 nM of each primer  was addehBMSCs were all adhered to the culture flasks. These cells were spindle shape in different passages . Three wrd followed by an increase until day 7th. The growth curve was plotted based on the average number of cells treated with 200 ng/mL of acetonic extract of Feijoa during a seven days period. An increasing trend in cell proliferation till day 3 was noted, while proliferation demonstrated a further decrease when compared to the control group  and anti-apoptotic proteins , while anti-apoptotic proteins are responsible for apoptosis through delaying the mitochondrial release of cytochrome-c, and that the proapoptotic proteins activate such releases.E. billardieri on MCF-7, A549, HepG-2 and HT-29 cell lines.31-33These results are consistent with other studies reporting that Bax and Bcl-2 play an important role in apoptosis, growth kinetics and cell differentiation. Our results resemble the \ufb01ndings of cytotoxic potential effects of rd. Therefore, when faster proliferation during a shorter time period is targeted, Feijoa can be safely added to the culture media in the first three days. This study also revealed that acetonic leave extract of Feijoa significantly induced apoptosis on day 7th by increasing expression of Bax and decreasing of Bcl-2 genes. However, more molecular studies should be undertaken to achieve a definite conclusion to determine the probable apoptotic pathways of the cytotoxic activity of Feijoa on day 7th. The modulatory activity of Feijoa may be responsible for its increasing effect on cell proliferation till day 3"}
+{"text": "Individuals undergoing TBP showed a trend towards a lower Breslow\u2019s thickness and a higher proportion of in situ melanomas compared to those without TBP. The number needed to excise one melanoma varied from 3:1 to 14.3:1 and was better for lesions that arose de novo than for tracked ones. The included studies were judged to be of unclear methodological concern with specific deficiencies in the domains \u201cflow and timing\u201d and \u201creference standard\u201d. The use of TBP can improve the early detection of melanoma in high-risk populations. Future studies are warranted to reduce the heterogeneity of phenotypic risk factor definition and the technical implementation of TBP. Artificial intelligence-assisted analysis of images derived from 3-D TBP systems and digital dermoscopy may further improve the early detection of melanoma.Early detection of melanoma is critical to reduce the mortality and morbidity rates of this tumor. Total body photography (TBP) may aid in the early detection of melanoma. To summarize the current evidence on TBP for the early detection of melanoma, we performed a systematic literature search in Medline, Embase, and the Cochrane Central Register of Controlled Trials (CENTRAL) for eligible records up to 6th August 2020. Outcomes of interest included melanoma incidence, incisional and excisional biopsy rates, as well as the Breslow\u2019s index of detected tumors. Results from individual studies were described qualitatively. The risks of bias and applicability of the included studies was assessed using the QUADAS-2 checklist. In total, 14 studies published between 1997 and 2020 with an overall sample size of  The incidence of cutaneous melanoma continues to rise steadily in Western countries with Caucasian populations each year ,2. SurgiIn Germany, the worldwide unique national skin cancer screening program was implemented in 2008. It involves a voluntary, standardized full-body examination by dermatologists or general practitioners specifically trained for this purpose. The costs are reimbursed by all German statutory health insurance companies every two years for members older than 35 years, while some health insurance companies also cover the costs for younger members . InsteadHowever, until now, the value of TBP in the early detection of melanoma has not been systematically investigated. Here we performed a systematic review to summarize the current publication landscape of TBP in the prevention of melanoma. Our results will assist dermatologists as well as patients in choosing an appropriate and individual screening and surveillance strategy.This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) , its extWe included studies investigating patients of any age with common melanocytic nevi (moles), congenital nevi, or dysplastic/atypical nevi undergoing 2-D TBP or 3-D TBP to detect melanoma early as part of screening for surveillance. Combination with other diagnostic measures, such as physical examination, self-examination, or SDDI was allowed but not obligatory for inclusion. In addition, studies investigating only TBP for keratinocyte cancer were excluded from the analysis.Regarding the study design, we included randomized controlled trials (RCTs), clinically controlled trials (CCTs), non-controlled prospective trials, prospective observational studies, case-control studies as well as retrospective studies. Narrative reviews and case reports or series were excluded. No language restrictions were set. In RCTs and controlled observational studies, usual care served as control. If no control group was specifically included, data from external control groups or the general population served as control.The outcomes of interest were all related to the early detection of melanoma. Thus, the incidence of melanoma, the number of excisions and biopsies, the number of changed or new nevi, as well as the benign-to-malignant ratio of excised lesions were defined as outcomes of interest. Additionally, the mean or median vertical invasion depth measured in mm (Breslow\u2019s index) of detected melanomas was considered as an outcome. As we expected heterogeneously reported outcomes, we extracted the data as reported in the publication and did not perform any re-calculations.We searched the electronic databases Medline, Embase (both via Ovid), and the Cochrane library CENTRAL until 6 August 2020 to identify all possibly relevant records . For ongThree authors  independently screened titles and abstracts for eligibility that were identified in the electronic database searches. For records that were considered relevant according to title and abstract screening, full-text articles were obtained, and inclusion and exclusion criteria were applied by the same three authors. Whenever discrepancies arose, a resolution was achieved by discussion with another independent author .Information for each included study regarding study design, baseline characteristics of the included population, risk factors for melanoma, imaging technique, and intervals of TBP, follow-up strategy, main outcomes, and study limitations were collected and summarized independently by two authors . Data were extracted to an internally piloted data extraction spreadsheet using Microsoft Excel 2010. If multiple reports of a primary study were identified, all available data were extracted. According to the principles of evidence-based medicine, we also decided to contact study authors for clarification in case of inconsistencies in reporting or overlapping study populations were identified. However, this was not the case for any of the identified records. The baseline characteristics and the outcomes of interest of each study were described qualitatively within the text, as a quantitative synthesis was not feasible due to the high heterogeneity of included studies.The risks of bias and applicability of the included studies were assessed by two reviewers  independently using a modified version of the QUADAS-2 checklist . QUADAS-n = 12082 (range 100\u20134692) were included in qualitative analysis  were detected in 16 patients. The age-adjusted incidence was calculated to 1835/100,000 person-years. Notably, 11 melanomas were detected due to comparison with baseline photographs.Kelly et al. assessed 278 patients with dysplastic melanocytic nevi in a prospective cohort study . PatientMoloney et al. performed a prospective observational study in Australia to examine the effect of regular full body examination in combination with dermoscopy and TBP . SDDI waNathansohn et al. established a pigmented lesions clinic based on a digital photography studio with TBP and dermoscopy. In the first 20 months of the prospective study, 895 patients were examined, 206 of them had follow-up visits ; 236 susTaken together in all six prospective studies 7 to 98 melanomas were detected. Two of these studies indicated that up to 11 out of 20 melanomas were detected due to TBP. Three studies specified the number needed to excise, ranging from 3:1 to 11.9:1. Altogether the number of biopsies ranged from 210 to 1152 biopsies .In the retrospective study by Drugge et al., 4692 patients were analyzed regarding patient characteristics, self-reported melanoma risk factors, and the usage of TBP ; 2473 ofn = 21 in situ; n = 6 invasive); 74% of the melanomas (n = 27) were biopsied due to changes from baseline while 19% were biopsied because they newly emerged.Feit et al. reported the results of a retrospective study including 576 patients ; 93 suspThe retrospective registry review of two cohorts by Rademaker et al. investigated demographic and histological data from 100 melanomas diagnosed through self-referred TBP and SDDI service compared to those diagnosed through traditional methods . Fifty-tRisser et al. compared biopsy numbers in 946 patients with multiple atypical nevi in their first year of care who received either total body skin examination alone or in combination with TBP . The mean = 53 in situ), comprising 8.5% of excised lesions. Sixty melanomas were tracked lesions  while 38 melanomas newly emerged .Salerni et al. analyzed the use of TBP and dermoscopy in 618 high-risk melanoma patients. A total of 1152 lesions in 407 patients were excised during the surveillance period, corresponding to a mean of 2.83 lesions per patient during a 10-year follow-up period  ,34; 98 mIn the retrospective study performed by Strunck et al., 121 melanomas were diagnosed among 1,955 patients receiving TBP  . Of the Truong et al. reviewed the records of 926 patients who received TBP and had two or more follow-up visits over two years or longer . PatientAltogether in seven retrospective studies or chart reviews the number of invasive melanomas were reported in six studies and ranged from 0 to 70. The number needed to excise was specified in two studies and ranged from 3.1:1 to 14.3:1. The number of biopsies in patients with TBP ranged from 53 to 921 biopsies .The reported depth of detected melanoma varied across all studies . HoweverOverall, the included studies were judged to be of unclear methodological concern . At leasn = 12,082 highlighting that individuals undergoing TBP showed a trend towards a lower Breslow\u2019s thickness and a higher proportion of in situ melanomas compared to those without TBP. In addition, our results show that the use of TBP can improve the early detection of melanoma in high-risk populations.In this study, we aimed to investigate the value of TBP in the early detection of melanoma. Overall, we have identified 14 studies with an overall sample size of For the early detection and thus secondary prevention of melanoma, there are two distinct strategies to select the target population. Firstly, untargeted mass screening programs can be performed (population-based screening) regardless of individual risk profiles. However, this procedure is only carried out in a few countries such as Germany and is costly due to the high number of screened individuals . In AustThe technical implementation and timing intervals of TBP were similarly heterogeneous as the definition of clinical risk factors. In most studies, 12\u201330 macroscopic images were acquired with conventional cameras to capture the entire skin surface in two dimensions (2-D TBP). As a limitation, multiple imaging systems were applied with distinct resolutions. Furthermore, the study protocols only referred to standard poses and often did not specify exactly how the images were acquired. Specific anatomic regions such as intertriginous areas, genitals, retroauricular area, or the flexor sides of the extremities may not always be adequately captured with 2-D TBP. Here, the use of 3-D TBP systems in defined postures can help to standardize the image acquisition as they generate a digital 3-D avatar of the imaged person that can be used to analyze and prospectively follow all nevi of interest ,37,42. CThe results of our study clearly show that 2-D TBP identified a higher proportion of in situ tumors and melanomas with a lower average Breslow thickness compared with the comparison groups without 2-D TBP. The number needed to excise one melanoma varied widely, ranging from 3:1 to 14.3:1. This ratio was better for lesions that arose de novo than for tracked ones. We conclude that the latter need to be assessed with additional methods such as dermoscopy and that the added value of TBP is highest for lesions that arose newly during the surveillance period.Artificial intelligence (AI) was not used in any of the included studies to evaluate the captured images. The image analysis was performed by the respective physicians in all reports. Several landmark studies have recently shown that AI performed on par with dermatologists in the distinction of nevi from melanoma based on dermoscopic images ,44,45. SOverall, our study shows that the use of 2-D TBP can improve the early detection of melanoma in high-risk populations. However, the definition of phenotypic risk factors, technical performance of TBP, and design of the included studies were highly heterogeneous. In the future, harmonized risk prediction tools should be developed to compare distinct populations. 3-D TBP with the generation of digital avatars is a game-changing technology and will enable standardized and gapless image acquisition."}
+{"text": "Perhaps the most characteristic property of both the adaptive and innate arms of our immune system is the ability to patrol basically the entire organism in search for potentially dangerous intruders, and respond once they are found. This response requires the activation of a complex machinery of intracellular pathways, the release of chemicals and proteins that act as chemoattractants to activate the migration of other cells of the immune system and finally, the coordination of a multicellular response that will lead to the destruction of the intruder. Immune cell mobility, immune cell activation and destruction of the intruder requires a precise spatiotemporal dynamics of the cells\u2019 cytoskeleton. For this, the cell\u2019s cytoskeleton must rearrange in a highly regulated fashion. Thus it is not surprising that these cells have developed an intricate network of cytoskeletal regulatory proteins which sometimes are exclusively expressed by cells of the immune system.ex vivo, suggesting that the function of the cytoskeleton is much more than just supporting migration to reach the intruder antigen, since a number of crucial immunoreceptor signaling functions are dependent on cytoskeletal organization and dynamics. However, the precise mechanistic roles that cytoskeleton plays in immune cell signaling, especially given that it is the same unit network that would perform all of the functions, remains a mystery.The cytoskeleton plays a versatile role in immune cells. A testament to this multifunctionality of cytoskeleton is that immune cells harboring mutations in cytoskeletal regulatory proteins often have compromised overall immune response. These defects exist even when the cells are challenged with potent activation signals In this special issue on the cytoskeleton in immune responses we have attempted to explore the reciprocal relationship between cytoskeleton, cellular signaling, and activation. In this issue there are original, opinion, and review articles that deepen our knowledge on the molecular regulation of the cytoskeleton as well as of cellular responses regulated by the cytoskeleton, with a goal to gain insights into the cytoskeleton\u2014intracellular signaling machinery interconnection. These articles address this topic at diverse locations  and scales .Dupr\u00e9 et al. describes how primary immunodeficiencies lead to distinct defects in immune cell function pointing to functional specializations of the cytoskeleton. Role of the actin microfilament NPF Wasp in antigen receptor signaling has been further elaborated in a review by Ngoenkam et al.. An article by Li et al. describes the role of WASP in regulating B cell actin architecture by forming actin-rich \u201cpods\u201d and cell spreading cells. Defects in WDR1, a regulator of cofilin activity, results in immunodeficiency. A report by Bolger-Munro et al. highlights the role of WDR-1 in actin remodeling and B cell responses.Immunopathies resulting from defects in cytoskeletal regulating proteins such as ArpC, HEM, WASP, WIP, and WDR1 serve as natural perturbations to study the regulation of the cytoskeleton. The review article by Ben-Shmuel et al. discusses NK cell cytoskeleton regulation and function, and dissects the role of individual cytoskeletal elements actin, microtubule, and NM-II in it. T cells require adhesion molecules such as integrin for activation, although their precise roles in signaling are not well understood. In a paper by Cassioli et al., the role of the integrin LFA1 at the T cell immunological synapse is explored using expression rescue experiments. The T cell line Jurkat E6.1 cells, which lack LFA1, showed restoration of architecture, signaling and activation when LFA1 was expressed at an adequate level.This special issue covers cytoskeleton regulation and signaling in different lymphocyte types. The review by Escolano et al., the authors find that altering the mechanical context of macrophage activation has profound effects on inflammasome signaling. Being a \u201cnovel\u201d field the readouts  are still limited. A report from Wohl and Sherman uses spectral analysis to quantify ATP-dependent mechanical vibrations of plasma membranes during T cell activation. Further quantitative tools to analyze mechanics in immune cells are discussed by Fritzsche in an opinion article and by Garlick et al. focusing on cutting edge super resolution techniques and on interdisciplinary approaches to extract maximum spatiotemporal signaling information from cells. Along the same theme of using the experimental system to better extract information from cells, a paper by Lee et al. describes a method for the analysis of cell migration as a marker for T cell proliferation potential. In their review article, Record et al. discuss molecular mediators of mechanical force transduction in immune cells and emphasize a need for understanding actin dynamics in not just the cytoplasm of the cell as has been conventionally done, but also in the nucleus of the cell.Mechanical regulation of immune cell activation has gained popularity, since recent studies support the idea that the mechanical context of molecular interactions can influence the signaling output. In a report by Mart\u00edn-C\u00f3freces et al. discusses the role of the CCT chaperonin in the remodeling and positioning of the centrosome, and consequently in the dynamics of the immunological synapse. Similarly, a paper by Iba\u00f1ez-Vega et al. analyses the role of the proteasome adaptor protein ECM 29 in proteosome localization and actin remodeling at the B cell immunological synapse. Another intriguing molecular player characterized here is Glial Matural Factor gamma (GMFgamma). A study by Deretic et al. uncovers a role of GMFgamma in cell spreading and actin remodeling and regtrograde flow at immunological synapse. Finally, a review by Kim et al. consolidates the knowledge about the role of transgelin in filamentous actin stabilization, and, consequently, in the stabilization of cellular microarchitectures such as microvilli.Regulation of the cytoskeleton in immune cells also involves uncharacterized and sometimes unconventional players. The precise mechanistic roles of these effectors are not completely clear. In the issue we publish articles that shed light on a few of these crucial players as regulators of subcellular localization and dynamics of filamentous actin. A review by Together, the issue covers a broad spectrum of articles on the cytoskeletal regulation in immune cells. In the future, we look forward to learn about additional players, novel mechanism on microscale architectures, emergent shape and its connection to cell functions."}
+{"text": "This study proposed an error-matching measurement and compensation method for curve mating and complex mating. With use of polynomial curve fitting and least squares methods for error analysis, an algorithm for error identification and error compensation were proposed. Furthermore, based on the proposed method, an online error-matching compensation system with an autorevising function module for autogenerating an error-compensated NC program for machining was built. Experimental verification results showed that the proposed method can effectively improve the accuracy of assembly matching. In a curve-type mating experiment, the matching error without compensation was 0.116 mm, and it decreased to 0.048 mm after compensation. The assembly accuracy was improved by 28%. In a complex-type mating experiment, the verification results showed that the error reductions after compensation for three mating shapes  were 81%, 87%, and 79%, respectively. It showed that the proposed method can improve the assembly accuracy for complex mating shapes, which would also be improved without losing production efficiency. In response to the needs of manufacturing applications, the international industry, academia, and research institution sustain the research on improving the machining accuracy of the machine through hardware improvement or software-based error compensation. For production lines with high-precision requirements, due to the tight tolerance zone design of each process, high-specification and high-precision machines are usually required for each process, resulting in higher hardware costs for the production line. Therefore, if the real-time error measurement and compensation in the manufacturing process enable the front and back processes to perform matching error compensation processing, then the matching between the processes can be greatly improved and the re-work rate and defect rate can be reduced, thereby reducing the manufacturing cost of the production line.In addition, in the production of precision industrial products, it is often necessary to individually manufacture components and then perform the precision assembly. The assembly matching accuracy depends on the requirements of individual processing accuracy and the selection of two components (part and counterpart) with the smallest matching error. At present, most of the production methods in the industry are mass production, and then suitable part and counterpart are manually matched and selected for assembly. However, due to the different manufacturing processes of the parts and the counterparts, and the different manufacturing tolerance ranges, the cost of maintaining manufacturing accuracy is high; additionally, the manual selection is time-consuming and inefficient. The online curve shape measurement and matching error compensation system proposed in this research is to parallel and efficiently measure the error of the part on the production line, and then convert it into the correction error of the counterpart to be machined, afterward automatically generate the numerical control (NC) processing program with the correction. With high-efficiency information communication, the suitable counterpart is machined, so that the matching accuracy between the part and its counterpart is improved in the low-cost and high-efficiency manufacturing process. Another advantage of this method is that there is no need to require strict tolerances in every manufacturing process, which makes the production line more robust and results in lower production costs.The matching shapes between the part and its counterpart can be divided into straight, triangle, and curve types. Our previous study proposed matching error compensation methods for straight and triangle matching ,2. This In the past, many studies have investigated error compensation methods to improve the machining accuracy of the machine. Wang et al.  proposedMonitoring and control of the manufacturing process are important for the development of manufacturing industries. Process monitoring is the manipulation of sensor measurement  in determining the state of processes. Different real-time monitoring techniques to monitor the manufacturing process have been investigated, such as Dinardo et al.  used vibFor high-precision products, measurement accuracy plays a key role. The Coordinate Measuring Machine (CMM) is widely used in industry to evaluate dimensional and geometric characteristics of complex high precision parts. However, due to the demand for shorter cycle times of measurement tasks, in such conditions, dynamic errors will certainly have a much more influence on the measurement accuracy. Echerfaoui et al.  investigThe previous research on error compensation mostly focused on the improvement of the processing accuracy of a single part and less focused on the assembly parts. This research focuses on the measurement and error compensation for high-precision assembly matching. The error of a part is measured in real time on the production line, then the error compensation value for its counterpart is calculated according to the error of the part. Furthermore, the error compensation value is compensated into the counterpart NC program. Finally, the counterpart is machined using a compensated NC program. Thereby, if an error exists on a part, the error is autocompensated to its counterpart to be machined, so that the high-precision matching assembly can be obtained. In the past, our research team has proposed compensation methods for straight and triangle matching errors . This paIn the matching error measurement and compensation method, the user needs to define the key nodes in the mating area of a part, then the coordinate of the key nodes is directly measured on the production line to create the actual size and geometric profile. The tolerance chain in the design was included in the calculation of the theoretical minimum and maximum dimensions of the part. Subsequently, the size and geometric profile obtained from the measured part are compared with the theoretical size and geometric profile to determine the size and profile error of the part, and then convert into the compensation value of the counterpart to be machined. Finally, a corrected counterpart NC program is autogenerated according to the compensation value, then machine the counterpart that matching to the part. The methodology includes: (1) the selection of the key nodes, (2) measurement methods, (3) curved contour reconstruction, (4) error comparison, and (5) error conversion and NC program autocorrection.Before calculating the error and compensation, the product needs to be measured by the measurement instrument. First, it is necessary to define the key measurement points in the part mating area, so that the complete measurement information of the actual size and contour profile can be obtained and reconstructed. There are three types of matching assembly shapes between the part and counterpart, namely straight line, triangle, and curved. The straight line matching focuses on straight and length. To construct a straight line, two points are needed. The triangle matching focuses on the triangle side length and the angle. To construct a triangle, it is necessary to create two lines and the angle between the line must be known. Therefore, four points are needed to create two lines and using dot product equation to obtain the length of two lines and the angle between the lines. For the curve shape, the required points to construct a hill or valley are at least three points. However, for large hills or valleys, it is recommended to add one point for every increase of 5 mm. To construct the curve shape, the polynomial regression and least squares methods were used. The number of points will influence the accuracy of the curve construction. More number of points will provide higher accuracy of curve reconstruction, but it will take a longer measurement time. Alternatively, a fewer number of points will result in lower accuracy of curve reconstruction, but shorter measurement time. The mating error of the part can be obtained by comparing the actual shape after machining and the theoretical shape from the CAD. Furthermore, this mating error value is used to modify the shape of its counterpart to obtain precision matching in assembly.The point-to-point measurement method in the Renishaw Equator 300 is used to measure the part. This method uses several points to reconstruct the shape of the part. First, the origin position of the machining coordinate system must be checked, then the coordinates of the key points must be measured according to this coordinate system; furthermore, the actual curve must be reconstructed based on the measured coordinates. Subsequently, the actual curved and theoretical curved (from CAD drawing) is compared, and then the deviation is calculated between the theoretical point and the actual point. For the curve shape, the point-to-point measurement method is used instead of the scanning measurement method. The advantage of using the point-to-point method is that it only needs several points to construct the curve, and the measurement time is short compared with the scanning method that uses numerous points and a longer measurement time. However, the number of points will affect the accuracy of the curve reconstruction. More points will produce higher accuracy of curve shape but longer measurement time is required as a consequence; on the other hand, fewer points will result in lower accuracy, but the measurement time is shorter. For example, ten points were taken while measuring the curve, as shown in As is known, the measurement has some degree of errors that may come from the instrument or loading/unloading. The Renishaw Equator 300 that used in this study is an automated flexible gauge that employs the comparator principle via RenCompare software to account for the influence of systematic effects associated with Coordinate Measurement System (CMS) ,25. The The uncertainty of Equator comparator measurement for touch-trigger probing (TTP) mode is less than 0.6 \u03bcm. The number of probing points has no significant influence on the comparator measurement uncertainty, and comparison uncertainty is less than 0.5 \u03bcm for the angular misalignment within a range of \u00b11 mm. A large number of contact points provides smaller measurement uncertainty. The length measurement uncertainty of the Equator comparator remains below \u00b12 \u03bcm when the fixture/component is relocated within an error range of \u00b11 mm (fixture requirement is according to the Equator system specification) [For the part-alignment in the measurement, the coordinate translation and rotation conversion as shown in 1, Y1), \u2026  and curve-fitted line. For a given coordinate, such as , a deviation Di exists between the corresponding value on the Y-axis and the Y value on the curve C. This deviation value can be positive, negative, or zero. The maximum order of the polynomial is dictated by the number of data points used to generate it, and can be calculated as m = N \u2212 1. The polynomial can be created passing through all the points when the polynomial of the degree is N \u2212 1. On the other hand, if the polynomial of the degree is less than N \u2212 1, the polynomial does not pass through any of the points, but overall approximates the data. The general form of polynomials function can be written as Equation (3).n is the order of the polynomials.The straight line fitting and geometric angle fitting are common problems in engineering applications. The least squares (LS) or total least squares (TLS) method can be used to fit the two-dimensional straight line. However, the LS or TLS method cannot be directly used to fit the three-dimensional spatial straight line due to the large number of equations to be solved; the solving process is relatively cumbersome, with poor practicability. Therefore, the three-dimensional problem is converted into a two-dimensional problem, then LS or TLS methods are used to fit the straight line. Polynomial curve fitting is a common method of data fitting. Polynomials can be used to fit data points in two ways. First, the polynomial passes through all the data points, and second, the polynomial does not necessarily pass through any of the points, but gives a good approximation of the data overall. The higher degree polynomials may give a larger error or are more likely to overfit. To obtain the best fit curve line to data points, the least squares method was used to minimize the sum of the squares of the residuals at all the data points. In addition, to avoid overfitting problems in curve construction using the polynomials method, regularization was used in the least squares to control the overfitting. The smaller the value, the better the fitting degree. The minimum sum of squares of error can be calculated using Equation (4).The polynomial curve fitting with the minimum sum of squares of error can be calculated using Equation (5).The theoretical coordinate of the key points of the workpiece can be obtained from the CAD drawing, whereas the actual coordinates of the workpiece can be obtained by measuring the machined workpiece. For instance, The actual measurement information is provided in CSV format. Therefore, a CSV data-parsing module was established to analyze the content of the source file and determine whether the data content belongs to the part or counterpart.X and Y; To easily understand the error conversion, an illustration assembly between part and counterpart is depicted in To achieve online automatic error compensation, the above-mentioned error conversion results need to be inserted into the NC machining program to correct the cutting tool path. Therefore, it is necessary to declare the processing block for error compensation in the system in advance. In the setting section on the human\u2013machine interface, the key measurement point and the corresponding NC program block is designed. The system will automatically search and identify the set block in the original NC program according to the key measurement setting, then integrate the error compensation amount into the X-, Y-, and Z-coordinate value of the block.The NC cutting process generally consists of G00, G01, G02, and G03. In this research, Regular Expressions in the C# programming language is used to identify a single block. Regular Expressions is a way to search a pattern with a certain rule string; therefore, this method is suitable for searching NC code in a single block. To automatically generate an error-compensated NC program for a counterpart, the first step is to find the block number to be compensated. After the deviation value is obtained by subtracting the actual coordinate from the theoretical coordinate of the part, then the coordinate of the counterpart is corrected according to the deviation value of the part. The detail of the auto-generated error compensation NC program can be seen in the previous work [The total time for the compensation can be calculated using Equation (7):Based on the proposed error compensation method, an online matching error compensation system for complex shapes was developed in visual C# language. The architecture of the system includes a login system, pre-setting system, and an intelligent error compensation system.Before performing error compensation, the user needs to define the required key point for measurement, and the corresponding NC processing program blocks information for automatic compensation. Experiments were designed to verify the effectiveness of the proposed method for the curve-mating and complex-mating types. Aluminum workpieces were used in the experiments. A Renishaw Equator 300 with a working range of 300 \u00d7 300 \u00d7 150 mm was used to in-line measure the part. A CNC vertical milling machining center YTM-763 with a travel range of 760 \u00d7 400 \u00d7 350 mm, maximum spindle speed of 20,000 rpm, and Delta NC300A controller was used to machine the counterpart. A computer with the specification of intel core i7-7700 and RAM 16 GB was used. The cutting parameters for experiment verification are shown in The verification for complex mating consists of straight line, triangle, and curve shapes, as shown in The actual distance between point 1 and point 2 of the straight line shape of the complex part was 17.649 mm, and the angle between L1 and L2 was 118.948\u00b0. Before compensation, the actual distance between point 1 and point 2 of the complex counterpart was 17.308 mm, thus the deviation was 0.341 mm. Meanwhile, the angle between L1 and L2 was 119.542\u00b0, thus the deviation was 0.594\u00b0. After compensation, the actual distance between point 1 and point 2 of the complex counterpart was 17.592 mm, which indicated that the error decreased from 0.341 mm to become 0.057 mm. Furthermore, the actual angle between L1 and L2 was 119.061\u00b0, which demonstrated an error reduction of 81%.In the triangle shape of the complex part, the actual angle between L2 and L3 was 63.948\u00b0. Before the compensation, the angle between L2 and L3 of the complex counterpart was 64.878\u00b0, which indicated an error of 0.93\u00b0. After the compensation, the angle between L2 and L3 of the complex counterpart was 63.83\u00b0, which showed that the error was reduced to 0.118\u00b0, and, therefore, an improvement of 87% as a consequence.The total time for this complex shape compensation can be calculated by using Equation (7). The given parameters data: key measurement points were 14, the velocity of touchpoint was 10 mm/s, the distance between the probe and key measurement point was 5 mm, and the calculation time of deviation was 1 s. After substituting these values into Equation (7), it showed that the total time for this complex shape compensation was 8 s.In this study, an error-matching compensation method for curve mating and complex mating was developed. Error analysis and algorithms for error identification and error compensation of curve mating and complex mating were proposed. Polynomial curve fitting method and least squares method were used for error analysis. Based on the proposed method, an online error matching compensation system was built. Experiment verification results showed that the proposed method can improve the precision of assembly matching. For curve mating, the matching error before compensation was 0.116 mm, but the error decrease becomes 0.048 mm after compensation so that the assembly accuracy increase to 28% as consequence. For complex mating, the experiment verification results showed error reduction after compensation for each shape, which are 81%, 87%, and 79% for straight line, triangle, and curve shape, respectively. Accordingly, the precision assembly of complex mating will also be improved."}
+{"text": "Intestinal obstruction is defined as a blockage or partial blockage of the passage of the intestinal contents. It is a potentially risky surgical emergency associated with high morbidity and mortality. Its pattern differs from country to country and even from place to place within a country. Therefore, this study aimed to find out the magnitude, pattern and management outcome of intestinal obstruction in Arba Minch General Hospital.A retrospective cross-sectional study was conducted in Arba Minch General hospital from January 09, 2015, to November 09, 2018. The data collection period was from December 15, 2018, to February 09, 2019. A simple random technique was applied to select 801 study participants. Then, the required data entered into Epi Info version 7.2.1.0 and exported to the statistical package for the social sciences software package version 20 for analysis. The binary logistic regression analysis has been done to determine crude statistical associations between independent variables and dependent variables. Linearity, Multivariate normality and multicollinearity were checked between independent and dependent variables by using scatter plot and Q\u2013Q plot respectively. Variables with a p-value of less than 0.25 in the binary logistic regression analysis were entered into multivariable logistic regression. Statistical significance factors were identified based on a p-value of <\u20090.05 and with a 95% confidence interval.This study revealed that the overall magnitude of intestinal obstruction was 40.60% with 95% CI (34.95\u201345.95). The magnitude of unfavorable management outcomes and deaths during the study period were 22.3% with 95% CI (18.00\u201327.00) and 7.1% with 95% CI (4.00\u201310.00) respectively. Persistent tachycardia 10.3 (3.28\u201332.42), Dehydration 13.7 (3.34\u201356.56), elevated serum creatinine 10.2 (1.89\u201354.94), gangrenous small bowel volvulus 2.7 (1.27\u20135.84), ischemic bowel 3.4 (1.17\u20139.81) and perforated bowl 7.68 (2.96\u201319.93) were significantly associated with the management outcome of intestinal obstruction.Intestinal obstruction was the most common among all acute abdomen cases and its management outcome highly associated with dehydration. Adequate early preoperative resuscitation and proper post-operative care with appropriate surgical techniques and wound care with sterile techniques would help to reduce further mortality. This could be achieved by increasing public awareness of health-seeking behavior. Moreover, health facilities capable of handling patients with small bowel obstruction should be available within the reach of the community. Intestinal obstruction (IO) is defined as a blockage or partial blockage of the passage of the intestinal contents. It is a potentially risky surgical emergency associated with high morbidity and mortality . It is aIn a global based report of the world health organization, about 3.2\u00a0million cases of bowel obstruction occurred in 2015 which resulted in 264,000 deaths . In mostStudies conducted in Ethiopia showed that the death rate after the management of intestinal obstruction cases were 13.6%, 9.2%, and 2.5% in South, East and Central Ethiopia respectively \u201314. In sAetiologies of bowel obstruction include sigmoid volvulus, small bowel volvulus, adhesions, hernias, inflammatory bowel disease, appendicitis, tumors, diverticulitis, ischemic bowel, tuberculosis and intussusception . The cauAnalysis of cases based on the specific causes of the acute abdomen has great value for early diagnosis and prompt treatment in clinical practice . DespiteA hospital-based retrospective cross-sectional study was conducted in Arba Minch General Hospital from January 09, 2015, to November 09, 2018. All patients with the diagnosis of non-traumatic acute abdomen cases who were admitted to the surgical ward of Arba Minch General hospital were included and patients who went home before completion of treatment, with lost cards and cards with incomplete data were excluded.The sample size was determined by the single population proportion formula. The assumptions considered to calculate the sample size were from the previous study in Ethiopia using a prevalence of 21.8% . ConsideA total of 5590 total surgical admissions were found during the study period. Among these, 1303 of them were admitted with conditions attributed to non-traumatic acute abdomen. During the procedure, the medical record numbers were sorted from smallest to largest code (1\u20131303). The required sample size was obtained by using a simple random sampling technique. So, 801 cards were selected randomly from 1303 cards. But, among the sample size, only 761 cards were found with complete data. Cards/Medical records with incomplete data were excluded from the study.Management outcome of IO (favorable or unfavorable).Socio-demographic characteristics , patient history , Physical examination findings , intraoperative findings , intraoperative procedure .The data were collected by using a structured checklist. The checklist was adapted from previous studies to enable us to collect important information. A checklist was developed in the English language to collect important information such as age, sex, ethnicity, admission clinical diagnosis, intraoperative findings, intra-operative procedures, duration of the presentation, causes of IO, postoperative complications and management outcome. For data collection, two clinical nurses were recruited.Acute abdomen: a patient\u2019s case which is labeled in their discharge diagnosis as one of the patterns of acute abdomen. Intestinal obstruction: a patient with acute abdomen who has abdominal pain, abdominal distension, vomiting, not passing gas and feces (obstipation) completely, not passing feces (constipation), with imaging diagnosis having obstructed dilated bowel loops and labeled in their discharge diagnosis note as intestinal obstruction. Management outcome: the condition of the patient after treatment has been carried out, after conservative or operative procedure that means whether discharged alive or died in the hospital or end up with complications. Favorable management outcome: discharged live and with no complication after management. Unfavorable management outcome: defined as a patient with intestinal obstruction developing one or more postoperative complications  and/or death.Data were cleaned, coded and entered into Epi-info version 7.2.1.0 and was exported to SPSS version 20 for analysis. Descriptive statistics were conducted and results have been presented using frequency tables, graphs and percentages. The binary logistic regression analysis has been done to determine crude statistical associations between independent variables and dependent variables. Linearity between independent and dependent variables was checked by scatter plot. Multivariate normality between variables was checked by a Q\u2013Q plot. And, multicollinearity was checked. Variables with a p-value of less than 0.25 in the binary logistic regression analysis were considered as a candidate to be entered into multivariable logistic regression. Multivariable analyses have isolated independent predictors of IO management outcome. Statistical significance factors were identified based on a p-value of <\u20090.05 with a 95% confidence interval.From the total study participants of 801, only 761 cards have complete data with a response rate of 95.12% where 507 (66.6%) cases were male, 478 (62.80%) were from a rural area (out of Arba Minch city) and 680 (85.42%) were less than 50\u00a0years old age. Among these 309 (40.60%) cases were found to have an intestinal obstruction which is the most common condition followed by Appendicitis 207 (27.20%), Hernia 200 (26.28%), ill determined acute abdomen conditions 9 (1.18%), urologic emergencies 8 (1.05%), peritonitis 7 (0.91%), intussusceptions, 6 (0.79%), cholecystitis 6 (0.78%), acute pancreatitis 5 (0.65%) and PPUD 4 (0.52%) respectively. Most of the cases from each were males  of them were males and 61 (19.74%) of them were females. The male:female ratio was 4:1. Among these, 243 (78.6%) of them were from a rural area (out of Arba Minch city), 64 (20.7%) of them were from urban (Arba Minch city) and 2 (0.6%) were with no information of residence. The age of patients ranged from 2\u00a0months to 100\u00a0years with a mean and standard deviation of (40.68\u2009\u00b1\u200917.88). Among these study participants, 195 (63.1%) were Protestants and 249 (80.9%) were Gamo in ethnicity  among non-traumatic acute abdominal cases. Of the total 309 intestinal obstructions, 302 (97.7%) of them were mechanical intestinal obstructions and the remaining 7 (2.3%) were a dynamic ileus/functional intestinal obstruction. According to the site of obstruction, 198 (64.1%) were small bowel obstructions, 108 (35.0%) were large bowel obstructions and the rest 3 (1.0%) of them were undetermined.The common clinical presentations were abdominal pain (95.8%), abdominal distension (88.0%), obstipation (78.3%), constipation (69.6%), vomiting (62.1%), rectal bleeding (2.6%) and history of abdominal surgery (7.1%). Dehydration, Persistent tachycardia, fever and shock were also seen in 5.8%, 10.7%, 12.6% and 2.6% of patients respectively. Hypertension, known cardiac disease, diabetic, CLD and CKD found in 4.9%, 1.3%, 1.9%, 1.3% and 1.3% of patients respectively. By laboratory diagnoses: hypokalaemia, leucocytosis, elevated serum urea, hyperkalaemia and elevated serum creatinine were found in 1.0%, 21.0%, 2.6%, 0. 3% and 3.9% of patients respectively.The time of arrival since the onset of disease was greater than 24\u00a0h for 253 (81.9%) of patients and less than or equal to 24\u00a0h for 56 (18.1%) of patients. The total hospital stays recorded was >\u20097\u00a0days for 170 (55.0%), <\u20097\u00a0days for 134 (43.41%) and equal to 7\u00a0days for 5 (1.6%) of patients. 63 (20.4%) of patients were managed only conservatively and 246 (79.6%) were managed surgically.From the operative procedure undertaken resection and anastomosis 118 38.2%) were the most common procedure done  were the most common complications. Surgical site infections 25 (8.1%), acute respiratory diseases 12 (3.9%), enter cutaneous fistula 9 (2.9%), anastomotic leakage 7 (2.3%) and wound dehiscence 4 (1.3%) were the remaining common complication types found respectively . However, only dehydration, persistent tachycardia, elevated serum creatinine, gangrenous small bowel volvulus, ischemic bowl and perforated bowl were significantly associated with the management outcome of intestinal obstruction in multivariable logistic regression Table .Table 4FThe magnitude of intestinal obstruction in this study is 40.45% at 95%, CI (34.95\u201345.95) which is consistent with a result of a study at Gonder University Hospital (43.4%) . But it The majority of intestinal obstruction cases were small bowel obstruction (64.1%) which is similar to the studies conducted in eastern and central Ethiopia , 22, 24.The commonest causes of intestinal obstruction found from intraoperative findings were small bowel volvulus 45.30%, sigmoid volvulus 21.35%, adhesion and bands 12.0% and intussusceptions 4.9%. This result is similar to studies conducted in Gelemso and Chiro General Hospital . But, diThe magnitude of post-management complication in this study is 22.0% with 95% CI (18.0\u201327.0). This result is consistent with the studies conducted in Nigeria (20.77%), Kenya (23.6%) and Adama 24.6%) in Adama , 25, 26..6% in AdSepsis and septic shock was the most common pattern of complication after management in the current study. This is different from the study in Gelemso General Hospital which found surgical site infection as the most common complication and Jogla General Hospital which found pneumonia as the most common complication type and septic shock next to it , 22. ThiIn this study, patients without dehydration were 13.73 times more likely to have favorable management outcome, [AOR with 95% CI (13.73 (3.34\u201356.56))], than those with dehydration which is similar to the study conducted in Nigeria.Persistent tachycardia is significantly associated with management outcome of intestinal obstruction, [AOR with 95% CI (10.31 (3.28\u201332.42))], in which patients without persistent tachycardia are 10.31 times more likely to have favorable management outcome than those with persistent tachycardia which is supported by a study conducted in Nigeria .Elevated serum creatinine is significantly associated with management outcome of intestinal obstruction, [AOR with 95% CI (10.19 (1.89\u201354.94))], in which patients without elevated serum creatinine are 10.19 times more likely to have favorable management outcome when compared with those patients who have elevated serum creatinine. This may be due to the fact that in patients with renal function impairment creatinine levels may increase .Perforated bowel, ischemic bowl and gangrenous small bowel volvulus are also significantly associated with management outcome of intestinal,  respectively, in which patients without perforated bowl, ischemic bowl and gangrenous small bowel volvulus are 7.68, 3.39 and 2.72 times more likely to have favorable management outcome than their counterparts respectively. This result is similar to study findings conducted in East Ethiopia , 24.Since this study was from secondary data, Limitations were incomplete documentation, missing charts and difficulty interpreting information found in patients' cards.In conclusion, intestinal obstruction was the most common pattern of non-traumatic acute abdomen conditions during the study period. Males were affected more than females. Intestinal obstruction was more common in rural residents. Small intestinal obstruction was more dominant than large bowel obstruction. Small bowel volvulus, sigmoid volvulus and adhesion and bands were the commonest patterns of intestinal obstruction respectively. Resection and anastomosis, derotation and adhesion removal were commonest procedures done respectively. Dehydration, persistent tachycardia, elevated serum creatinine, gangrenous SBV, ischemic bowl and perforated bowl were significantly associated with the management outcome of intestinal obstruction. Therefore, designing a strategy addressing these factors would be helpful to decrease the likelihood of unfavorable surgical management outcomes for the patients attending hospital with IO.Based on our findings we suggest that health professionals in the hospital should increase public awareness on IO by providing appropriate health information.Physicians should diagnose and intervene on time before the intestine develops a complication.Health professionals should give attention to appropriate surgical techniques and wound care with sterile techniques to decrease surgical site infection which is the most common complication.Cardroom staff should improve record-keeping in the hospital because some medical records were incomplete.Further research using a prospective study design is recommended as a way to overcome the limitations of secondary data in the current retrospective research that preclude generalization to the whole population."}
+{"text": "Small bowel obstruction is a common and dangerous surgical emergency which is associated with high morbidity and mortality if not managed appropriately and timely.  To determine the causes and management outcome of small bowel obstruction in Nekemte Referral Hospital, Nekemte, Ethiopia. p \u2264 0.25 were selected as a candidate for multivariate logistic regression analysis. The level of statistical significance was set at p \u2264 0.05.  Institution-based retrospective cross-sectional study design was used. Three-year data  were collected from July 1 to August 30, 2017. Data were collected from medical records and checked for any inconsistency, coded, and entered into SPSS version 20 for analysis. Descriptive, binary, and multivariate logistic regression analyses were used. On binary logistic regression analysis, variables with  With 100% response rate, records of 211 patients with small intestinal obstruction were retrieved for analysis. One hundred thirty-seven (64.9%) were males. The commonest cause of small bowel obstruction was adhesion (35.1%). More than a quarter (26.5%) participants developed postoperative complications, and wound infection was the commonest postoperative complication (49.2%). A majority (84.8%) of patients improved and were discharged, and the rest 15.2% of patients died. Sex , duration of illness before surgical intervention , level of hematocrit , types of intestinal obstruction , and length of hospital stay  were independent predictors of the management outcome of patients with small bowl obstruction.  Small bowel obstruction is a commonly encountered surgical emergency. Adhesion, small bowel volvulus, and intussusception were the leading causes of small bowel obstruction, respectively. Small bowel obstruction (SBO) is defined as blockage of the passage of small intestinal contents from the proximal to distal segment. It is one of the most common conditions resulting in hospital admissions .Small bowel obstruction is a common and dangerous surgical emergency which is associated with high morbidity and mortality if not managed appropriately and timely. The prevalence and causes of SBO differ internationally and locally \u20135, but iThe most common risk factor for adhesive obstruction is violation of the peritoneal cavity following laparotomy. It is possible that talc or starch of the surgical gloves in routine use in our environment played a role in adhesion formation in some of the patients .Primary small bowel volvulus is one of the other common causes of SBO in parts of Africa. It was more common during the rainy seasons, that is, through June to October , 6.The pattern of the disease changes from time to time and needs periodic studies to evaluate the causes and prevalence of the disease. The causes of SBO are several, and their relative incidence varies across different populations and countries. It has also shown variation over the decades . The knoThe management outcome of the diseases may be a good indicator of how well a country's surgical services are doing. Several factors contribute to the poor outcome of patients with SBO. Some of these factors may include poor health-seeking behavior, ignorance, poverty, and poor clinical judgment , 3. ThusA hospital-based retrospective cross-sectional study was conducted at NRH, Nekemte town. Nekemte town is located in Oromia regional state, western Ethiopia, which is 333 kilometers from Addis Ababa. The hospital was established in 1923 EC, providing service for approximately 3 million people in the catchment area including surgery. The study was conducted from July 1 to August 30/2017.All patients admitted to NRH and managed for bowel obstruction were source populations. All patients admitted with a diagnosis of small intestinal obstruction at NRH from January 1, 2014, to December 30, 2016, were study populations.Patients who were clinically diagnosed with SBO and managed conservatively without operationPatients who were clinically diagnosed with SBO and managed operativelyPatients who died after presenting with the clinical diagnosis of small intestinal obstruction without surgeryPatients with mechanical and nonmechanical (adynamic) intestinal obstructionIncompletely documented chartsPatients whose charts were lostAll 211 patients admitted to the surgical ward of NRH with the diagnosis of SBO and treated from January 1, 2014, to December 30, 2016, were included.Favorable management outcome: if patients did not develop either postoperative complication or death after conservative or operative management.Unfavorable management outcome: if the patient developed one or more postoperative complications and/or death.The checklist was first developed by English language and translated to Amharic language and then retranslated back to English to maintain its consistency. Patients that were admitted to the surgical ward of NRH with the diagnosis of SBO and treated were initially identified from admission logbooks of surgical wards and operation theater of NRH from which the chart number of patients was obtained. Then, charts of the patients were retrieved from the card room. Relevant information was collected from these charts.p value \u22640.25 was used as a candidate for multivariate logistic regression analysis. A statistical significant association was tested at a p value <0.05.The collected data were coded and entered into SPSS version 20 for data analysis. Association between the management outcome of SBO and independent variables was checked by using the binary and multivariate logistic regression. On binary logistic regression, a Training for data collectors was given for 2 days regarding the purpose of the study, on how to fill the prepared checklist, and the importance of data quality. In order to avoid interpersonal variation between data collectors, data were collected by the same data collectors throughout the study period. Regular daily supervision was conducted to check the consistency and completeness of the checklists.A total of 301 patients were admitted and managed for intestinal obstruction from January 1, 2014, to December 30, 2016, and of whom, 211 (70.7%) were SBO. From the total SBO cases, 41.7% were aged 21\u201344 years with a mean age of 27.2 (SD\u2009\u00b1\u200919.2). From the total participants, 64.9%, 34.6%, 24.2%, and 68.7% were males, illiterates, farmers, and from rural area, respectively .From a total of patients participated, 144 (68.2%) came within 24 hours of manifestation, and 159 (75.4%) of the patients were with hematocrit of >36%. In addition, 113 (53.6%) of participants came with referral, and 78 (37%) had previous abdominal operation. Eighty-one (38.4%) of the participants were with preoperative complication.In cases managed during the study period, dynamic bowel obstruction was the leading cause (90.5%), and adhesion, SBV, and intussusceptions (74 (35.1%), 51 (24.2%), and 50 (23.7%)) were the commonest intraoperative findings, respectively .Laparotomy was the most common method of small intestinal obstruction management in this study (95%), and 5% were improved by conservative management. Bowel resection and anastomosis were the commonest intraoperative procedures done. From a total of operated cases, 26.5% developed postoperative complication with SSI being the most common complication (49.2%), 61.6% stayed in the hospital for less than seven days, and 84.8% were improved and discharged.A total of ten variables  were found to have an association with the management outcome of SBO on bivariate analysis.In the multivariable regression model, male sex , duration of illness before surgical intervention , level of hematocrit , types of intestinal obstruction , and length of hospital stay  were significantly associated with the management outcome of small bowel obstruction .Small bowel obstruction is a commonly encountered emergency condition in hospitals worldwide and a leading cause of emergency room visits. This postures a challenge to the surgical trainee. Its treatment requires cautious preoperative preparation, great surgical judgment, and postoperative care which are frequently very demanding , 4. ThisThe study shows small intestinal obstruction is more prevalent in males (62.1%) than in females 37.9%). This is comparable with other studies conducted in Larkana, Rwanda, Nigeria, and southern Ethiopia 7.9%. Thi.In this study, duration of obstruction before surgical intervention has a statistical significant association with the management outcome of patients. Patients who presented after 24-hour duration of obstruction are 4.4 times more likely to have unfavorable result as compared with patients who came to the hospital within 24 hours. This is in agreement with the studies conducted in Rwanda, Adama, Ethiopia, and Niger , 9, 11.With early diagnosis and suitable management, most of patients enduring from intestinal obstruction can be spared. Most of the time, the situation is quite different. Some patients come after subjecting themselves to relatively long periods of observation, and they usually present to us when they really feel very sick . In thisThis study also shows that patients who stayed in the hospital for shorter than 7 days after surgery were about 5 times more likely to have favorable outcomes when compared with those who stayed for longer than or equal to 7 days after the surgery. This is similar with the result of studies conducted in Ethiopia , 9, 10 aIn this study, lower hematocrit level was associated with an increased occurrence of unfavorable outcomes. Participants with hematocrit greater than or equal to 36% were 4.25 times more likely to have favorable outcomes when compared with those who had hematocrit less than 36%. It is in line with the study conducted in Ethiopia .The most frequently occurred postoperative complications were wound infection 49.1%), which is consistent with other studies conducted in Gondar, Adama, and Sudan .1%, whic, 13. MosOne hundred seventy-nine 84.8%) patients improved and were discharged after they obtained service in the hospital, whereas 32 15.2%) died after being operated, and the result is comparable with studies done in Gondar 13.3%), Sudan (15.7%), Nigeria (14%), and Tanzania (14.3%), respectively [4.8% pati.3%, Suda.2% died Adhesions are the single most common cause for small bowel obstruction . Our stuThe limitation of this study was that secondary data were used.Adhesion, small bowel volvulus, and intussusceptions were the main causes of small bowel obstruction. Laparotomy was the most common means of intestinal obstruction management, and bowel resection and anastomosis were the commonest intraoperative procedures done. The most frequently occurred postoperative complication was wound infection. Sex, duration of illness before surgical intervention, hematocrit level, types of intestinal obstruction, and period of hospital stay were predictors of the management outcome of patients with small bowl obstruction."}
+{"text": "In this study, Xie et al. investigated the mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation in mammalian embryos, and report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. Their findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development. Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator  Upon fertilization, one of the earliest requirements in the development of a new organism is the formation of a totipotent zygote, which possesses the capacity to generate the entire embryo and all extraembryonic structures. In mice, only the zygote and two-cell stage embryo possess totipotency , with sucis-regulatory information to mammalian genomes, providing transcription factor binding sites, enhancers, and promoter sequences -like cells , marked gulators .Dux knockout in embryos has overall mild effects, implying the existence of parallel and redundant mechanisms to activate MERVL and ZGA in vivo that remain to be discovered  and Kap1/Trim28 proteins  that show reduced output and abrogated Dux repression compared with ESCs. Direct disruption of nucleolar structure and function by RNA polymerase I inhibition (iPol I) or by perturbation of nucleolar liquid\u2013liquid phase separation is sufficient to rapidly release Dux from perinucleolar regions, activate its expression, and convert ESCs into a 2C-like state. In vivo, short-term iPol I prevents the formation of mature, Ncl-positive nucleoli from NPBs, activates Dux, and impairs developmental progression past the two- to four-cell stage. Our study reveals a direct link between rRNA transcription, nucleolar-based repression, and cell fate during early mammalian development.Here, we investigated the impact of nucleolar dynamics and its link to +) . We confirmed that 2C-GFP/CD4+-enriched cells (\u201c2C-pos\u201d) express markers of bona fide 2C-like cells, including high levels of MERVL and 2C-specific transcripts , similar to FACS-purified 2C-like cells -like cells can be identified from within ESC cultures by expression of a stably integrated fluorescent reporter  . These c+) A. With tke cells . 2C-GFP/ocenters E,F, all ocenters . Thus, 2Rpl/Rps RNA expression and protein synthesis . To confirm that these changes are not an artefact of CD4-based enrichment, nascent transcription and translation rates were profiled in unsorted, bulk 2C-GFP reporter ESCs .We previously discovered that a ribonucleoprotein complex comprising LINE1 RNA, together with Nucleolin (Ncl) and Kap1, is essential for both ribosomal RNA (rRNA) expression as well as 2-cell exit . Since Nynthesis . We measynthesis A. We disynthesis D as wellter ESCs . In agreSubsequently, we investigated whether these changes are reflected at the two-cell stage in vivo. Following fertilization, one- to two-cell embryos possess immature nucleolar precursor bodies (NPBs)\u2014largely uncharacterized structures that are initially transcriptionally silent and lacking distinct compartments . In contSupplemental Fig. S3A), and by 4 h induces morphological nucleolar remodeling, generating singular ring-like structures in ESCs resembling 2C-like nucleoli and embryo NPBs . Strikingly, we found that following overnight nucleolar reprogramming, iPol I causes a significant increase to 20% of 2C-like cells within ESC cultures . The 2C-specific protein Zscan4 is also highly elevated following iPol I treatment, compared with purified 2C-GFP/CD4+ cells as a positive control . Knockdown (KD) of other nucleolar proteins such as Sirt7 has a limited effect, suggesting a reliance on specific factors for repression of the 2C-like state. Conversely, KD of Npm3, a negative regulator of ribosome biogenesis . Collectively, these data reveal an intriguing link between nucleolar rRNA synthesis and 2C repression to maintain ESC identity.These results support the hypothesis that the development of functionally mature nucleoli may play a role in the repression of the two-cell transcriptional program and exit from the two-cell stage. To investigate this, we asked which nucleolar proteins are most important for repression of the 2C-like state in ESCs and performed an siRNA miniscreen for nucleolar components in 2C-GFP ESCs. Similar to the effects of Ncl loss, we found that depletion of RNA Pol I and Fbl also causes a notable increase in 2C-like cells  and found that Dux is significantly induced as early as 4 h following nucleolar disruption . By 8 h, Dux targets are highly up-regulated among all significantly altered genes following iPol I . Next, to test whether 2C-like gene induction is dependent on Dux, we performed iPol I experiments in Dux\u2212/\u2212 ESCs . We subsequently investigated whether iPol I can also prevent nucleolar maturation and Dux silencing in mid-two-cell embryos  and causes significant rRNA reduction and concomitant Dux activation in L2C\u20134C embryos . Together, these results indicate that nucleolar disruption rapidly leads to Dux derepression and induction of the two-cell state.Next, we asked how rRNA synthesis and nucleolar function are mechanistically linked to repression of the 2C-like state. We focused on Dux, which is a potent MERVL and 2C-like gene activator and is up-regulated upon nucleolar protein knockdown H. We perg iPol I B. Moreovg iPol I  demonstr\u2212/\u2212 ESCs  compared embryos E, which  embryos . We foun embryos G,H. We o CX-5461 G,H, simi CX-5461 A. FinallDux activation, we first tested whether this is dependent on nucleolar stress. Although iPol I does not induce typical markers of nucleolar stress , levels of total and activated p53 (phospho-p53) are increased upon iPol I, similar to the effect of the topoisomerase II inhibitor etoposide, used as a positive control . Interestingly, etoposide treatment also increases the proportion of 2C-GFP+ cells in culture , suggesting that p53 activation can activate the 2C-like state. Indeed, a recent study reported DNA damage-dependent activation of Dux/DUX4 and the 2C-like state via p53 . Furthermore, iPol I treatment is still able to cause significant Dux activation in the absence of p53  of DAPI staining in ESCs versus 2C-like cells. These experiments revealed a reduction in perinucleolar chromatin fibers in the 2C-like state . Thus, it is only nucleolar-localized Dux alleles that are affected by iPol I. Subsequently, we used RNA FISH to monitor the appearance of Dux mRNA in single cells and found that Dux movement is closely linked to reactivation of Dux RNA expression . We next scored the location of putative nascent RNA foci  .In ESCs but not 2C-like cells, we previously reported that the elevance . We nextote NPBs A,B. Thesne marks  and is lne marks . We therke state . Reducedke state D, suggesr BMH-21 E,F. UsinDux is closely tied to its repression and suggest that iPol I induces global disruption of nucleolar chromatin organization and gene expression.Nucleolar-associated DNA regions (NADs) have been previously identified by isolation and sequencing of DNA associated with purified nucleoli (NAD-seq) , which hDux locus release and derepression. The membrane-less nucleolus is held together by liquid\u2013liquid phase separation (LLPS), which is driven by the association of rDNA with nucleolar proteins and moreover is dependent on continual rRNA synthesis , an aliphatic alcohol used to disrupt liquid-like condensates . Interestingly, D4Z4 repeats containing human DUX4 have also been proposed to reside within a NAD , but not other nucleolar proteins, are particularly important.Importantly, this mechanism of Dux regulation appears separate from nucleolar stress-mediated p53 activation, which is capable of directly inducing Dux . We fountivation . Insteadtivation  and thattivation . Indeed,tivation  indicatein a NAD  and to bin a NAD . StudyinDux nucleolar association and repression. The nucleolus is self-organized into its three subdomains by phase separation, driven by the interaction between nucleolar proteins and rDNA , late two-cell (48\u201349 h after hCG), morula (3 d after hCG), or blastocyst (4 d after hCG) stage.All animal experiments were performed according to a UK Home Office Project License in a Home Office-designated facility using 4- to 6-wk-old female and 2- to 6-mo-old male CD1 mice (Charles River). Animals were maintained on a 12-h light/dark cycle and provided with ad libitum food and water in individually ventilated cages. Female mice were superovulated by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin , followed by 5 IU of human chorionic gonadotropin  46\u201348 h later, and then placed immediately with males. Zygotes were collected from oviducts \u223c22\u201324 h after hCG in M-2 medium (Sigma M7167), isolated from cumulus cells with 200 \u00b5g/mL hyalurionidase (Sigma H3506), washed through successive drops of M-2, and then cultured in pre-equilibrated KSOMaa (Sigma MR-106-D) in microdrops overlaid with mineral oil (Sigma M5310) or in four-well dishes. Zygotes were cultured in a humidified incubator at 37\u00b0C and 5% CO2 on 0.1% gelatin-coated plates in ES-FBS culture medium . Experiments comparing wild-type versus Dux\u2212/\u2212 E14 ESCs were performed in FBS/LIF media as above, supplemented with 2i  as in Supplemental Table S2) were added to ESCs at the indicated concentrations and durations unless otherwise explicitly mentioned in the figure legends.Mouse E14Tg2A (E14) ESCs  were used for all experiments  and to d+ surface expression. E14 ESCs were nucleofected with 4 \u00b5g of linearized 2C-GFP plasmid, plated at low density in 10-cm2 plates, and then selected with 250 \u00b5g/mL G418 (Mirus) for 8 d. Individual colonies were picked and expanded, with a single colony that showed high specific expression of GFP expanded and used for subsequent validations and experiments. For 2C-GFP/CD4 isolation, cells were trypsinized, washed, resuspended in FACS buffer , and then isolated either by MACS using CD4 (L3T4) microbeads (Miltenyi Biotec) or with the EasySep mouse CD4-positive selection kit II (Stem Cell Technologies) according to the manufacturer's protocols in each case. Apart from Supplemental Figure S1, all purification experiments were performed with the EasySep kit. Flow-through cells were collected as the 2C-negative population. For flow cytometry analysis, ESCs were pelleted and resuspended in FACS buffer containing 1:8000 Sytox Blue (Thermo Fisher Scientific) to enable exclusion of dead cells.The 2C-GFP reporter construct  was modiZ-scores were calculated as the percentage 2C-GFP value for each factor minus the average 2C-GFP level for the entire plate, divided by the plate standard deviation. Other siRNA transfections or validation experiments were performed as above, scaling up cell numbers, Lipofectamine, and siRNA amounts accordingly for ESCs cultured in 12- or 24-well plates, with cells harvested at the indicated time points.The nucleolar miniscreen was performed with a Cherry Pick custom library plate of OnTargetPlus siRNAs (Horizon Discovery) consisting of 20 wells of different gene targeting siRNA pools and three siControl wells. 2C-GFP ESCs  at a denNascent transcription (EU) and translation (HPG) assays were carried out as described previously  using Clg for 20 min to remove insoluble material. Proteins were quantified by the BCA assay (Pierce), and equal amounts were loaded onto 4%\u201312% Bolt Bis-Tris plus SDS-PAGE gels (Thermo Fisher) to separate proteins and then transferred onto PVDF membranes. Blocking was performed in 5% milk/PBS-T for 1 h, and then membranes were incubated overnight with primary antibodies at 4\u00b0C in milk/PBS-T. The next day, membranes were incubated with the appropriate HRP-conjugated secondary antibodies  for 1 h, and then proteins were detected by ECL reagent on an Amersham Imager 680.Whole-cell extracts were prepared from ESCs by scraping in ice-cold RIPA buffer containing protease inhibitors , incubating for 30 min at 4\u00b0C, and then pelleting at 16,000Supplemental Table S2.RNA was isolated directly from ESCs by scraping in RLT lysis buffer (Qiagen) containing 1:100 \u03b2-mercaptoethanol (Sigma), or RLT was added to equal numbers of ESCs for CNN approaches. RNA was purified and DNase I-treated according to the manufacturer's instructions using RNeasy mini kits (Qiagen). For embryo inhibitor experiments, two-cell embryos were flushed at 46 h after hCG and cultured in KSOMaa medium containing either 1 \u00b5M CX-5461, 1 \u00b5M BMH-21, or 0.1% DMSO in a four-well dish. Culture in inhibitors began after 1 h for a period of 8 or 24 h. Equal numbers of embryos per experimental condition were lysed in 75 \u00b5L of buffer RLT prepared as above, and the RNA was isolated according to RNeasy micro kits (Qiagen). In ESCs and embryos, cDNA synthesis was performed with up to 1 \u00b5g of DNase-treated RNA using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific), and qRT-PCR was performed with SYBR Green (KAPA) on a QuantStudio 5 qPCR machine (Thermo Fisher Scientific). qRT-PCR data were normalized to two housekeeping genes (Rpl7/H2A), unless a cell number normalization (CNN) approach was used as detailed in the legend for Supplemental Table S1. RNA-seq data have been uploaded to GEO, accession GSE185424.For RNA-seq, RNA was extracted using the RNeasy mini kit as for qRT-PCR, and then three biological replicates per condition of DNase-treated total RNA spiked with ERCCs (Thermo Fisher) were used for RNA-seq library preparation and sequencing at the MRC London Institute of Medical Sciences Genomics Core facility. RNA quality was assessed using the Agilent 2100 RNA 6000 Nano assay, and libraries were prepared using the NEBNext Ultra II directional RNA library preparation kit with NEBNext poly(A) mRNA magnetic isolation module following the manufacturer's instructions. Library quality was evaluated using the Agilent 2100 high-sensitivity DNA assay, and their concentrations were measured using the Qubit dsDNA HS assay kit. Libraries were pooled in equimolar quantities and sequenced on an Illumina NextSeq 2000 to generate a minimum of 40 million single-read 50-bp reads  per sample. Reads were trimmed and aligned to reference genome mm10 plus ERCCs using TopHat2. Default settings were used apart from the specification \u201cg -1\u201d to map each multimapping read to one random TE or gene in the genome. Reads were counted using the Subread package FeatureCounts for each gene or TE family. Data were filtered to exclude rows with counts per million (cpm) >0 in fewer than three samples. To account for any global decreases in RNA amounts due to iPol I, we used our previously described cell number-normalized (CNN) approach to normalize reads to the abundance of ERCC spike-ins  using LiSupplemental Table S2. The next day, samples were washed with PBS and incubated in secondary antibodies  for 1 h at room temperature, followed by a wash for 30 min in PBS plus DAPI and two more washes in PBS, and then samples were mounted in VectaShield mounting medium containing DAPI. Confocal images were taken on a Leica SP5 fluorescent microscope under an oil immersion 63\u00d7 objective.ESCs were allowed to adhere to Matrigel-coated eight-well chambers or 10-mm glass coverslips for 1\u20132 h, fixed in 4% PFA for 10 min, and then stored in PBS until staining. Blocking and permeabilization were carried out in one step in immunofluorescence (IF) buffer  plus 0.4% Triton X-100 for 30 min. Primary antibody incubations were carried out overnight at 4\u00b0C using the indicated antibodies and dilutions in IF buffer as described in Embryos were fixed in 4% PFA in PBS containing 0.1% Triton X-100 for 30 min, followed by three washes in PBS containing 0.1% PVA (PBS-PVA). Samples were permeabilized in PBS containing 0.5% Triton X-100 for 30 min, followed by blocking in 5% BSA in PBS for 1.5 h. A 1:100 dilution of primary antibody  was prepared in blocking solution, and embryos were incubated in 10-\u00b5L drops in a humidified Terasaki plate (Greiner Bio-One) overnight at 4\u00b0C. Embryos were washed three times in PBS containing 0.1% Tween-20 and 0.1% PVA (PBST-PVA) for 5 min. A 1:500 dilution of secondary antibody  was prepared in blocking solution, and embryos were incubated in 10-\u00b5L drops in a humidified Terasaki plate for 1 h in the dark. EU and HPG assays were performed as for ESCs, except that embryos were incubated for 1 h in pre-equilibrated KSOM  prior to incubation in KSOM containing 500 \u00b5M HPG or 1 mM EU for 2 h. Embryos were fixed in 4% PFA in PBS containing 0.1% Triton X-100 for 15 min and permeabilized in 0.5% Triton X-100 in PBS for 20 min prior to Click-iT reactions. Prior to mounting, all samples were washed three times in PBST-PVA for 5 min, followed by a 30-min incubation in 1:1000 DAPI in PBS and a further three washes in PBS-PVA. Embryos were mounted in VectaShield (Vector Laboratories) under a 20-mm \u00d7 20-mm #1.5 coverslip (Agar Scientific) supported at the corners by Dow Corning high-vacuum silicone grease (Sigma-Aldrich) and sealed with nail polish. Confocal images were captured using a Leica SP5 or SP8 fluorescence microscope using an oil immersion 40\u00d7 objective and acquired in 1-\u00b5m Z-stacks. All steps were carried out in 500-\u00b5L volumes at room temperature unless otherwise noted.Dux oligo probes used to detect Dux foci were used and labeled with Cy3 fluorphores (Amersham PA23001) as described previously  software  using default parameter settings. Channel alignment was performed using calibrations obtained from a multicolored bead slide, acquired with equivalent acquisition settings.Purified 2C-GFP/CD4-negative and -positive cells or wild-type ESCs incubated for 8 h with or without iPol I were adhered onto Matrigel-coated \u00b5-slide eight-well glass-bottom dishes (Ibidi 80827), fixed with 4% paraformaldehyde in PBS for 10 min, and then stored in PBS at 4\u00b0C before immunolabeling. Cells were rinsed three times in PBS, incubated for 15 min in 20 mM glycine in PBS, rinsed, permeabilized with 0.1% Triton X-100 for 10 min in PBS, blocked for 1 h with 4% BSA in PBS, and then incubated with mouse anti-B23 to detect nucleoli in 4% BSA in PBS overnight at 4\u00b0C. The next day, samples were washed three times for 60 min with 2% BSA in PBS, then incubated for 2 h with Alexa secondary antibodies (1:250), and washed again as above. Finally, samples were washed and counterstained with 1 \u00b5g/mL DAPI for 30 min and rinsed successively in PBS before coverslips were mounted in VectaShield. The long incubation times used allowed for antibody accessibility throughout the cells, providing the highest sensitivity for SIM imaging. Multicolor SIM imaging was performed using a Zeiss Elyra S.1 (Carl Zeiss Microimaging) and a plan-apochromat 63\u00d7/1.4 oil lens. Raw SIM images were acquired with an sCMOS camera (pco.Edge 4.2) using five phase shifts and three grid rotations, with a Multicolor image single snapshots (IF and EU/HPG assays) or Z-stacks  were acquired with a laser-scanning confocal microscope with a pinhole diameter of 1 airy unit . Different channels were imaged sequentially to avoid bleedthrough and cross-excitation and then exported as TIFF files for further image analysis. RNA and DNA FISH image Z-stacks were also acquired on an Olympus spinning-disk confocal system based on an IX83 inverted microscope stand . Raw .LIF images were processed into TIFF files and merged, each channel manually thresholding or filtering with the same setting in Fiji software for data analysis.Dux FISH loci were related to each individual nucleus and contained nucleoli, and distance to the nearest nucleolus and nucleus border were measured.Dux FISH 3D spatial analysis was undertaken using custom-written scripts in Fiji for nucleus and nucleolus segmentation, and Imaris  for Dux FISH probe identification and distance measurements. Briefly, identification of LaminB1-labeled nuclei and B23-labeled nucleoli was performed in Fiji in combination with the MorphoLibJ plugin to perform 3D segmentation. Labeled image masks generated were combined with the original image stack and imported into Imaris for use with the \u201cCells\u201d package to facilitate interactive review of 3D segmentation results and Dux FISH probe identification. Identified https://cellprofiler.org; software version 4.1.3). To describe and quantify the number of \u201cring-shaped\u201d nucleoli , the fluorescence intensity distribution over four concentric layers in each nucleolus was measured. The mean fluorescence intensity of the outer layer was divided by the mean intensity of the innermost layer, and any nucleolus with a ratio >1.4 was counted in the \u201cring-shaped\u201d category. Nucleolar circularity (\u201cFormFactor\u201d) was calculated within CellProfiler according to 4\u03c0 \u00d7 (area)/(perimeter)2, excluding very small nucleoli, which were liable to give unreliable measurements .The analysis of nucleolar morphology and fluorescence intensities was carried out with a custom-written CellProfiler pipeline (n) and the reported error either as standard deviation (SD) or standard error of the mean (SEM). All statistics are P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****), with the relevant test performed described in the figure legends and corrections for multiple testing applied where necessary. Welch's correction was applied to t-tests when the variance was unequal between conditions.All statistical analyses were carried out in Prism 7 or above (Graphpad) or R (RNA-seq data). Details of individual tests are outlined in each figure legend, including number and type of replication performed (RNA-seq data have been uploaded to GEO, accession GSE185424.https://github.com/mpercharde/RNAseq and/or are available on request.RNA-seq data were analyzed with standard packages and programs, as detailed in the Materials and Methods. Code for data processing and analysis are available at"}
+{"text": "Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while pro-amniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings.  \u2022Increased-size embryos present a delay in embryonic morphogenesis\u2022Embryo size determines the mechanism of pro-amniotic cavity formation\u2022Apoptosis is required for the formation of a single cavity in enlarged embryos\u2022Cavity formation and size regulation have distinct molecular underpinnings In this article Shahbazi, Zernicka-Goetz, and colleagues explore how increasing embryo size affects the mechanism and timing of embryonic morphogenesis. Enlarged embryos present a delay in pro-amniotic cavity formation, an essential morphogenetic event that happens concomitantly with the regulation of embryo size. However, whereas pro-amniotic cavity formation requires apoptosis of embryonic cells lacking basement membrane contact, size regulation occurs independently of cell death. Early mammalian development is regulative, as embryos are able to compensate for perturbations to the normal developmental program to ensure the formation of a viable organism . When, fAt implantation, mouse and human embryos comprise three cell lineages: embryonic epiblast that will give rise to the future organism; extra-embryonic primitive endoderm that will give rise to the yolk sac; and trophectoderm that will build the placenta. After embryo implantation, these three lineages interact to undertake the first morphogenetic step, remodeling of the epiblast from a group of apolar cells to a polarized epithelium that lines the incipient pro-amniotic cavity . This isin\u00a0vivo and in\u00a0vitro revealed that pro-amniotic cavity formation does not require cell death but takes place through cell polarization, apical membrane repulsion, and exocytosis  as a model for pro-amniotic cavity formation suggested that formation of the lumen entails death of the cells located in the center of the tissue by apoptosis . Howeverocytosis , a proceocytosis . During ocytosis . The\u00a0mecogenesis . This exHere, we address this question by generating double-sized embryos and ESC aggregates of different sizes as two complementary model systems. To our knowledge, our results provide the first evidence for a size-dependent mechanism of pro-amniotic cavity formation in the mouse embryo, in agreement with the regulative nature of mammalian development.in\u00a0vitro and in\u00a0vivo. We first confirmed that at the blastocyst stage, double embryos displayed twice the normal number of cells in all three lineages: the epiblast, the primitive endoderm, and trophectoderm  blastocysts into pseudo-pregnant recipients and recovered them at early post-implantation stages. To account for the asynchrony within each litter and across different experiments, we scored embryos according to their morphological features  , the epiSince it has been shown that increasing the density of Madin-Darby canine kidney (MDCK) cells results in cysts that open a lumen through apoptosis rather than cell polarization , we hypo+) and the number of epiblast cells positive for cleaved CASPASE-3 . These comprise both epiblast and primitive endoderm, and recapitulate the mechanism of amniotic cavity formation in the absence of trophectoderm derivatives: primitive endoderm cells secrete laminin, which triggers epiblast polarization and lumenogenesis . To isolic cells R and 2S,in\u00a0vivo  aggregates of mouse ESCs as a model of epiblast of varying size. We previously showed that upon exit from naive pluripotency, single ESCs embedded in Matrigel are able to polarize and open a lumen through membrane repulsion, a process that recapitulates lumenogenesis in\u00a0vivo . To mimiAfter 24\u00a0h we collected the aggregates of different sizes, classified them into small, medium, and large based on their size D\u2013S1K, anAfter 48\u00a0h in culture, nearly 90% of structures formed from small ESC aggregates showed a single cavity E and 3G in\u00a0vitro culture, as their size plateaued after 3\u00a0days (data not shown). By 96 h, all aggregates displayed single lumens irrespective of their size  and allowed them to develop in 3D Matrigel. We first confirmed that the difference in size was still maintained after 48 and 72\u00a0h of culture O and S1PTo identify the dynamics of lumen formation, we imaged this process following induction of the expression of a GFP-tagged PODOCALYXIN (GFP-PODXL). To monitor cell death in living samples, we added SYTOX to the medium . The anaGFP-PODXL (gray), SYTOX (green). Scale bar, 50\u00a0\u03bcm.GFP-PODXL (gray), SYTOX (green). Scale bar, 50\u00a0\u03bcm.GFP-PODXL (gray), SYTOX (green). Scale bar, 50\u00a0\u03bcm.Since our results suggested that apoptosis might be involved in fusion of smaller cavities into a unified lumen, we next sought to test whether inhibiting the apoptotic pathway would affect the ability of large aggregates to form single lumens. With this aim, we used the pan-caspase inhibitor Z-VAD-FMK and cultured ESC aggregates of varied sizes in 3D as previously. We found that following Z-VAD-FMK treatment, small, medium, and large ESC aggregates showed fewer dead cells compared with control aggregates, even though apoptosis was not fully inhibited A\u2013S4D. InBcl-2, which suppresses apoptosis without affecting the ability of ESCs to self-renew or to differentiate  with pre-implantation mouse embryos. To test the effect of apoptosis in enlarged embryos, we first generated both single and double embryos at the 8-cell stage, as previously, then aggregated them with either 3\u20134 or 8\u201310 H2B-GFP BCL-2 OE ECSs, respectively  and aggregation of two E2.5 embryos in drops of KSOM. Single embryos also underwent a brief incubation in acidic Tyrode's solution to remove the zona pellucida. Single and double embryos were cultured in KSOM  covered by mineral oil at 37\u00b0C in 5% CO2. For transfer experiments, early blastocysts  were transferred into F1 foster females at E0.5 or E2.5 of pseudo-pregnancy and recovered at E5.5. All embryos were recovered at the same time (10 a.m.), but due to their heterogeneous development we were able to classify them into different morphological stages. Post-implantation embryos were dissected out from the decidua and immediately fixed.Mice were bred in the Animal House of the Gurdon Institute in accordance with national and international guidelines. Experiments were approved under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. For embryo production, F1 (C57Bl/6\u00a0\u00d7 CBA) females were superovulated by injection with 7.5 IU of pregnant mare's serum gonadotropin (Intervet) followed by injection with 7.5 IU of human chorionic gonadotropin (Intervet) 48\u00a0h later and mating with F1 males. Pre-implantation embryos were collected at E2.5 from the oviducts and uteri in drops of M2 medium. Double embryos were generated by removal of in\u00a0vitro cultured single and double embryos were incubated with anti-mouse whole serum  and sera complement guinea pig  diluted to 20% in M2 medium for 45\u00a0min each. After elimination of trophectoderm cells by pipetting with a narrow glass capillary in M2 medium, ICMs were seeded in individual hanging drops of pre-warmed IVC-1 medium  in N2B27 supplemented with 2iLIF. On the following day all aggregates were collected, mixed, and washed with PBS to remove any remaining 2iLIF. They were then plated in 8-well ibiTreat  dishes previously coated with 40\u00a0\u03bcL of growth factor reduced Matrigel  in N2B27. Upon attachment to the gel, the medium was replaced with N2B27 containing 5% Matrigel, and aggregates were cultured for 24\u201396\u00a0h with media change every 48 h. To generate mouse ESC aggregates with defined size, we manually picked ESC aggregates containing two, four, or eight cells and plated them using the same method as described above. For apoptosis inhibition, Z-VAD-FMK  was used at 20\u00a0\u03bcM. For plating of mouse ESC aggregates in agarose, 1% low-melting-point agarose  was dissolved in PBS. Cell aggregates were resuspended and plated in a single drop of agarose in 8-well ibiTreat dishes covered with N2B27 supplemented with naive pluripotency factors (2iLIF) or N2B27 alone.All relevant data are available from the corresponding authors upon reasonable request.L.C.O., V.S.R., M.N.S., and F.A. designed and performed the experiments with help from C.K. W.M. performed embryo transfer experiments. L.C.O., V.S.R., and M.N.S. analyzed the data. L.C.O., V.S.R., M.N.S., and M.Z.-G. wrote the paper. H.M.-S. supported the work of V.S.R. M.N.S. and M.Z.-G. supervised the study. M.Z.-G. conceived the project."}
+{"text": "Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2\u2032 cleavage sites in S protein synthesis and processing as well as virus assembly and infection.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2\u2032 sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three \u03b1-helices contribute to the spike\u2019s conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2\u2032 sites are very important, like the S2\u2032 cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants. The spike (S) protein of coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a single-pass type I membrane protein and the major determinant of virus entry, host range, tissue tropism, pathogenesis, and virulence , 2. The 9\u2013For SARS-CoV-2 and SARS-CoV entry, the proteolytic cleavage of coronavirus S protein by one or more host proteases, such as cysteine proteases cathepsin B and L or the transmembrane serine protease 2 (TMPRSS2), is essential to trigger membrane fusion , 13. TMP16\u2013In structural analyses of the SARS-CoV-2, the S proteins were found to freely exist on the viral envelope, and the S head was connected to the viral membrane with the stalk domains with three flexible hinges in the S2 subunit , 20. Pre6VSB [The conservation analysis of S protein showed that the amino acid similarity among 27 SARS-CoV-2 isolates and SARS-CoV (CUHK-W1 strain) is 99.7 to 99.9%, and these SARS-CoV-2 strains share amino acid sequence similarities of only 74% with MERS virus (HCoV-EMC/2012) . Especially the region between the S1/S2 and S2\u2032 cleavage sites of S protein , the ami6VSB ), which To investigate the influence of these amino acid residues between the S1/S2 and S2\u2032 cleavage sites on the synthesis and function of S protein, a series of the S mutants was generated through site-directed mutagenesis , includiTo explore the regular pattern, we mutated the Y756 to other amino acid residues: tryptophan (W), alanine (A), glycine (G), histidine (H), or glutamate (E). We found that the mutation of Y756A, Y756G, or Y756E caused the S proteins to be produced as uncleaved forms, but the mutated S proteins with Y756W or Y756H were partially cleaved . OverallWe next evaluate the consequences of mutation of tyrosine 756 on SARS-CoV-2 infection using a lentivirus (HIV-1 NL4-3 strain) pseudotyping system . First, To further confirm pseudovirus infection, a HEK293T cell line stably expressing human ACE2 (293T-hACE2) was prepared through the lentiviral transduction system and blasticidin pressure selection. The ACE2-positive proportion of the 293T-ACE2 cell line was quantified as above 98% by flow cytometry, and the expression of ACE2 was detected by Western blotting . Then, tIn addition, we investigated the effect of L753, F759, or T998 mutation on SARS-CoV-2 spike-mediated virus infection. The three mutations significantly reduced the pseudovirus infection, especially the L753G mutation , consistSince these mutations, including R685, L753, Y756, or R815, affect the expression and processing of S protein, the subcellular localization of S protein may also be affected. For visual analysis of subcellular localization, the fusion expression plasmids of S or its mutants with a C-terminal enhanced green fluorescent protein (EGFP) tag and tyrosine protein kinase Lck with a C-terminal red fluorescent protein (RFP) tag were constructed and cotransfected into HeLa cells, and Lck was used as a subcellular localization marker; Lck localizes to the plasma membrane and pericentrosomal vesicles . As specBased on the results showing the effects of L753 and Y756 mutations on spike synthesis and processing in our experiments, we have boldly speculated that these mutations may affect the yield or quality of the SARS-CoV-2 pseudotyped viruses. During the packaging of S-enveloped pseudotyped lentiviruses in cell culture, the expression of S and its mutations was confirmed to be consistent with the data shown in In addition, except that no pseudovirus particles bearing Y756G S were observed, the characteristics of the particles formed by the WT S or its other mutants were observed under a negative-staining electron microscope. The virions embedded by WT S or its mutants with Y756F, R685A, D614G, Y756A, or Y756H exhibited similar morphologies and a diameter of \u223c200\u2009nm, but the virions embedded by the Y756C, Y756W, Y756E, and R815A S mutants were irregular in shape . AmazingWe next evaluated the impact of the mutation of residue L753, F759, or T998, structurally related to Y756, on pseudovirus production with SARS-CoV-2 S protein. Pseudoviruses with each mutant S protein were produced, concentrated, and detected by Western blotting as described above. Similarly, the quantification of pseudovirus particles was performed using p24 protein as a reference. The expression of WT or mutant S protein was detected by anti-spike RBD or S2 antibody in the producer cells . For theThe next question is whether the above-mentioned locus mutations affect the production of authentic SARS-CoV-2 virions. The SARS-CoV-2 virion, as is well established, is composed of four structural proteins: spike, membrane, envelope, and nucleocapsid protein. Recent studies have shown that the coexpression of four structural proteins in HEK293T cells could result in self-assembly into virus-like particles (VLPs) , 28, 29.For the VLPs, the WT or D614G mutated S proteins incorporated into SARS-CoV-2 VLPs composed of M, E, and N . HoweverFurthermore, in the pellets, compared to VLPs with WT S protein, F759G or T998G mutant VLPs only contained lower levels of cleaved S1 or S2 products, especially F759G mutant, whereas L753G spike did not appear in the precipitation . These rThe above-described results confirmed that although the R685A mutation causes the S protein to be incompletely cleaved, it can be assembled into pseudovirions with the typical spike structure of the SARS-CoV-2 prototypic virus. So then, an important question is raised: does this mutation affect antiviral therapy targeting the S protein? The neutralization potency of the monoclonal antibodies and horse or rhesus monkey antiserum targeting the S protein receptor-binding domain was assessed using the pseudotyped viruses bearing the WT S or D614G variant and vesicular stomatitis virus G protein (VSV G)-enveloped pseudotyped virus as a nonspecific control. As expected, the anti-RBD monoclonal antibodies efficiently neutralized both viruses pseudotyped with the WT S and D614G variant but not VSV G-enveloped pseudotyped viruses , which wAt the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a novel betacoronavirus broke into people\u2019s lives; this virus had highly transmissible and pathogenic characteristics and caused a pandemic of acute respiratory disease named coronavirus disease 2019 (COVID-19) , 32. As 39\u2013The SARS-CoV-2 virion mainly consists of four structural proteins, including the spike, envelope (E), membrane (M), and nucleocapsid (N) proteins. For SARS-related coronaviruses, the S protein is synthesized and processed, including posttranslational modification and cleavage, in the endoplasmic reticulum (ER) and Golgi, respectively , 45. MorMoreover, other proteases\u2019 S1/S2 cleavage is processed beyond the furin proteolytic enzyme . For SAR6VSB) (The S2 subunit consists of the fusion peptide FP), a second proteolytic site (S2\u2032), an internal fusion peptide (IFP), and two heptad-repeat domains (HR1 and HR2) preceding the transmembrane domain (TM) , 45. Fur6VSB)  and F. FP, a secoIt is noteworthy that the changes in the expression and subcellular localization of the S protein affect the assembly of virus particles and viral transduction to a certain extent. Indeed, we observed very low or even no infectivity of Y756-mutated S-based pseudovirus compared to that with WT S protein under the conditions of our experiments , which mTaken together, our results show that the S protein of SARS-CoV-2 as the primary receptor binding and membrane fusion mediator mediates virus entry into host cells and is also one of the main structural proteins for virus assembly. Therefore, the correct expression, structural stability, and precise subcellular localization of the S protein are critical to the correct assembly of the virus and indirectly affect the infection of the progeny virus. In this work, we confirm for the first time that the tyrosine residue at position 756 and the associated L753 and F759 are critical to the correct expression and subcellular localization of the S protein, regulate virion assembly and production, and, in turn, affect the S protein-mediated virus entry, using a pseudotyped virus or virus-like particle model. In addition to the resolved cryo-EM structure of the S protein, we found that the hydrogen bond formed by Y756 is also important for the stability of the S protein structure. In terms of significance, this study provides a new potential target for novel interventions, including antiviral drugs or CRISPR-mediated gene mutation therapy, by analyzing the key amino acid sites of the S protein. In the future, we will cooperate with other teams to carry out authentic SARS-CoV-2 verification and small-molecule drug screening research due to biosafety factors.NC_045512.2) and then inserted into the eukaryotic expression vector pCMV-3\u00d7Flag-14 and fused with 3\u00d7Flag for expression. The S mutants, including D614G, R685A, R815A, and series mutation of Y756, L753, F759, or T998, were engineered and constructed by PCR with a Q5 site-directed mutagenesis kit , using the S gene of the Wuhan-Hu-1 strain as the parent and WT sequence. The WT S gene and its mutants fused with EGFP at the C terminus were cloned into the pEGFP-N3 vector for subcellular localization assays. Human ACE2 was amplified using pcDNA3.1-ACE2-Flag  as a template and cloned into pLenti 6.3/V5-DEST  via homologous recombination to construct a cell line stably expressing human angiotensin-converting enzyme 2 (ACE2). The lentiviral backbone plasmid pNL4-3.Luc.R-E- was provided by Ningyi Jin. For the coexpression of SARS-CoV-2 M and E proteins, the M-IRES-E fusion fragment was composed of the M and E genes fused with Flag tag in the N and C termini, respectively, and M and E were linked by the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The pcDNA3.1-SARS-CoV-2-N-Myc was purchased from Beyotime (China). All plasmids were verified by DNA sequencing.The full-length SARS-CoV-2 spike (S) gene was synthesized  according to the sequence of SARS-CoV-2 Wuhan-Hu-1 strain  (GenBankDYKDDDDK) tag (no. 20543-1-AP), ACE2 (no. 21115-1-AP), GAPDH (no. 10494-1-AP), and \u03b2-actin (no. 20536-1-AP), and CoraLite 488-conjugated ACE2 monoclonal antibody (no. CL488-66699) were purchased from Proteintech (China). Horse anti-RBD/SARS-CoV-2 spike serum and cynomolgus monkey anti-RBD serum were gifts from Ningyi Jin.Rabbit monoclonal antibody against the SARS-CoV-2 receptor-binding domain (RBD) (no. 40592-R001), rabbit polyclonal antibody against SARS-CoV-2 nucleocapsid (no. 40588-T62) and spike S2 (no. 40590-T62), and HIV-1 p24 protein  antibody (no. 11695-T48) were purchased from Sino Biological (China). Rabbit polyclonal antibodies against Flag . The expression of hACE2 was examined by Western blotting with anti-ACE2 antibody, and the hACE2+ cell percentage was identified by flow cytometry.Human lung epithelial (A549) cells were maintained in Ham\u2019s F-12K (Kaighn\u2019s) medium  or supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin . HEK293T, HEK293, and HeLa cells were maintained in Dulbecco\u2019s modified Eagle medium  containing 10% FBS and 1% penicillin-streptomycin. Human hepatoma (Huh7) cells were cultured in DMEM supplemented with 10% FBS, 1% GlutaMAX , and 1 mM sodium pyruvate . These cells were cultured at 37\u00b0C in a 5% COCells were lysed with a buffer of 20\u2009mM Tris (pH 7.5), 150\u2009mM NaCl, 1% Triton X-100, and EDTA-free protease inhibitor cocktail  and phenylmethylsulfonyl fluoride . The extracted proteins were subjected to SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane , and protein bands were probed with the indicated primary antibodies.6 HEK293T cells in a 10-cm dish were cotransfected with 8\u2009\u03bcg of S and its mutant expression plasmids pCMV-S-3\u00d7Flag including the WT, D614G, R685A, R815A, or Y756 mutant or L753G, F759G, or T998G and 8\u2009\u03bcg of pNL4-3.Luc.R-E- using Lipofectamine 3000  to prepare various SARS-CoV-2 S-pseudotyped lentiviral luciferase reporter viruses. Six hours posttransfection, the supernatants were replaced with fresh DMEM supplemented with 2% FBS. The supernatants containing the pseudoviruses were harvested 48 h after transfection and purified by filtration with a 0.45-\u03bcm filter. In accordance with the manufacturer\u2019s protocol, the pseudoviruses were titrated with the Lenti-X reverse transcription-quantitative PCR (qRT-PCR) titration kit . For SARS-CoV-2 S-pseudotyped virus entry assays, Huh7 or 293T-hACE2 cells were infected with a fixed dose of pseudoviruses for 48 h and then washed and lysed to detect luciferase signal with the ONE-Glo luciferase assay system  according to the instructions.A total of 5\u2009\u00d7\u200910The supernatants containing the pseudoviruses with SARS-CoV-2 S protein or its mutants were obtained as described above and then layered onto 20% (wt/vol) sucrose cushions and purified and concentrated by ultracentrifugation  using an Optima XPN-100 ultracentrifuge (Beckman Coulter). Furthermore, the pellets were resuspended in sterilized 1\u00d7 phosphate-buffered saline (PBS) and prepared for SDS-PAGE and Western blot analysis using the corresponding primary antibodies against RBD, Flag, or HIV p24.The resuspended pellets containing the pseudovirions obtained as described above were added to a glow-discharged 230-mesh copper grid covered with carbon-coated collodion film. After standing for several minutes, the excess liquid was absorbed with a filter paper from the edge of the copper mesh. Then, the copper mesh was stained with phosphotungstic acid  for 5\u2009min and then sucked dry with a filter paper. Samples were analyzed with a JEM-1200EXII transmission electron microscope (JEOL).Analysis of the subcellular localization of C-terminally EGFP-tagged S or its mutants in HeLa cells was done using a laser scanning confocal microscope . The tyrosine protein kinase Lck was used as a subcellular localization marker. Briefly, HeLa cells were cultured on glass coverslips to cotransfect with S or its mutant expression plasmids and Lck marker for 48 h, then fixed in 4% paraformaldehyde and PBS for 30\u2009min, and then permeabilized with 0.25% Triton X-100 according to the specific requirement. The nuclei were stained with 4\u2032,6-diamidino-2-phenylindole . Images were taken under a fluorescence microscope or a Leica TCS SP8 confocal microscope.HEK293T cells in a 10-cm dish were cotransfected with the plasmids encoding the SARS-CoV-2 M, E, N, and S or its mutants. In the transfection mixtures, 6\u2009\u03bcg of each plasmid M-IRES-E, pcDNA3.1-SARS-CoV-2-N-Myc, and pCMV-3\u00d7Flag-S or the plasmid with S mutant were added into 1\u2009mL of Opti-MEM and then mixed with 18\u2009\u03bcL of PEIpro transfection reagent . The transfection mixtures were incubated for 15\u2009min at room temperature and dropped into HEK293T cells. At 72 h posttransfection, the culture medium was collected, filtered through a 0.45-\u03bcm filter, layered onto 20% (wt/vol) sucrose cushions, and purified and concentrated by ultracentrifugation  using an Optima XPN-100 ultracentrifuge (Beckman Coulter) as described above. Moreover, the pellets containing VLPs were resuspended in sterilized 1\u00d7 PBS, aliquoted, and stored at \u221280\u00b0C freezer.The VLP pellets and the producer cell lysates were boiled with 5\u00d7 SDS-PAGE loading buffer for 10\u2009min at 100\u00b0C to identify the components of VLPs. The cell lysates were used to normalize loading conditions for SARS-CoV-2 structure proteins. These samples were resolved by a 12.5% SDS-PAGE separating gel and subsequently transferred to a nitrocellulose membrane for Western blot analysis with the indicated antibodies against SARS-CoV-2 S RBD S2 or N Flag tag. GAPDH was the loading control.4/well overnight. Threefold serial dilutions of a commercially available rabbit anti-RBD monoclonal antibody or anti-RBD serum samples were prepared and mixed with an equal volume of pseudoviruses. The mixture was incubated at 37\u00b0C for 1 h before adding 293T-hACE2 cells. At 48 h after infection, cells were lysed to detect luciferase signal with the ONE-Glo luciferase assay system  to determine the neutralization levels.The SARS-CoV-2 pseudovirus neutralization assay was used to test the effect of mutation on S antigenicity. The horse or rhesus monkey anti-RBD serum samples collected after two immunizations were gifts from Ningyi Jin and were inactivated at 56\u00b0C for 30\u2009min. 293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1\u2009\u00d7\u200910t test or one-way analysis of variance (ANOVA) with Dunnett\u2019s multiple-comparison test.Data are presented as means \u00b1 standard deviations (SD) from 3 to 6 replicates. Data are representative of those from three independent experiments. Statistical analyses of the data were performed by using GraphPad Prism version 8.4.3. Statistical comparisons were made using a two-tailed paired or unpaired Student"}
+{"text": "Consequently, there is a need for further research to analyze and comprehend the real biological synthesis-dependent processes. This is currently an untapped hot research topic that required more investment to properly leverage the green manufacturing of metallic nanoparticles through living entities. The review covers such green methods of synthesizing nanoparticles and their utilization in the scientific world.Current advancements in nanotechnology and nanoscience have resulted in new nanomaterials, which may pose health and environmental risks. Furthermore, several researchers are working to optimize ecologically friendly procedures for creating metal and metal oxide nanoparticles. The primary goal is to decrease the adverse effects of synthetic processes, their accompanying chemicals, and the resulting complexes. Utilizing various biomaterials for nanoparticle preparation is a beneficial approach in green nanotechnology. Furthermore, using the biological qualities of nature through a variety of activities is an excellent way to achieve this goal. Algae, plants, bacteria, and fungus have been employed to make energy-efficient, low-cost, and nontoxic metallic nanoparticles in the last few decades. Despite the environmental advantages of using green chemistry-based biological synthesis over traditional methods as discussed in this article, there are some unresolved issues such as particle size and shape consistency, reproducibility of the synthesis process, and understanding of the mechanisms involved in producing metallic nanoparticles  This is a crucial feature of the green chemistry developing industry  is vital to attain this aim. Green production of metallic nanoparticles has been utilized to accommodate diverse biological components  . Among the current greenways of synthesis for metal/metal oxide nanoparticles, the use of plant extracts is a straight forward technique to generate nanoparticles at large scale in comparison to bacteria and/or fungal assisted synthesis. These compounds are known together as biogenic nanoparticles. Here, we present an update on recent breakthroughs in the synthesis of biological nanoparticles and outline their future development and potential uses  leaves. During the examination, silver and gold nanoparticles were polydispersed and flat plate-like in shape. After being exposed to Au3+ and Ag + ions in solution, bimetallic Au core-Ag shell NPs were formed, which were verified by TEM analysis and further showed that the Ag NPs served as adsorbents onto the gold NPs, resulting in the core-shell structure  nitrate trihydrate, and double-distilled water made CuO NPs. The solution was homogenized for 2\u20135\u00a0min using a magnetic stirrer. Next, a pre-heated muffle furnace was utilized to execute a combustion reaction on the mixture to yield CuO NPs at a temperature of 400 \u00b0C. The ash component of the plant extracts was removed by filtering the resulting combination. Methanol was used to eliminate contaminants from the solution after rinsing with distilled water. Researchers reported the synthesis of copper nanoparticles supported on sodium borosilicate glass using extract of Acalypha indica L. 6 (ndica L. . The nan4Fe(CN)6 .Centella asiatica L. leaf extract , then crush it before extracting the resulting crystals from the solvent . FollowiFungal biomass and associated metabolites are used to synthesize NPs in a relatively young field of nanotechnology known as \u201cmyconanotechnology\u201d . Micro- in vivo or in vitro. Most of the harmful transition metal ions are converted to a non-toxic form in the mycelia of the fungus during the in vivo method, which exploits this process to make NPs intracellularly. It is possible to directly use washed mushroom mycelia to produce NPs intracellularly in this method. Mycelia must undergo additional treatment to remove the NPs from the mycelia before being used again   . Anothertigation .P. citrinopileatus, the P. eous, the P. cystidiosus, the P. ostreatus, P. eryngii, and the P. flabellatus were all examined. Tests for Pulmonarius were carried out as well  extract as the reducing agent . Chemical change of peptidoglycan of of metal . A factoPseudomonas stutzeri AG259, which was isolated from a silver mine, internal accumulation of silver NPs, as well as some silver sulphide, with sizes ranging from 35 to 46\u00a0nm, was observed , and a further undetermined structure  with sizes ranging from 6 to 13\u00a0nm. These NPs stayed steady in the dark over 8\u00a0months. The generation and stability of silver nanoparticles seemed to depend on the temperature, pH, or kind of bacteria from which the supernatant was obtained, among other factors. Researchers observed that A. kerguelensis supernatant could not produce silver nanoparticles at the same temperature that P. antarctica could produce the same nanoparticles. This work provided significant evidence that the components in cell-free culture supernatants that encouraged the production of silver NPs varied from one bacterial species to another and that this was true across all bacterial species  outside the cell. Several shapes and sizes of AgNPs may be found in extracellularly produced AgNPs. These include hexagonal, spherical, triangular, circular, and cuboidal, depending on the culture medium utilised for the growth of bacteria   in the medium and periplasm  . AgNPs  citrate as an electron acceptor, S. algae bacteria can grow anaerobically. When H2 gas was used as an electron source, they showed that S. algae resting cells could reduce ions (1\u00a0mM) into elemental gold in 30\u00a0min at 25\u00b0C throughout a pH range of 2.0\u20137.0 . Extracellular gold NPs were deposited when the pH of the solution fell to <2.8. There was a wide range of biogenic gold NPs (15\u2013200\u00a0nm) on the bacterial cells at this pH. Biogenic gold NPs (20\u00a0nm) were coated on the bacterial cells, and bigger gold particles (350\u00a0nm) were deposited extracellularly in a solution with pH 2.0. So it may be inferred that pH significantly impacts the form and location of biogenic gold nanoparticles . Furthermore, H2 gas did not chemically reduce Au (III) in a sterile control condition devoid of S. algal cells. Thus, in the presence of molecular H2 as an electron donor, S. algae resting cells converted soluble Au (III) into insoluble gold , the reaction mixture changed color more slowly. With the rise in pH, the reduction process became more visible. With increasing concentrations of silver nanoparticles, the antifungal activity against Aspergillus fumigatus, Candida albicans, and Fusarium sp. was observed to rise.ia sinus  algae sparticles . After 1Pithophora oedogonia, a fresh water green alga, has been used to synthesize silver nanoparticles diameter 25\u201344\u00a0nm. Carbohydrates, saponins, steroids, and proteins were shown to decrease AgNO3 to silver nanoparticles by IR spectroscopy and quantitative analysis of the extract. Compared with Gram-positive bacteria, they were shown to be more efficacious  silver nanoparticles having face-centered cubic shape. Using a combination of 14 bacteria and microalgae, silver nanoparticles were created. Even in the dark, extracellular polysaccharides were producing nanoparticles. Silver nanoparticles of varying sizes and morphologies were found, ranging from species to species  reduction of Au+3 to Au0; and 3) release of gold nanoparticles into the solvent. This theory has been widely accepted. As a result of their distributed nature, they don\u2019t collect in one place. Their dimensions range from 10 nm to several hundred nanometers. This shows that all algae have a tolerance limit and a certain ability to decrease metal ions to protect them from the poisonous impact of Au3+/Au0, since an AuCl3 concentration of 103\u00a0M is deadly to E. gracilis.Living cells of the d others . Like otStoechospermum marginatum biomass has been shown to produce gold nanoparticles biogenically , for example  was used to biosorb and bioreduce Au, resulting in nanoparticles of varied sizes and morphologies  (Sargassum myriocystum)  exhibit biosynthesized Au nanoparticles ranging in size from 7 to 11\u00a0nm (Padina pavonica). The biosynthesis of gold nanoparticles by two freshwater algae species has also been shown  , and Staocystum) , while Socystum) . Green aarticles . A brownathogens . For exato 11\u00a0nm  and fromen shown ; 2014b. Bifurcaria bifurcata). Nanoparticles of cuprous oxide (Cu2O) and cupric oxide (CuO) were generated in a simple technique. A few nanoparticles were elongated, but the vast majority of the particles were spherical. An average particle size of 22.6\u00a0nm was discovered for the samples, which varied from 5 to 45\u00a0nm. Copper oxide nanoparticles were shown to be effective against both Enterobacter aerogenes and Staphylococcus aureus in subsequent antibacterial experiments  was also described in a recent work by Khanehzaei et al. Stabilized copper cored-cuprous oxide nanoparticles with a mean particle size of 53\u00a0nm were synthesized in the presence of seaweed. Nanoparticle surfaces were also discovered to be capped by pairs of electrons, some hydroxide and sulphur groups from the water-soluble sulphated polysaccharides present in seaweed cell walls  nanoparticles using brown seaweed was recently shown in work by Mahdavi and others (Sargassum muticum). To make Fe3O4 nanoparticles, an aqueous seaweed extract was combined with an aqueous ferric chloride solution. Reduction and capping are both accomplished by the amino, carboxy, and hydroxyl functional groups produced from the water-soluble polysaccharide cell walls  solution and an aqueous extract of marine microalgae (Tetraselmis suecica) were used to synthesize and characterize gold nanoparticles (AuNPs). There was a distinct band in the UV\u2013Vis spectrum that corresponded to the formation of AuNPs. However, the most common 79\u00a0nm diameter with a polydispersed and crystalline structure , and an AFM study showed that they were between 53 and 67\u00a0nm in size  and rendering naturally coloured solutions (Rhodamine B and Sulforhodamine) into colorless solution in the presence of NaBHcatalyst . Becausecatalyst . NanoparAvena sativa biomass   was linked to an increase in the reduction of Ag+. Smallerrnatants . The scirnatants . It has r 60 \u00b0C) . Plant mSilver has a long history of usage as an antibacterial, and its nanoforms are significantly superior and more biocompatible antimicrobial agents now than their conventional counterparts. Research on silver consumption in nanotechnology has been going on since the dawn of the age of nanotechnology, and this section covers a lot of that work. Silver nanoparticles have received a great deal of attention in recent years for their potential to combat infectious diseases by closing the gaps in current antimicrobial formulation techniques, eradicating drug-resistant microorganisms, and establishing a foothold for the emerging field of conjugated silver nanoparticles. This research could help us better understand the role of silver nanoparticles in future antimicrobial treatments. Because of the additive action of nanosilver and the large variety of phytoconstituents with intrinsic antimicrobial capabilities, silver nanoparticles may be readily manufactured from plant extracts with extraordinary stability and environmental friendliness and demonstrate a broad spectrum of antimicrobial activities.Klebsiella pneumonia, Escherichia coli, and Salmonella typhimurium. Furthermore, the Minimum inhibitory concentrations (MICs) recorded were: 3.9, 3.9, 7.8, and 3.90\u00a0\u03bcg/ml . All of the investigated substances were strongly inhibited by the produced nanoparticles. M. tuberculosis MIC values were determined to be 4\u00a0\u03bcg/ml and 1\u00a0\u03bcg/ml for M. bovis and M. tuberculosis correspondingly. M. tuberculosis MDR strain has a MIC value of 16\u00a0\u03bcg per litre. However, MIC values of 1\u201316\u00a0\u03bcg/ml and 4\u201332\u00a0\u03bcg/ml have been recorded for M. tuberculosis and M. bovis clinical isolates, respectively. According to the findings of this investigation, silver nanoformulations may have the antitubercular potential , amoxicillin (2048\u00a0\u03bcg/ml), and erythromycin (64\u00a0\u03bcg/ml). This research reveals that AgNPs have more promise and effectiveness in treating hospital-acquired illnesses than traditional antibiotics  produced from Puerh tea leaves have been discovered to have high antibacterial efficacy against Gram-ve pathogenic pathogens such as 90\u00a0\u03bcg/ml . Silver research . Selim eotential . Using sibiotics .E. coli, Avicennia marina-based AgNPs were shown to have bactericidal effects because the proton motive force may have been dissipated . i et al. .Xanthomonas citri, S. aureus, and Erwinia carotovora causes ROS levels to rise beyond critical levels. The study found that ROS levels rose when AgNPs were added to bacterial suspensions, resulting in bacterial suppression. AgNPs caused the rise in ROS when exposed to resistant strains of E. coli and S. aureus, according to E. coli and S. aureus  in resistant bacteria is one method of inhibiting their proliferation. Antimicrobials are often used to raise ROS levels. However, the introduction of AgNPs to resistant microorganisms elevates ROS levels, leading to resistant species, according to a recent study. Based on the findings of Khan and Ali , it seem. aureus .S. aureus in terms of antibacterial activity against E. coli and Pseudomonas aeruginosa   (gamma-irradiated AgNPs)  . CurvulaC. aromaticus leaf extracts were utilised to create the Au NPs, then employed as antibacterial agents. Temperatures  were used to control the generation of NPs. It was shown that C. aromaticus-prepared Au NPs had strong antibacterial activity (ABA) when tested against various strains of bacteria  could not grow in the presence of Au nanoparticles, measured to be 21.52\u00a0nm in size. A. nigra, according to the findings, may be able to produce nanoparticles of gold (Au NPs) that serve as antibacterial agents   .P. atlantica leaf extract, and the antibacterial properties of the NPs were also tested. Particles with a diameter of 50\u201360\u00a0nm were used. Diffusion technique was employed to assess the antibacterial activity of Au NPs, and the broth dilution method was used to determine the MIC and Minimum Bactericidal Concentration (MBC). The ABA of the Au NPs was encouraging, and the MIC value was also relatively low, equivalent to that of a conventional antibiotic  leaf extract  nanosheets containing holes. The bacteria on the porous rGO matrix may be tracked using Ag ions released from the nanocomposite directly and quickly. Antibacterial activity against E. coli, Lactococcus lactis, and Klebsiella pneumonia was maximized when polyvinylpyrrolidone (PVP) nanoparticles (PVP/PtNPs) were combined as nanocomposites  in the liver, spleen, and bone marrow is more effective in absorbing particles larger than 200\u00a0nm after administration  The liveFor nanoparticle behaviour and interaction with proteins and cells, surface characteristics of nanoparticles are crucial . For nanin vitro, but there is a dearth of data on their effects in vivo, as shown by the literature. There is still a lot of work that needs to be done in the subject of nanomaterials in order to make significant advancements across a variety of industries. However, despite the many advantages of using fungal-mediated metal nanoparticles, there are still a number of issues that must be addressed in order for this technology to become a reality on the market today.In a nutshell, work on myconanotechnology is ongoing. The use of nanoparticles will continue to expand, but we must first investigate their toxicity, environmental buildup, and impact on human and animal health. Nanoparticles may also be utilized to cure a variety of serious ailments and open up new avenues in the biomedical area in the future, according to recent research. AgNPs might be used to power gadgets, which could help alleviate the current energy shortage. Nanoparticles have been extensively studied in vivo medicinal applications.Controlling nanostructure dispersity, which strongly influences electrical and optical characteristics, and isolating and purifying plural form are major issues in microbial nanobiosynthesis. The size distribution of a nanoparticle population is a critical feature that affects the particle\u2019s behaviour in fluids. Methods including freeze-thawing, osmotic stress, and centrifugation may modify nanoparticle structures and cause aggregation and precipitation. Using appropriate methodologies might enhance microbial nanoparticle biosynthesis. The application of genetic engineering techniques and suitable microbial strains might assist overcome the disadvantages of slower production rate and polidispersity  . A cappiMany efforts have developed novel green synthesis processes during the previous several decades. Living creatures have a lot of potential for making nanomaterials that may be used in various sectors, including biomedicine. To manufacture nano-objects of the appropriate size and form, organisms ranging from primary bacteria to very sophisticated eukaryotes may all be employed. Prokaryotes are the most basic biomasses, making them easier to genetically alter to create more desirable synthesis chemicals. However, bacterial culture and large-scale manufacturing remain difficult compared to alternative methods. Bacteria were investigated as the first nano-factories to manufacture noble metal nanoparticles as a first step. The poor synthesis rate and restricted number of size and form distributions accessible, on the other hand, steered the research towards fungi and algae.Fungi may be used to produce green nanoparticles on a massive scale. They\u2019re simple to work with in downstream processing, and they release a lot of enzymes that help with the reduction. They also have metal filament tolerance, a high binding capacity, and intracellular uptake. However, eukaryotes have a considerably more difficult time genetically manipulating individual enzymes to increase production.via biological entities. Therefore, there is a need for further research to analyze and comprehend the real biological synthesis dependent processes. This is a vastly untapped subject that needs much more research investment to properly leverage the green manufacturing of metallic nanoparticles through living entities.Plant extracts have lately been the subject of several studies, and the number of research papers published in this area has exploded in the past 2\u00a0years due to their widespread availability, environmental friendliness, and cost-effectiveness. This green chemistry strategy of utilizing living entities is in stark contrast with traditional chemical and physical processes that commonly involve hazardous compounds that have the potential to cause environmental toxicity, cytotoxicity, and carcinogenicity. Whilst biological entities have been extensively used to produce nanoparticles, the use of plant sources offers a straightforward, clean, non-toxic, and robust procedure that does not need any special culture preparation or isolation techniques that are normally required for bacteria and fungi-based techniques. In particular, the utilization of plant extracts for manufacturing nanoparticles is affordable, readily scaled up, and environment-friendly. Plant extracts have the ability to generate nanoparticles with a specified size, shape and content. Plant produced nanoparticles have the potential to be extensively employed in current medical processes utilizing nanoparticles such as fluorescent labelling in immunoassays, targeted administration of therapeutic medications, tumour death by heating (hyperthermia), and as antibacterial agents in bandages. On another front, plant produced nanoparticles have the potential to be exploited for the delivery of anti-microbiological chemicals for use as insecticides for agricultural crops. Moreover, agricultural crop wastes and food industry wastes are also ideal prospects for delivering supplies of plant-based bio-chemicals with the ability to synthesis metallic nanoparticles and related products. Despite the environmental advantages of using green chemistry based biological synthesis over traditional methods as discussed in this article there are some unresolved issues such as particle size and shape consistency, reproducibility of the synthesis process, and understanding of the mechanisms involved in producing metallic nanoparticles"}
+{"text": "Emerging zoonotic infections present a serious global health threat . It is eIt is reasonable to anticipate that the number of outbreaks and pandemics could increase in the near future. This assumption is based on multiple factors such as increased frequency of travel, particularly to remote areas, climate change, intensified global trade, susceptibility to pathogen exposure and several more . In addiThe SARS-CoV-2 pandemic has emphasized global weaknesses in the detection and control strategies aimed to minimize the human health and socioeconomic impact of exposure to zoonotic pathogens . It is cA major obstacle in our progress towards developing novel therapeutics and vaccines is the limited knowledge of the reservoir host disease dynamics, virus-recipient host interactions and the detailed processes that permit the establishment of infection . This isIn this Special Issue, we presented manuscripts  providing an insight on the distribution, spread, and protein functions of SARS-CoV-2, Puumala orthohantavirus and Ebola viruses. In the original research manuscript, Au and colleagues addressed the important issue of detecting and analyzing SARS-CoV-2 virus transcription in infected cells . AlthougIn another original study by Davidyuk et al., the genetic diversity of Puumala orthohantavirus (PUUV) in the Republic of Tatarstan (RT) was analyzed . PUUV prThis Special Issue also features review manuscripts on Ebola virus protein function, structure, and immune properties. The manuscript by Jain et al. presents a detailed analysis and discussion of the multi-functional features of each Ebola virus (EBOV) protein . EBOV beIn the final review, Jain et al. present a concise description of the cell types targeted by EBOV . In addiCollectively, this Special Issue highlights the proficiency with which viruses are able to cause severe disease and the complexities of virus\u2013host interactions that impact both disease pathology and outcome. The knowledge attained from the articles covered here could aid the development of more specific peptide-based antivirals, monoclonal antibodies, and novel vaccines that protect against infections in the future. This area of research is absolutely essential and is urgently needed for better preparation for future pandemics.We wish to express our appreciation to all the authors who have participated in this Special Issue and the reviewers for their comments."}
+{"text": "One of the topical problems of materials science is the production of van der Waals heterostructures with the desired properties. Borophene is considered to be among the promising 2D materials for the design of van der Waals heterostructures and their application in electronic nanodevices. In this paper, we considered new atomic configurations of van der Waals heterostructures for a potential application in nano- and optoelectronics: (1) a configuration based on buckled triangular borophene and gallium nitride (GaN) 2D monolayers; and (2) a configuration based on buckled triangular borophene and zinc oxide (ZnO) 2D monolayers. The influence of mechanical deformations on the electronic structure of borophene/GaN and borophene/ZnO van der Waals heterostructures are studied using the first-principles calculations based on density functional theory (DFT) within a double zeta plus polarization (DZP) basis set. Four types of deformation are considered: uniaxial /biaxial  stretching and uniaxial /biaxial  compression. The main objective of this study is to identify the most effective types of deformation from the standpoint of tuning the electronic properties of the material, namely the possibility of opening the energy gap in the band structure. For each case of deformation, the band structure and density of the electronic states (DOS) are calculated. It is found that the borophene/GaN heterostructure is more sensitive to axial compression while the borophene/ZnO heterostructure is more sensitive to axial stretching. The energy gap appears in the band structure of borophene/GaN heterostructure at uniaxial compression by 14% (gap size of 0.028 eV) and at biaxial compression by 4% (gap size of 0.018 eV). The energy gap appears in the band structure of a borophene/ZnO heterostructure at uniaxial stretching by 10% (gap size 0.063 eV) and at biaxial compression by 6% (0.012 eV). It is predicted that similar heterostructures with an emerging energy gap can be used for various nano- and optoelectronic applications, including Schottky barrier photodetectors. The stacking of atomically thick 2D materials in the form of a vertical layered heterostructure opens up wide opportunities for obtaining new materials with unique properties ,2. SincePromising results in the design of van der Waals heterostructures with the desired properties are expected from borophene. Borophene is an experimentally obtained new 2D material consisting of boron atoms ,15. The 2 heterostructures [4, C2H4, C2H2, CH3OH, and HCHO organic molecules on borophene in the borophene/MoS2 van der Waals heterostructure was studied by the DFT method [2 heterostructures can be used as an excellent gas sensor for organic molecules such as C2H2 and HCHO.Computer simulation methods are used to search for new configurations of borophene-based vertical heterostructures, predict their properties, and analyze the possible applications. Using ab initio methods, Kochaev et al. predicted the appearance of a vertical heterostructure based on borophene with a periodic perforation and graphenylene . The resructures . It was T method . The cal2  to form a metal-semiconductor contact. Scientists have shown that deformed \u03b212/MX2 heterostructures can be used to implement tunable Schottky barriers [One of the hot research topics in the last few years is the implementation of a metal-semiconductor contact based on borophene heterostructures. In 2020, Katoch et al. performed an ab initio study of the prospects for using borophene in combination with MXbarriers . The elebarriers . It has barriers . The resbarriers ,27.In this paper, attention is drawn to the new atomic configurations of borophene-based 2D van der Waals heterostructures, namely borophene/GaN and borophene/ZnO. These configurations include a buckled triangular borophene, which has the highest energy stability among other allotropic forms of borophene . Previou\u22125 eV. To minimize the energy of the electronic subsystem, an efficient Broyden\u2013Pulay mixing scheme was used [z-axis of the supercell was set to be greater than 20 \u00c5. The real-space grid cutoff was chosen to be 300 Ry.Calculations of buckled triangular borophene/GaN and buckled triangular borophene/ZnO van der Waals heterostructures were carried out using the DFT method implemented in the Siesta 4.1.5 software package ,32. The was used . In ordeb, which was calculated as the difference between the total energy of the heterostructure and the total energy of the isolated monolayers. The binding energy Eb was ~\u221250 meV/atom for the supercell of the borophene/GaN heterostructure and ~\u221280 meV/atom for the supercell of borophene/ZnO. x and Ly, as well as the distance between the layers along the Z axis. The supercell translation vectors were Lx = 3.35 \u00c5 and L = 6.10 \u00c5 for the borophene/GaN heterostructure and Lx = 3.28 \u00c5 and Ly = 5.83 \u00c5 for the borophene/ZnO heterostructure. The distance between the borophene and GaN monolayers along the Z axis was 2.91 \u00c5, and between the borophene and ZnO monolayers was 2.51 \u00c5.The atomistic models of borophene/GaN and borophene/ZnO heterostructures were built within our previous study and tested for the thermodynamic stability . The thex). A linear dispersion law is observed between the \u0393\u2013X, S\u2013Y, and \u0393\u2013S points of the Brillouin zone. Near the valence band maximum (VBM) between the \u0393\u2013X, S\u2013Y, and \u0393\u2013S points of the Brillouin zone, the dispersion law is close to the isotropic parabolic. Between the X\u2013S and Y\u2013\u0393 points of the Brillouin zone (in the direction of the wave vector ky), the flat energy bands are observed near the VBM and the conduction band minimum (CBM). As can be seen from The band structure was calculated for the supercells of the borophene/GaN and borophene/ZnO heterostructures. In order to explain the features of the electronic structure of borophene/GaN and borophene/ZnO van der Waals heterostructures, we analyzed the projected densities of the electronic states (PDOS) distributions. The calculated PDOS distributions are shown in e|, and from borophene to the ZnO monolayer is 0.22 |e|. The close values of the transferred charge for both heterostructures are explained by the proximity of Ga and Zn in the fourth row of the periodic table, and N and O in the second row of the periodic table.The significant contribution of borophene to the electronic properties of the borophene/GaN and borophene/ZnO heterostructures also follows from the Mulliken population analysis. The calculated electron charge density distributions over the supercell atoms are shown in After analyzing the regularities of the electronic structure of borophene/GaN and borophene/ZnO van der Waals heterostructures, we proceeded to identify the ways to control their electronic properties, including studying the possibility of opening an energy gap in the band structure. As such ways, various types of mechanical deformations were considered, namely, uniaxial and biaxial stretching/compression, which are often used in a real experiment. The selected ranges of the uniaxial and biaxial deformations are due to the computational complexity of the quantum calculations performed for the heterostructures under study. At the same time, the problem of determining the ultimate strength of borophene/GaN and borophene/ZnO van der Waals heterostructures under stretching/compression was not solved in this paper.y as compared to an undeformed structure. Consequently, under uniaxial compression, the contribution of the GaN monolayer to electronic structure of the borophene/GaN heterostructure becomes more significant. This is especially noticeable for the case of compression by 14%  for the cases of compression by 1% and 14% . It can y (between the X\u2013S and Y\u2013\u0393 points of the Brillouin zone) change to parabolic ones, as in the case of a uniaxial compression . The calculated PDOS distributions clearly explain the gapless band structure of the borophene/GaN heterostructure under uniaxial stretching. The fragments of these distributions near the Fermi level are shown in The band diagrams of the borophene/GaN heterostructure at minimum (1%) and maximum (14%) uniaxial stretching are shown in pression . In the Let us turn to the consideration of the band diagrams of the borophene/ZnO heterostructure under uniaxial compression and stretching. The cases of minimum (1%) and maximum (14%) uniaxial compression are shown in We present the PDOS distribution  in the cy between the X\u2013S and Y\u2013\u0393 points of the Brillouin zone. In the direction of the wave vector kx, the edges of the valence and conduction bands touch not only between the \u0393\u2013X points (as in the case of stretching by 1%), but also between the S\u2013Y points of the Brillouin zone. Both of these changes in the conduction band of the borophene/ZnO heterostructure upon stretching by 14% can be explained from the PDOS plot shown in The band diagrams of the borophene/ZnO heterostructures at minimal (1%) and maximum (14%) uniaxial stretching are shown in The opening of the energy gap between the valence and conduction bands in the band structure of the borophene/ZnO heterostructure was found for the case of stretching by 10%. Since the energy gap is quite small, we present a fragment of the DOS distribution near the Fermi level to show it more clearly . The ploThus, we can conclude that the borophene/GaN and borophene/ZnO heterostructures react differently to different types of uniaxial deformation. The borophene/GaN heterostructure is more sensitive to uniaxial compression, while the borophene/ZnO heterostructure is more sensitive to uniaxial stretching.Next, we turned to the consideration of the biaxial deformation of the borophene/GaN and borophene/ZnO heterostructures. In order to understand the reasons for the appearance of the energy gap in the band structure of the borophene/GaN heterostructure, let us analyze the distributions of the PDOS, as for the uniaxial deformations. The band diagrams of the borophene/GaN heterostructure at biaxial stretching by 2%  and 6%  are shown in The PDOS plot for the case of 6% biaxial stretching  shows thLet us consider the effect of biaxial compression/stretching on the electronic structure of the borophene/ZnO heterostructure. The band diagrams of the borophene/ZnO heterostructure for cases of 2% and 6% biaxial stretching are shown in Thus, it was found that of the two considered types of biaxial deformation, the two-basic biaxial compression deformation is more effective in terms of controlling the electronic structure of the studied van der Waals heterostructures.(1)The different distances along the Z axis between the borophene and GaN/ZnO monolayers in van der Waals heterostructure supercells: the distance between the borophene and GaN was 2.91 \u00c5, the distance between the borophene and ZnO was 2.51 \u00c5. At shorter distances, the interaction between the layers in heterostructure will be more noticeable and, therefore, will lead to more noticeable changes in the properties of the heterostructure as compared to the properties of its constituent monolayers. This is also indicated by difference in the calculated binding energies for the heterostructure supercells. Due to the shorter distance between borophene and ZnO, the binding energy of the borophene/ZnO heterostructure (\u22120.80 meV) will be greater than the binding energy of the borophene/GaN heterostructure (\u22120.50 meV). In addition, a shorter distance between borophene and ZnO causes more noticeable changes in the atomic structure of the ZnO monolayer in the borophene/ZnO heterostructure as compared to changes in the atomic structure of the GaN monolayer in the borophene/ZnO heterostructure. In particular, the dihedral angles of the GaN and ZnO monolayers differ greatly: for GaN, it is negative and amounts to \u22127.21, while for ZnO, it is positive and amounts to 19.18.(2)The differences in the features of the band structure of graphene-like GaN and graphene-like ZnO monolayers. The band structure of graphene-like GaN has an indirect band gap, while graphene-like ZnO has a direct band gap ,39. In aBased on the first-principles calculations, it was found that borophene/GaN and borophene/ZnO heterostructures demonstrate the different sensitivity of the electronic structure to consider the types of axial deformations. These differences can be explained by the following reasons:Thus, based on the first-principles calculations, the regularities of the influence of uniaxial and biaxial stretching/compressive deformations on the electronic structure of borophene/GaN and borophene/ZnO van der Waals heterostructures have been revealed. It has been found that the borophene/GaN heterostructure is more sensitive to axial compression, while the borophene/ZnO heterostructure is more sensitive to axial stretching. The differences are due to the different values of the distance between the 2D monolayers in borophene/GaN and borophene/ZnO heterostructures, as well as the features of the band structure of the GaN and ZnO monolayers, in particular, the different positions of VBM and CBM relative to the points of the Brillouin zone. For the appearance of an energy gap in the band structure of borophene/GaN and borophene/ZnO heterostructures, it is necessary that the zero DOS near the Fermi level be present in the PDOS of both borophene atoms and GaN/ZnO atoms. The p-orbitals of boron and nitrogen atoms make the decisive contribution to the band structure of the borophene/GaN heterostructure, and the p-orbitals of boron and oxygen atoms make the decisive contribution to the band structure of the borophene/ZnO heterostructure. It can be assumed that borophene/GaN and borophene/GaN heterostructures with electronic properties tuned by stretching/compression have prospects for an application as a material for nano- and optoelectronics, including devices with a Schottky barrier."}
+{"text": "Anal canal cancer (ACC) has been reported to be an uncommon cancer in Japan, as in the USA, Europe, and Australia. This retrospective multi\u2010institutional study was conducted to clarify the characteristics of ACC in Japan. First, the histological ACC type cases treated between 1991 and 2015 were collected. A detailed analysis of the characteristics of anal canal squamous cell carcinoma (SCC) cases was then conducted. The results of the histological types revealed that of the 1781 ACC cases, 435 cases (24.4%) including seven cases of adenosquamous cell carcinomas were SCC and 1260 cases (70.7%) were adenocarcinoma. However, the most common histological type reported in the USA, Europe, and Australia is SCC. Most ACC cases are adenocarcinomas and there is a low incidence of SCC in Japan which is different from the above\u2010mentioned countries. Moreover, we reclassified T4 into the following two groups based on tumor size: T4a (tumor diameter of 5\u00a0cm or less) and T4b (tumor diameter of more than 5\u00a0cm). The results of the TNM classification of SCC revealed that the hazard ratio (HR) to T1 of T2, T3, T4a, and T4b was 2.45, 2.28, 2.89, and 4.97, respectively. As T4b cases had a worse prognosis than T4a cases, we propose that T4 for anal canal SCC in Japan be subclassified into T4a and T4b. Most anal canal cancers in Japan are adenocarcinomas, the squamous cell carcinomas rate is low. In the T4 factor based on the depth of tumor invasion, there was a difference in prognosis depending on the tumor diameter and therefore we propose creating the subclassifications T4a and T4b for anal canal SCC in Japan. ACCanal canal cancerAJCCAmerican Joint Committee on CancerAPRabdominoperineal resectionCIconfidence intervalCRTchemoradiotherapyCTchemotherapyHIVhuman immunodeficiency virusHPVhuman papillomavirusHRhazard ratioJCCRCJapanese Classification of Colorectal Appendiceal and Anal CarcinomaJSCCRJapanese society for cancer of the colon and rectumOSoverall survivalRTradiotherapySCCsquamous cell carcinomaUICCUnion for International Cancer Control1Anal canal cancer (ACC) is an uncommon tumor that represents 4% of all cancers in the lower gastrointestinal tract.Previously, the anatomical definition of anal canal tumors in the literature varied, leading to confusion regarding diagnosis and treatment. At present, the definition of the anal canal has been revised in the TNM classification (8th edition), and the perianal skin (excluding the vulva) within 5\u00a0cm from the anal verge has been added along with the conventional surgical anal canal which corresponds to the tubular structure extending from the superior border of the puborectal sling to the anal verge,The Japanese society for cancer of the colon and rectum (JSCCR) has provided the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma (JCCRC)As a result, the JSCCR conducted a retrospective multi\u2010institutional study aiming to clarify the characteristics of ACC including adenocarcinoma and SCC in Japan. In the present study, we describe the characteristics of anal canal SCC and propose a new classification of T factor for anal canal SCC in Japan.2The patient records with malignant tumors of the anal canal who were treated between 1991 and 2015 were collected from 47 medical institutions in Japan. The anal canal is defined as the conventional surgical anal canal (P region) and the perianal skin (excluding the vulva) within 5\u00a0cm from the anal verge (E region) based on the TNM classification (8th edition).A total of 1781 patients were registered at the above\u2010mentioned 47 JSCCR affiliated institutions. Of these, 435 patients were SCC including seven adenosquamous cell carcinoma cases. There were 124 males and 311 females, with a mean age of 66.1\u00a0\u00b1\u00a012.0\u00a0years and prognosis analysis was performed for 295 anal canal SCC patients. 54 patients with unknown invasion depth, 105 with unknown maximum tumor size, 14 with unknown lymph node metastasis, nine with unknown survival time, and 10 with stage0 were excluded from the analysis.T stage is based on the TNM classification (8th edition), but T4 was subclassified into two groups: T4a is defined as a T4 tumor with a diameter of 5\u00a0cm or less and T4b is as those with a diameter of more than 5\u00a0cm.Consent to conduct the research was provided by the JSCCR ethical committee.2.1Survival curves were estimated using the Kaplan\u2013Meier method and the stratified Cox proportional hazard model was used to estimate the hazard ratio (HR) and the 95% confidence interval (CI). The estimation using the Stratified Cox proportional hazard model was considered the following variables; age, sex, tumor location (P/E), treatment methods , N factor (N0/N1), and M factor (M0/M1). The variables that violated the proportional hazards assumption were used as stratification factors. All statistical analyses were performed using EZR , which is a graphical user interface for R .33.1A total of 1781 patients who were treated for ACC between January 1991 and December 2015 were registered from 47 JSCCR affiliated institutions. Of these, 428 patients (24.0%) were SCC, 7 (0.4%) were adenosquamous cell carcinoma, 1260 (70.7%) were adenocarcinoma and 16 were undifferentiated carcinoma , radiotherapy (RT) in 28 (6.4%), chemotherapy (CT) in 5 (1.1%), surgical treatment in 132 (30.3%), and no treatment in 11 (2.5%). Among surgical treatment, APR was performed in 79 patients, total pelvic exenteration in 8, local resection in 42, and other surgical procedures in 3. There has been a significant change in treatment over the past 25\u00a0years. CRT, RT, or CT were performed in 14.3% of the cases between 1991 and 2000, in 62.2% of the cases between 2001 and 2010, and in 84.3% of the cases between 2011 and 2015.p\u00a0=\u00a00.036), 2.28 , 2.89 , and 4.97 , respectively  rate for T1, T2, T3, T4a, and T4b cases was 85.0%, 73.4%, 65.2%, 65.4%, and 51.0%, respectively  for stage IIA, 4.74  for stage IIB, 6.27  for stage IIIA, 6.18  for stage IIIB, 9.81  for stageIIIC, and 16.26  for stage IV  for T4aN0M0, 8.50  for T4bN0M0, 8.29  for T3N1M0, 9.65  for T4aN1M0, and 11.98  for T4bN1M0  was 93.1% for stage I, 81.1% for stage IIA, 60.0% for stage IIB, 71.1% for stage IIIA, 71.6% for stage IIIB, 61.0% for stage IIIC, and 45.2% for stage IV. The HR was 1 for stageI, 4.14  and T4b (tumor diameter of more than 5\u00a0cm). The results revealed that the T4b cases had a worse prognosis than T4a cases.Anal cancer is an uncommon cancer, accounting for 0.43% of all malignancies, and the incidence rates in the United states have increased from 0.8 to 1.7 cases per 100,000 persons per year from 1975 to 2011.As risk factors for developing SCC of the anus, anal carcinoma is associated with HPV infection; a history of receptive anal intercourse or a sexually transmitted disease; a history of cervical, vulvar, or vaginal cancer; immunosuppression after solid organ transplantation or HIV infection; hematologic malignancies; certain autoimmune disorders; and smoking.Regarding the treatment of anal canal SCC, there has been a transition from surgical treatment to CRT,In the TNM classification, the description of ACC was presented from the Union for International Cancer Control (UICC) 4th edition (1987) and the American Joint Committee on Cancer (AJCC) 3rd edition (1988), and the definition of T factor had not changed until the current 8th edition  .On the other hand, the prognosis of stage IV in this study was relatively good, and the main reason for this was considered to be the good prognosis of patients with extraregional lymph node metastasis of anal canal SCC. The regional lymph nodes in ACC are the perirectal, internal iliac, external iliac, and inguinal lymph nodes; however, the inferior mesenteric trunk nodes, inferior mesenteric root nodes, and common iliac nodes are the extraregional lymph nodes. The AJCC has reported on a prognosis survey by stage for anal canal SCC,Since this is a retrospective study, all information obtained was recorded using the criteria in place at the time of diagnosis and treatment. Ideally, it is best to collect cases prospectively by predetermining the examination criteria , but since ACC is an uncommon cancer, a long period of time is required to collect cases. ACC has been also historically subjected to various changes in staging and treatments, but there is little information on its prognosis, which needs to be investigated. ACC is an uncommon cancer, and this study showed that there are areas where SCC is common and areas where adenocarcinoma is common. Therefore, it is important to accumulate more data through studies from various perspectives.In conclusion, we demonstrated that most ACC cases are adenocarcinomas and that there is a low incidence of SCC in Japan which is different from that in the United States, Europe, and Australia. Moreover, we propose subclassifying T4 for anal canal SCC in Japan into T4a and T4b based on tumor size.Kazutaka Yamada and the co\u2010authors have no conflicts of interest to declare.Kazutaka Yamada and Yasumitsu Saiki carried out concept; acquisition and performance of the analysis; drafting of the text, tables, and figures; responsibility for the overall content. Koji Komori, Akio Shiomi, Masashi Ueno, Masaaki Ito, Koya Hida, Seiichiro Yamamoto, Manabu Shiozawa, Soichiro Ishihara, Yukihide Kanemitsu, Hideki Ueno, Tatsuya Kinjo, Kotaro Maeda, Junichiro Kawamura, Fumihiko Fujita, Keiichi Takahashi, Tsunekazu Mizushima, Yasuhiro Shimada, Shin Sasaki, Eiji Sunami, Fumio Ishida, Keiji Hirata, Shinobu Ohnuma, Kimihiko Funahashi, Jun Watanabe, Yusuke Kinugasa, Shigeki Yamaguchi, Yojiro Hashiguchi, Masataka Ikeda, Takeshi Sudo, Yoshito Komatsu, Keiji Koda, Kazuhiro Sakamoto, Masazumi Okajima, Hideyuki Ishida, Yuichi Hisamatsu, Taiki Masuda, Shinichiro Mori, Kazuhito Minami, Seiji Hasegawa, Shungo Endo, Akinori Iwashita, Madoka Hamada, and Yoichi Ajioka were involved in acquisition and interpretation of data. Koichiro Usuku and Tokunori Ikeda performed the statistical analysis. Kenichi Sugihara was involved in the drafting of the article. All authors reviewed the final document and approved it for publication.This study was approved by the Institutional Review Board of the JSCCR. This study was also approved by the Hospital Review Board of each hospital. Informed consent was obtained using an \u201copt\u2010out\u201d method under the approval of the ethics committee. The study was performed in accordance with the Declaration of Helsinki."}
+{"text": "Reinke's edema is chronic laryngeal disease in which the superficial layer of the lamina propria is expanded by thick mucus, giving it a gelatin aspect. The disease is directly related to smoking and more frequent in women, who end up having a lower tone of voice. Its histological characteristics cannot always distinguish it from other benign lesions of the larynx for which additional histological techniques are necessary.to study the immunoexpression of fibronectin, collagen IV and laminin in Reinke's edema by immunohistochemical technique. Prospective study.histological blocks of 60 cases of surgical Reinke's edema were saved, submitted to new cross-sections and to immunohistochemical reactions for fibronectin, laminin and collagen IV by the Avidin-Biotin-Peroxidase method. Fragments of five normal vocal folds were used as control, removed during autopsy. All patients were chronic smokers and adults\u2013 50 women and 10 men.the immunoexpression of fibronectin, collagen IV and laminin was more important in the endothelium of blood vessels  and less relevant in the basement membrane .the immunoexpression of fibronectin, laminin and of collagen IV in the basal membrane of Reinke's edema was not relevant, with a predominance of these antibodies in the endothelium of blood vessels. Reinke's edema is a chronic laryngeal disease in which the superficial layer of the lamina propria, also called Reinke's space, occupied by a thick mucous, providing the vocal folds with a gelatinous and myxomatous aspect. It happens to chronic smokers, predominantly to women - the reason is unknown so far. For some authors, such fact is due to the great concern of women with the progressive transformation that happens to their voices, which becomes lower, often times being confused with male voicesReinke's edema has been included among the benign exudative lesions of the larynx, together with vocal polyps and nodules, because of histopathology similarities between these lesions, in which there is a predominance of submucosal edema, justifying the homogeneity found in pathology reportsMany etiological factors are involved in the development of benign laryngeal lesions, making it possible to identify the predominance of some of them, such as smoking, in the development of Reinke's Edema and speech trauma in vocal nodules. Regarding vocal polyps, the identification of a more predominant etiological factor is more difficultHistological studies have exhaustively tried to differentiate each one of the benign laryngeal lesions; nonetheless, the alterations found are common to the three lesions, without specificity, and there are varied degrees of epithelial hyperplasia, basal membrane thickening, corium edema, inflammatory infiltrate and vessel prolipherationVolic et al.The lack of histological specification among the many laryngeal lesions has led to many ultrastructural studies, aiming at finding structural details in order to differentiate these disorders. Therefore, a marked and constant finding in vocal nodules is the deposit of subepithelial heterogeneous material, rarely seen in other lesions. Other marked ultrastructural findings, however without establishing specificity among the lesions, are the focal areas of basal membrane detachment and interruptions and the widening of cell junctions, causing structural alterations in the desmosomes. All these findings are interpreted by the authors as a response to speech trauma by the epithelium, being clearer in the lesions associated to high vocal demand, as it happens with vocal nodulesIn an immunohistochemical assay, GrayAs we saw, the results from the morphological analysis showed that vocal nodules have peculiar traits which differentiate them from the other lesions, thus being necessary to carry out additional studies involving the other laryngeal lesions so as to better understand its morphological characteristics.This research project was approved by the Ethics in Research with Human Beings Committee of the institution where it was carried out under protocol number 190/06. After its approval, we carried out a retrospective review of the medical charts of all the patients with clinical and endoscopic diagnosis of Reinke's Edema, previously seen at the Voice Disorders Ward of this institution, from 2001 through 2007, and we excluded those charts with incomplete data. Thus, we selected 72 cases of Reinke's Edema, of which 60 had been submitted to surgery. Data review on the charts of these 60 patients revealed that 50 of them were females and 10 were males, nine of them were between 20 and 40 years of age, 44 were between 41 and 60 years and seven had more than 61 years of age. All the patients were chronic smokers and only 12 drank alcoholic beverages frequently. Vocal abuse was noticed in 20 cases, nasosinusal symptoms in 16 and gastroesophageal symptoms in 21 cases.The histology diagnostics were revised; the paraffin blocks of specimens were submitted to new cross-sections and to immunohistochemistry dyes for antibodies against fibronectin, laminin and collagen IV. Normal larynxes of five adult cadavers , without macroscopic lesions were used as controls in the immunohistochemistry study, after making sure these patients were non-smokers and had not been submitted to tracheal intubation.The paraffin blocks of the 60 patients were submitted to 3 micron cross-sections. The specimens were then placed on silanized slides and submitted to immunohistochemical reactions by the Avidin Biotin Peroxidase11 method, using anti-fibronectin polyclonal antibody , monoclonal antibody  and anti-collagen IV monoclonal antibody . After preparing the material, the histology slides were examined under light microscopy , by an experienced pathologist, with different magnifications and image capture by a digital camera. Results were interpreted depending on the degree of color intensity into mild, moderate or intense. The immunoexpression sites were: basal membrane, corium and vessel endothelium. The normal larynxes used as control were submitted to the same immunohistochemical reactions.For histology analysis, the statistical method used was the chi-squared, with a significance level of 5%.Immunohistochemical analysis of the larynxes used as control was very similar in the five fragments of vocal folds assessed, in other words, we identified mild fibronectin pigmentation, especially on the basal membrane , althougOn Reinke's edema is considered benign for most of the authors, classified among exudative lesions on Reinke's space, together with vocal nodules and polypsBecause of the direct relationship between Reinke's edema and chronic smoking, it is important to carefully assess its morphological aspects. In a recently published study, we presented the histological findings and those from electron scanning and transmission microscopies of Reinke's edema, reporting on different levels of epithelial dysplasia and one case of in situ carcinomaThrough immunohistochemical dyes using anti-fibronectin, anti-laminin and anti-collagen IV antibodies, the lamina propria vessels can be well highlighted, since they are in large number. In the present investigation we noticed that the anti-fibronectin antibodies deposit on the basal membrane was not a frequent finding in Reinke's edema. According to Gray et al.Neves et al.Additional studies have shown that besides the morphological alterations shown above, the distribution of collagen fibers seem to be also altered in Reinke's edema. Sakae et al.As we have seen, the many morphological studies about Reinke's edema add information which help unveil the lesion pathophysiology and explain the alterations in the voice quality of these patients. Considering the immunoexpression of the basal membrane fibronectin, laminin and collagen IV did not show revelevance in immunohistochemical studies, these antibodies prevail in the endothelium of the lamina propria vessels."}
+{"text": "Membrane contact sites (MCS) are critical for cellular functions of eukaryotes, as theyenable communication and exchange between organelles. Research over the last decadeunravelled the function and composition of MCS between a variety of organelles includingmitochondria, ER, plasma membrane, lysosomes, lipid droplets, peroxisome and endosome, toname a few. In fact, MCS are found between any pair of organelles studied to date, withcommon functions including lipid exchange, calcium signalling and organelle positioning inthe cell. Work in the past year has started addressing the composition and function ofnuclear-mitochondrial MCS. Tether components mediating these contacts in yeast have beenidentified via comprehensive phenotypic screens, which also revealed a possible linkbetween this contact and phosphatidylcholine metabolism. In human cells, and in theprotozoan parasites causing malaria, proximity between these organelles is proposed topromote cell survival via a mitochondrial retrograde response. These pioneering studiesshould inspire the field to explore what cellular processes depend on the exchange betweenthe nucleus and the mitochondrion, given that they play such central roles in cellbiology. Cellular compartmentalisation into organelles is an essential trait of eukaryotes whichenables multi-level control of critical cellular functions. This role has two faces: on onehand organelles act as a separate biochemical microenvironment that host distinct pathways.On the other hand, communication and exchange between organelles is required to enableshared biosynthetic pathways, inter-organelle signalling and organelle positioning in thecell. These essential interactions between organelles are mediated by the so-called MembraneContact Sites (MCS). Over the past decade the cell biology field had seen a growing focus onthe composition and function of these sites. It seems that MCS function between any twoorganelles studied to date, and in some cases the same organelle pair has different types ofMCS mediated by different tethers and performing different functions. Over the past year anew contact has sprung to the attention of the cell biology field: that of the nucleus andthe mitochondrion (nmMCS) which we review herein.The first MCS tether identified , and likSaccharomyces cerevisiae, which led to the identification of thefirst nmMCS tether  biosynthesis pathway. Thisfinding points to a possible link between the Cnm1 mediated nmMCS and PC metabolism. Insupport of such a link, disruption of the PC biosynthesis pathway affects Cnm1 levels aswell as the extent of the observed nmMCS. Whether this new nmMCS directly regulates PCmetabolism and through what mechanism remains to be studied.The new focus on nmMCS raises the exciting question of what other cellular functions may besupported or controlled through an exchange between those two organelles. Two recent papersdescribe enhanced proximity between mitochondria and nucleus that is linked to stressresistance and cell survival. Mitochondria are known to play an active role in reprogrammingof cells, whereby mitochondrial damage is communicated to the nucleus leading to change ingene transcription. Since the traditional way of thinking about eukaryotic cell biology isthat signals move from the nucleus to the rest of the cell, this mitochondrial to nuclearcommunication is often referred to as mitochondrial retrograde response (MRR). Both studiespostulate a putative role for nmMCS in supporting MRR .The first study hypothesised that nmMCS might catalyse MRR in the context of pro-survivalpathways in cancer cells, via the mediation of cholesterol, reactive oxygen species andcalcium .The autPlasmodiumfalciparum, the causative agent of malaria (Plasmodium too. Importantly, the proximity seen in this studydescribes a distance between the contacting membranes that is larger than most MCS describedto date, and the observed proximity is yet to be analysed by electron microscopy.Furthermore, no tether has been proposed to mediate this contact. Thus, while the proposedfunction for a putative nmMCS in Plasmodium is intriguing, the existence ofa bona fide nmMCS in this organism remains to be fully validated.The second study focuses on the eukaryotic unicellular parasite  malaria . This stin vitro (Finally, a recent study aiming to understand routes of translocation of the mitochondrialpyruvate dehydrogenase complex into the nucleus, pointed to another potential nmMCS . In thisin vitro . These o2O2 which, if it accumulates, is damaging to the cell, andits elimination by degradation and diffusion to the mitochondrial matrix or to the cytoplasmis slower than the rates of its production. It was shown that the calcium uptake atER-mitochondria MCS mediates H2O2 release via aligned cristae junctionat the contact. The suggested mechanism is that H2O2 generated byrespiration in the cristae space, along with calcium uptake, induces a compression of thecristae which forces their volume through the aligned cristae junctions and ER-mitochondrialcontact to the interface between the two organelles (In conclusion, the new focus on nmMCS should inspire the field to explore what cellularfunctions may be served through the exchange between those organelles. The critical role ofmitochondria in controlling cell fate, triggered the three studies summarised above tohypothesise a role in mediating pro-survival and proliferative signalling. Moreover, theauthors of the study performed in cancer cells also raise the important point thatmitochondria-produced reactive oxygen species, whose rate of diffusion in the cytosol isslow, would gain a \u201cfast-track\u201d for nuclear accumulation via the nmMCS. This rationaleprovides further support for a role in retrograde signalling and is in line with how otherMCS work. One example for MCS mediated proximity that enhances the natural mobility rate ofa signal is the case of calcium exchanged between the ER and the mitochondrion at the porinmediated contact . This coganelles . Thus, tToxoplasma gondii, provided evidence that points at aVDAC mediated ER-mitochondrial MCS, however in this organism VDAC depletion has no effect oncalcium homeostasis. Moreover, IP3R is not found in Toxoplasma suggesting adifferent ER partner for this MCS (Trypanosoma brucei,another divergent protozoan found in a different eukaryotic clade to that of mammals andyeast, and to the clade of Toxoplasma, VDAC and IP3R function as tetherbetween the mitochondrion and the acidocalcisome rather than the ER (The identification of tethering components is a critical step in defining MCS, as itprovides means to study function, via genetic manipulation and phenotypic analysis. Theidentity of tethers, or of other proteins that affect the abundance of the MCS, could alsoprovide hints about function, as demonstrated by the Cnm1 study, where involvement of thenmMCS in PC metabolism is exposed as a possibility. Interestingly, Cnm1 is a yeast specificprotein, highlighting a likely case of organism specific MCS. This finding thus adds anexample to a growing body of evidence supporting a dogma whereby MCS are highly divergentbetween different organisms and is in agreement with the hypothesis that MCS evolvedindependently in different lineages . The ER-this MCS . Interesn the ER . Thus, t"}
+{"text": "Tactile processing plays a pivotal role in the early stages of human development; however, little is known about tactile function in young children. An understanding of how tactile processing changes with age from early childhood to adulthood is fundamental in understanding altered tactile experiences in neurodevelopmental disorders, such as autism spectrum disorder.In this cross\u2010sectional study, 142 children and adults aged 3\u201323 years completed a vibrotactile testing battery consisting of 5 tasks, which rely on different cortical and cognitive mechanisms. The battery was designed to be suitable for testing in young children to investigate how tactile processing changes from early childhood to adulthood.Our results suggest a pattern of rapid, age\u2010related changes in tactile processing toward lower discrimination thresholds (lower discrimination thresholds = greater sensitivity) across early childhood, though we acknowledge limitations with cross\u2010sectional data. Differences in the rate of change across tasks were observed, with tactile performance reaching adult\u2010like levels at a younger age on some tasks compared to others.While it is known that early childhood is a period of profound development including tactile processing, our data provides evidence for subtle differences in the developmental rate of the various underlying cortical, physical, and cognitive processes. Further, we are the first to show the feasibility of vibrotactile testing in early childhood (<6 years). The results of this work provide estimates of age\u2010related differences in performance, which could have important implications as a reference for investigating altered tactile processing in developmental disorders. Tactile processing is essential in early child development and continues to develop throughout childhood and adolescence. Here, we examine the development of tactile performance across 5 tasks in 142 children and adults aged 3\u201323 years. All tasks show a pattern of rapid age\u2010related improvement that plateau by adulthood; however, there are pronounced differences in the rate of change. For example, we show a rapid decrease in reaction time (left) while simultaneous amplitude discrimination performance (right) develops more slowly. One of the primary means children use to explore and learn is through tactile experiences Cascio, . While uSensory processing in children has been investigated using various methods. Questionnaire\u2010based assessments\u2014either self\u2010report or parent\u2010report\u2014have been commonly used across a large age range to examine sensory function in healthy and clinical conditions   of somatosensory processing and can be designed to provide insight into multiple cortical functions , and its alterations in clinical disorders.22.1N = 45 recruited), late childhood , adolescence , and adulthood   were delivered pseudo\u2010randomly to the left middle or index finger, and participants were asked to respond by either pressing the spacebar of the Chromebook or a button on a separate wired mouse with the right hand \u201cas quickly as possible\u201d upon feeling a stimulus Figure . For eac2.3.2In the sequential amplitude discrimination (sqAD) task, two stimuli  were delivered sequentially to the two fingers , with one stimulus having a higher amplitude Figure\u00a0. One fin2.3.3Two stimuli  were delivered to the left index and middle finger, separated temporally by a starting ISI of 150 ms . The ISI was decreased by 15% for correct trials and increased by 15% for incorrect trials. Participants were asked to distinguish which finger received the first stimulus Figure\u00a0. The tem2.3.4In the duration discrimination (DD) task, two stimuli  were delivered sequentially to the middle and index finger (ISI: 500 ms), with one finger receiving a longer duration stimulus . The duration of the comparison stimulus was decreased by 25 ms for each correct answer and increased by 25 ms for each incorrect answer. Participants were asked which finger received the longer duration stimulus Figure\u00a0. The DD 2.4Data analysis was performed using custom\u2010written R , MATLAB , and SPSS  routines. Data for an individual task was excluded when it was reported by the experimenter that the task had not been executed properly  and/or visual inspection of the profile of the staircase showed large deviations from the expected profile , as may occur with fatigue or loss of focus.2.52 and then age3 terms were added and evaluated using linear regression. The higher order terms were removed if they did not better explain the data. Results were considered significant if uncorrected p\u2010values were below 0.05. We subsequently modeled data with power function models that do not have any data distribution assumptions to better characterize nonlinear associations with age.Initially, to examine the relationship between age and task performance, linear models were developed and subsequently age2.5.1f(x) = a \u00d7 exp(b \u00d7 x) + c \u00d7 exp(d \u00d7 x)) and power (f(x) = a \u00d7 x^b + c) models.For each task, individual vibrotactile measures were plotted as a function of age in years across groups. The polynomial models computed earlier were tested and compared with linear, exponential, and power models, where the model with the best fit is presented. The best fit for each individual task was determined by squared estimates of error (SSE), R\u2010square, and root mean square error (RMSE) values. The fitting approaches do not have any assumptions about the underlying data distribution. Final data models used were: exponential  under the assumption that the young adults in this study represent stabilized tactile performance development. A task performance correlation matrix was also formed for participants <18 years. To investigate the developmental trajectory of these task performance correlations, pairs of tasks were summarized as a ratio  and plotted against age. For the task pairs, RT was paired with all other tasks  and then related tasks (amplitude discrimination tasks sqAD/smAD and timing\u2010related tasks TOJ/DD) were also visualized. These age relationships were assessed with linear regression.33.1Compared to older participants, fewer participants in the early childhood data collection group were able to perform each task Figure\u00a0. The lowN = 8 excluded ; sqAD: N = 2 excluded (ages 3 and 5 years); TOJ: N = 4 excluded ; DD: N = 5 excluded . Table\u00a0The data from three 3\u2010year\u2010old participants were completely excluded as they were judged by the experimenter to not have understood any of the vibrotactile tasks. Additional data exclusions by task are as follows: RT: As can be seen from Figure\u00a03.22, and age3 terms , while smAD was best modeled with only a linear age term and TOJ was best modeled with age and age2 terms. The final linear model and goodness of fit measures are summarized in Table\u00a0All tasks showed a significant relationship in performance with age. Most tasks were best explained with by models including age, age3.3Power and exponential models showed the best fits of the age versus performance for each task Figure\u00a0. A power3.3.1r = 0.45, p = 0.004, see Figure\u00a0The correlation matrix of task performance in the adult subgroup showed no tasks to be correlated with the exception of sqAD and TOJ, 4In the present study, we explored age\u2010related changes in various measures of tactile processing from early childhood to adulthood using a vibrotactile testing battery that includes five different tasks. Although vibrotactile testing has been extensively used in older children and adults , with rapid changes occurring across early childhood, suggesting that this is an important period for tactile processing development. For some tasks, such as sqAD and smAD as well as DD, tactile performance reaches adult\u2010like levels in late childhood (ages 8\u201310 years). For other tasks, such as reaction time (RT) and TOJ, performance continues to improve until later in adolescence (ages 15\u201317 years). The pattern of rapid changes in vibrotactile thresholds during early childhood is likely a reflection of the profound brain development that occurs during this period  with age to reach adult\u2010like levels during late childhood. The same pattern was observed in reaction time variability. This pattern is consistent with the proposed U\u2010shaped relationship between age and reaction time across the lifespan; increases in age throughout childhood are associated with decreases in reaction time and its variability, while increases in age throughout adulthood are associated with greater reaction time and its variability , rapid improvement (decrease) in discrimination thresholds was observed, with adult\u2010like levels reached in late childhood. On average, smAD thresholds were higher than sqAD thresholds, which can be explained by the greater difficulty of the former task. GABAergic lateral inhibition has been shown to play a pivotal role in separating tactile stimuli . GABA mediates lateral and feed\u2010forward inhibition . As such there is a common working memory task in these two perceptual tasks. Interestingly, the task correlation matrix including the participants under 18 showed multiple correlated measures. When examining how age affects the relationships between paired tasks, a couple age\u2010related associations of paired task performance were found. RT/RTVar showed a strong age\u2010relationship; however, these two measures are from the same task and thus are interrelated. RT/TOJ also showed age\u2010related changes, but this was a weak effect, and the significance would not survive correction for multiple comparisons. We therefore suggest that task performance correlations that are seen when pooling across age actually reflect the rapid task performance improvements across childhood and not that relationships in task performance deteriorates with age.Multiple factors that may affect performance across discrimination tasks and particularly relevant in this developmental study are working memory, attention, and the development of related brain networks. For example, the frontoparietal and salience networks are engaged by demanding tasks and develop profoundly throughout childhood and adolescence. As such, working memory also matures across this age range . This may assist to define therapeutic strategies that are targeted by developmental stage. Future investigations of tactile processing in early childhood with neuroimaging and longitudinal assessments could also provide new insight into the neural basis of these age\u2010related effects and alterations in clinical disorders.Mark Tommerdahl is co\u2010founder of Cortical Metrics, LLC. Cortical Metrics is licensed by the University of North Carolina to distribute the tactile stimulator used in this study.https://publons.com/publon/10.1002/brb3.2644The peer review history for this article is available at Supporting informationClick here for additional data file."}
+{"text": "Moreover, 88% of the patients showed a pain reduction of at least 10 points  from pre- to post-treatment. Our retrospective study using the in-office procedure of ultrasound-guided combination of intra-articular and intraosseous infiltrations of PRGF is a safe and efficacious approach for the treatment of grade 3\u20134 knee osteoarthritis.The aim of this study was to explore and assess office-based ultrasound-guided intraosseous and intra-articular infiltrations of plasma rich in growth factors (PRGF) in patients with moderate and severe knee osteoarthritis (KOA). Seventy-nine patients (30 women and 49 men) with grade 3\u20134 KOA according to the Kellgren\u2013Lawrence classification participated in the study. All patients were treated with a minimally invasive technique using local anesthesia WALANT  in the ambulatory setting. A PRGF intra-articular infiltration and two intraosseous infiltrations in the tibial plateau and femoral condyle were performed weekly for a total of three sessions. The evaluation of the results was carried out using knee injury and osteoarthritis outcome score (KOOS) at baseline and post-treatment. After a follow-up period of 11 months (median) , all the KOOS domains showed statistically significant improvement ( Knee osteoarthritis (KOA) is a painful, chronic low-grade inflammatory disease in which the whole synovial joint tissue homeostasis is dysregulated , with thRecently, mechanism-oriented therapeutic products and delivery strategies have emerged in the field of regenerative pain medicine to treat KOA pain and disabilities, among which autologous blood-derived products  are deliIn line with this, a growing number of clinical studies have demonstrated favourable pain relief and improvement in the quality of life using a combination of intra-articular and intraosseous infiltrations of platelet-rich plasma (PRP) for the treatment of moderate and severe KOA ,23,24,25The aim of this study was to explore and assess this office-based ultrasound-guided intraosseous and intra-articular infiltrations of plasma rich in growth factors (PRGF) treatment in patients with moderate and severe KOA using the baseline and post-treatment knee injury and osteoarthritis outcome score (KOOS) as a means of evaluating the therapy.Thus, the hypothesis of our study was that intraosseous and intra-articular infiltrations of PRGF improve pain and quality of life in patients with KOA on a clinically relevant basis.This retrospective study was reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines  27]. Th. Th27]. The inclusion criteria for the study were as follows: (1) patients over 18 years old from both sexes; (2) patients diagnosed with grades 3\u20134 KOA according to the Kellgren\u2013Lawrence classification; (3) patients treated with office-based intraosseous and intra-articular infiltrations of PRGF; (4) patients who completed the KOOS (knee injury and osteoarthritis outcome score) evaluation scale prior to PRGF intervention and at the end of the follow-up; and (5) a minimum follow-up of six months. The absence of complete data on the variables to be studied was the exclusion criterion.PRP was prepared according to PRGF-Endoret technology . Briefly, peripheral venous blood was withdrawn into eight 9-mL collection tubes containing 0.4 mL of 3.8% (wt/vol) trisodium sodium citrate as an anticoagulant. Next, the blood was centrifuged at 580 g for 8 min at room temperature. The upper volume of the plasma was discarded. The 2 mL plasma fraction (F2), located just above the sediment red blood cells but not including the buffy coat, was collected. PRGF (F2) has a moderate enrichment of platelets and is free of leukocytes and erythrocytes, so it can be classified as pure PRP (P-PRP). Based on the aforementioned features of PRGF, and in accordance with the latest coding system , PRGF isThe office-based intraosseous PRGF infiltration procedure was recently described by Rios et al. . The entThe complete treatment, namely, the intra-articular infiltration and the two intraosseous infiltrations in the tibial plateau and femoral condyle, was performed weekly for a total of three sessions.The evaluation of the results was carried out using patient-reported outcomes. We used the validated KOOS (knee injury and osteoarthritis outcome score) scale, which was developed in the nineties and first published in 1998 by Ross et al. . It is cThe safety of the procedure was also evaluated. All complications and adverse events were also gathered from the medical records of the patients.p < 0.05. Statistical analyses were performed using GraphPad Prism, version 9 .Descriptive data were presented as frequencies and percentages. The results of the questionnaires were presented as medians (interquartile range (IQR)). All data were tested for normality using the Shapiro\u2013Wilk test. The box-and-whisker plots were generated following the Tukey style: the boxes show the median and the IQR, while the whiskers indicate the 25th percentile-1.5 \u00d7 the IQR and the 75th percentile-1.5 \u00d7 IQR. The round points outside the whisker range indicate the outliers . The WilThis study included 79 patients (30 women and 49 men), and 85 knees were treated with three weekly sessions of a combination of three intraosseous and intra-articular infiltrations of PRGF in the ambulatory setting assisted with the WALANT technique. The severity of KOA assessed by the Kellgren\u2013Lawrence OA grade was grade 3 in 38 patients (44.7%) and grade 4 in 47 patients (55.3%). The median follow-up period was 11 months, with an interquartile range of 7\u201314 months. The baseline demographic characteristics of patients who participated in this study are shown in p < 0.001), symptoms (p < 0.001), activities of daily living (p < 0.001), sports/recreational activities (p < 0.001), and quality of life (p < 0.001) according to the KOOS score  and articular cartilage, two tissues that are sometimes inefficiently targeted by systemic drug delivery. Moreover, the intra-articular route circumvents systemic toxicity and its side effects, presents an excellent bioavailability, and does not entail molecular size limitation, in contrast to the systemically delivered molecules entering the joint via the capillaries of the subsynovium ,41. PRGFImportantly, this in-office procedure of intraosseous infiltrations with PRGF represents a step forward in the cost-effectiveness of the treatment of knee osteoarthritis that healthcare systems and surgeons should take into account as a significant therapeutic tool to safely treat moderate and severe KOA ,71,72.However, this study presents several limitations. The first limitation is inherent in the study design, as this was a retrospective study based on the usual clinical practice. Another limitation was the mid-term follow-up of 11 months. In order to consider this treatment as a disease-modifying approach, the follow-up should have been longer and time-homogeneous for all the patients participating in the study. However, it was reported that bone marrow lesions disappeared after this treatment ,36, and In summary, considering the limitations of this retrospective study, this study shows that intra-articular and intraosseous PRGF infiltrations performed in an office setting are safe and effective in the treatment of moderate-to-severe KOA. Further randomized clinical trials are needed to confirm the data obtained in the present usual clinical practice study."}
+{"text": "In this study, we investigated the potential of electrochemical skin conductance (ESC) measurements gathered from home-based devices to detect circadian-like patterns. We analyzed data from 43,284 individuals using the Withings Body Comp or Body Scan scales, which provide ESC measurements. Our results highlighted a circadian pattern of ESC values across different age groups and countries. Our findings suggest that home-based ESC measurements could be used to evaluate circadian rhythm disorders associated with neuropathies and contribute to a better understanding of their pathophysiology. However, further controlled studies are needed to confirm these results. This study highlights the potential of digital health devices to generate new scientific and medical knowledge. The digital revolution has transformed the medical profession in numerous ways. It has introduced new concepts such as virtual reality  and has Withings  recentlyIntegrating ESC measurement into a connected scale gave us access to a wide range of real-world data at a scale that had never been reached before. This integration, along with previous research , 31, ledPositive results would motivate us to set up a clinical study to have a better understanding of the individual profiles of ESC throughout the day. It would also help us to compare normal profiles vs. neuropathic profiles to understand how SFN may affect circadian patterns of ESC and allow us to integrate chronobiology as a biomarker if associated with any condition.To build our dataset, we selected a subset of users of the Withings Body Comp and Body Scan scales, which are the devices providing the ESC measurements. We selected only patients aged 18\u2009years and over with a minimal weight of 30\u2009kg. The minimal weight is a threshold to exclude measures taken by young children using adult accounts. Then, we summarized our measurements at the minute level, meaning that if one individual performed several measurements within the same minute, then all the results were regrouped as one average. Returned, defective, and factory QA samples were also removed to avoid technical outliers.The conditions we applied for data selection led us to keep 1,905,580 measures from 43,284 individuals, including 26,915 men and 16,369 women, from Germany\u2014DE (34%), United States\u2014US (25%), France\u2014FR (21%), United Kingdom\u2013GB (11%), Switzerland\u2014CH (6.8%), and Austria\u2014AT (3%). The average age of our population was 46.8 (13.9). Details can be found in The overall values of ESC are presented in Electrochemical skin conductance (ESC) is a physiological measurement that can reflect the conductance of the sweat glands in the skin. The Withings Body Scan and Body Comp scales are non-invasive medical devices designed to be used at home daily to assess sweat gland function and diagnose autonomic neuropathies. They measure the electrochemical conductance of the feet, providing insights into the integrity of the autonomic nervous system. They have multiple functions (weight and pulse wave velocity), are approved by the U.S. Food and Drug Administration , and have medical CE approval (ECM22MDR001). Data were collected by our application and then sent to a specific medical cloud in accordance with the legal constraints of each country.The technique is based on the principles of reverse iontophoresis and chronoamperometry, similar to Sudoscan . The devThis approach distinguishes itself from traditional electro dermal activity (EDA) measures such as skin conductance response (SCR) , 36 by dt-test, while circadian patterns were checked using the Circacompare V0.1.1 package , generative AI has started to become an important tool for various research jobs. To follow the ethical guidelines about that subject, we will state how we used it here. In our case, we used gpt-4  to writeThe following paragraph is the text that was given to ChatGPT to generate the abstract after feeding it the full publication (excluding the abstract):I want you to act as a lead scientist working in neuropathies medical research. Your team wrote a publication and they need you to write the final abstract. I\u2019m going to give the full publication. There are no figures and tables, including only their associated legends. The abstract should be less than 300 words and understandably summarize the work.\u201d\u201cData were collected within the device\u2019s intended use without any action from Withings toward the individuals; thus, our study is neither interventional nor clinical. No individual or medical data from specific individuals was presented in this study, and all the data were aggregated, making them non-identifiable. For the data presented individually, we did not provide the age, gender, or data localization, making them also unidentifiable.We first checked how the data were globally distributed within the day . Then, wThen, we checked the pattern of the median value throughout the day for all the data  and throp\u2009<\u20092.2e\u221216) and then performed a t-test. The results are shown and plotted in Next, we decided to split the ESC values by time categories to test whether there were differences between the daily values and the other time categories. We defined the time categories as 0\u201310\u2009h (night-morning), 10\u201319\u2009h (day), and 19\u201324\u2009h (evening). We checked the normality of the data within our time categories  with a specific set of parameters:Finally, we checked if our median values could be modeled by a cosine function (circadian pattern-like), and the results were positive , and the period is 24\u2009h. The results can be checked in where user 1 and user 2) who did that at the moment of data collection. They are illustrated in Following the global perspective, we decided to check on specific individuals who took many measures per day for several days and not around the same hour. As we worked directly on individual data, we did not have the luxury of getting many of them using the scale multiple times a day. Indeed, we only found two individuals .The data analyzed in this study is subject to the following licenses/restrictions: data are available only for research purposes. Datasets are available on demand through our API (Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants\u2019 legal guardians/next of kin in accordance with the national legislation and the institutional requirements.BV processed the data, created the graphics, and wrote the main part of the publication. VC, R-YY, PV, ST, SM, and PG provided corrections and meaningful insights and polished the writing. All authors contributed to the article and approved the submitted version."}
+{"text": "The view that bone and energy metabolism are integrated by common regulatory mechanisms is broadly accepted and supported by multiple strands of evidence. This includes the well-characterized role of the PPAR\u03b3 nuclear receptor, which is a common denominator in energy metabolism and bone metabolism. Little is known, however, about the role of PPAR\u03b1 nuclear receptor, a major regulator of lipid metabolism in other organs, in bone.KO) and mice with osteocyte-specific PPAR\u03b1 deficiency (\u03b1OTKO) in order to parse out the various activities of PPAR\u03b1 in the skeleton that are of local and systemic significance. This study included transcriptome analysis of PPAR\u03b1-deficient osteocytes, and analyses of bone mass and bone microarchitecture, systemic energy metabolism with indirect calorimetry, and differentiation potential of hematopoietic and mesenchymal bone cell progenitors. These analyses were paired with in vitro studies of either intact or silenced for PPAR\u03b1 MLO-A5 cells to determine PPAR\u03b1 role in osteocyte bioenergetics.A side-by-side comparative study of 5-15 mo old mice with global PPAR\u03b1 deficiency  is age-dependent. In younger mice, osteocyte metabolism contributes positively to global energetics, however, with aging the high-energy phenotype reverts to a low-energy phenotype and obesity develops, suggesting a longitudinal negative effect of impaired lipid metabolism and mitochondrial dysfunction in osteocytes deficient in PPAR\u03b1. However, bone phenotype was not affected in \u03b1OTKO mice except in the form of an increased volume of marrow adipose tissue in males. In contrast, global PPAR\u03b1 deficiency in \u03b1KO mice led to enlarged bone diameter with a proportional increase in number of trabeculae and enlarged marrow cavities; it also altered differentiation of hematopoietic and mesenchymal marrow cells toward osteoclast, osteoblast and adipocyte lineages, respectively.In osteocytes, PPAR\u03b1 controls large number of transcripts coding for signaling and secreted proteins which may regulate bone microenvironment and peripheral fat metabolism. In addition, PPAR\u03b1 in osteocytes controls their bioenergetics and mitochondrial response to stress, which constitutes up to 40% of total PPAR\u03b1 contribution to the global energy metabolism. Similarly to \u03b1PPAR\u03b1 role in bone is multileveled and complex. In osteocytes, PPAR\u03b1 controls the bioenergetics of these cells, which significantly contributes to systemic energy metabolism and their endocrine/paracrine function in controlling marrow adiposity and peripheral fat metabolism. PPARs are also responsible for regulation of glucose and fatty acid metabolism, with PPAR\u03b3 being a prominent target for TZDs, antidiabetic drugs, and insulin sensitizers, whereas PPAR\u03b1 is a pharmacologic target for fibrates, a class of drugs used to treat dyslipidemia. The third member of this family, PPAR\u03b2/\u03b4 is emerging as an essential factor in regulation of energy metabolism in muscles, and therapies to target this nuclear receptor to combat sarcopenia are in development. All three PPARs are expressed in bone cells and, by a virtue of the fact that bone and energy metabolism are inherently linked, they may play an important role in regulation of bone homeostasis.While the role of PPAR\u03b3 in regulation of bone homeostasis has been extensively studied in animals and humans, the role of PPAR\u03b1 in bone has not been systematically studied and available findings are not conclusive. Studies by Wu et\u00a0al. did report observations of enlarged marrow cavities in PPAR\u03b1-null mice; however, differentiation of bone marrow stroma cells toward osteoblasts or adipocytes was not found to be affected . ClinicaThe PPAR\u03b1 nuclear receptor monitors intracellular non-esterified fatty acid (NEFA) levels, which upon binding activate its transcriptional activities regulating expression of a number of genes involved in lipid metabolism and storage. PPAR\u03b1 is a major regulator of energy production in the process of fatty acid \u03b2-oxidation. It is highly expressed in metabolically active tissues such as those of the liver, heart, and muscle, which rely on fatty acid catabolism to fulfill energy requirements. In the liver, PPAR\u03b1 promotes fatty acid uptake and ketogenesis by regulating expression of genes involved in clearance of very low-density lipoprotein, fatty acid activation and transport into the mitochondria, and peroxisomal and mitochondrial \u03b2-oxidation, as well as several genes coding for enzymes in the mitochondrial respiration chain , 6. The It is well documented that bone metabolism relies on glucose, which provides energy to osteoblasts in the process of aerobic glycolysis . HoweverHere, we present a study on the role of the PPAR\u03b1 nuclear receptor in bone and the first piece of evidence for an essential contribution of osteocytes, under PPAR\u03b1 control, to systemic energy metabolism and the development of bone marrow adipose tissue.22.1KO mouse model, PPAR\u03b1 was deleted specifically in osteocytes using the Cre-Lox system. 10kb DMP1cre mice (B6N.FVB-Tg (Dmp1-cre)1Jqfe/BwdJ), in which Cre expression is driven by the DMP1 promoter in osteocytes, were obtained from the Jackson Laboratory . PPAR\u03b1fl/fl mice, which have loxP nucleotide sequences flanking exon 4 of PPAR\u03b1, were obtained from the University of Toulouse, France  consisted of mice with either the PPAR\u03b1fl/fl or the DMP1-Cre genotype, after it was established that these two parental strains did not differ in analyzed parameters relating to bone and metabolic phenotype. Global PPAR\u03b1 KO (PPAR\u03b1 \u2212/\u2212 129S4/SvJae-PPAR\u03b1 tm1Gonz/J) and adequate WT mice were purchased from the Jackson Laboratory. In PPAR\u03b1 KO (\u03b1KO) animals, NeoR cassette replaced 83 bp of coding sequence in exon 8.To generate the \u03b1OT, France . Mice frKO and \u03b1OTKO mice were maintained at the University of Toledo Health Science Campus. All animals were housed under a 12-h dark\u2013light cycle and had free access to standard chow . Animal treatment and care protocols conforming to the National Institutes of Health Guidelines were followed under the University of Toledo Institutional Animal Care and Utilization Committee protocol. All presented experiments were performed on 5- to 15-month-old skeletally mature male and female mice.The colonies of \u03b12.2Microcomputed tomography (micro-CT) of the tibiae was performed using the \u03bcCT-35 system , as previously described . BrieflyFor lipid evaluation, decalcified bone specimens were stained for 1 h in solution containing 2% osmium tetroxide prepared in 0.1 M sodium cacodylate buffer, pH 7.4, according to the protocol . Stainin2.3To enable dynamic bone histomorphometry, mice were intraperitoneally injected with 20 mg/kg calcein (Sigma) 8 and 2 days before sacrifice, and undecalcified tibiae were fixed in 70% ethanol, embedded in methyl methacrylate, and sectioned. Images were recorded using a Zeiss Axiovert 40 CFL fluorescence microscope  equipped with a Micropublisher 3.3 Megapixel Cooled CCD color digital camera . Analysis of BFR was confined to the secondary spongiosa of the proximal tibia and was performed using the Nikon NIS-Elements BR3.1 system. The measurements were collected with a 40\u00d7 objective lens  from 6 representative fields per bone sample. The terminology and units employed were those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research . For sta2.4Genomic DNA samples were isolated from bone, liver, muscle, iWAT, and gWAT using Tri Reagent  and according to the protocol provided. 0.25 \u00b5g of genomic DNA was used for PCR amplification with a thermocycling protocol consisting of 3 min at 94\u00b0C, 35 cycles of 30 s at 94\u00b0C, 1 min at 50\u00b0C, and 1 min at 72\u00b0C. As in a previous study performed by Montagner et\u00a0al.  to detec2.5via infrared scanning using the Odyssey system (LI-COR Biosciences). Antibodies against PPAR\u03b1 (sc-1982) and Hsp90 (sc-8262) were obtained from Santa Cruz Biotechnology .Proteins isolated from femoral cortical bone using Tri Reagent were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 1 h in TBS  containing 3% BSA, then incubated with primary antibody overnight at 4\u00b0C. After 3 washes in TBST (TBS plus 0.1% Tween 20), membranes were incubated with infrared anti-rabbit , anti-goat , or anti-mouse  secondary antibodies  at 1:10,000 dilution in TBS for 2 h at 4\u00b0C. Immunoreactivity was visualized and quantified 2.65/cm2 onto a 48-well plate with the medium supplemented with M-CSF (50 ng/ml) and RANKL (50 ng/ml) . After 6 days of growth, cultures were stained for TRAP5b using the Leukocyte Acid Phosphatase (TRAP+) kit (Sigma)  cultures were established from femur marrow aspirates and differentiated as described . In brie (Sigma) , 28.2.7via sequential collagenase digestion of femoral bone according to a previously described protocol  and processed with the StepOne Plus System . The thermocycling protocol consisted of 10 min at 95\u00b0C, 40 cycles of 15 s at 95\u00b0C, 30 s at 60\u00b0C, and 20 s at 72\u00b0C, followed by a melting curve step at temperatures ranging from 60 to 95\u00b0C to ensure product specificity. Relative gene expression was measured via the comparative CT method, using 18S RNA levels for normalization. Primers were designed using Primer-BLAST  and are listed in Half microgram of the total RNA (by \u03bcg) was converted to cDNA using the Verso cDNA synthesis kit . PCR amplification of the cDNA was performed 2.9KO animals according to the protocol described in section 2.7 were plated on collagen-coated 100 mm plates supplemented with 5% FBS, 5% CS, and 1% PS media. After 6 days of growth, old media were replaced with fresh media. On 10th day, osteocytes were collected via scraping, while the media enriched with osteocyte secretome (conditioned media) were used to grow WT BMSC for analysis of their pro-adipocytic effect in recipient cells. From donor osteocytes, RNA was extracted using the Qiagen RNeasy mini kit as per the manufacturer\u2019s recommended protocol. The samples were processed and sequenced using a NextSeq 500 instrument at Scripps Research Institute, Jupiter, Florida. The overall quality of the reads was high, with no decrease over the course of sequencing. Principle Component Analysis (PCA) of the top 500 genes differentially expressed in the experimental groups showed biological variation between groups. Differentially expressed transcripts (p<0.05) were identified through pairwise comparison of samples from both groups using R version 3.4.0 and deseq2 version 1.16.1. To reduce false positives, the more conservative threshold of adjusted p-value < 0.05 was applied (padj). Analysis of the raw data acquired from NGS in terms of differentially expressed transcripts considered base mean (transcript number), fold change (relative expression), and raw and adjusted p-values (significance). Raw data files are deposited in the NCBI Gene Expression Omnibus (GEO) repository, submission number: GSE225595. DAVID  Bioinformatic Resources 6.8 was used for functional annotation and gene functional classification of up- or downregulated genes.Osteocytic fractions isolated from femora of 6-month-old WT or \u03b12.10Ppar\u03b1 siRNA (Santa Cruz Biotechnology), with DsiRNA NC1  as a negative control. RNA oligonucleotides were delivered to MLO-A5 cells using the X-treme Gene siRNA Transfection Reagent  according to a protocol provided by the manufacturer. \u03b1A5KD cells and their scrambled (Scrl) control were used for assays 36 h after transfection.PPAR\u03b1 silencing was achieved using 2.11KD cells and their scrambled controls were plated 6 h before the assay onto Seahorse cell culture microplates . The four corner wells of the plate were used as negative controls. The assay medium consisted of Seahorse XF base medium without phenol red , supplemented with glucose (25 mM), sodium pyruvate (1 mM), and glutamine (2 mM). Oligomycin (1 \u00b5M), FCCP (2 \u00b5M), and rotenone/antimycin (0.9 \u00b5M) were used as stressors . Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at basal level and after addition of different stressors using a Seahorse XFe96 analyzer (Agilent Technologies). Assay operation software Wave version 2.6.1 was used to run the assay and collect the data. For normalization, live cells stained with Hoechst 33342   were counted using a Cytation 5 plate reader (BioTek Instruments - Agilent). Values obtained from the Mito Stress test were normalized per 1,000 cells.For Mito Stress testing, \u03b1A52.12Mice were evaluated in calorimetric cages  for 4 days with free access to food and water. The mice were housed individually under controlled room temperature and humidity, and exposed to an alternating cycle of 12 h of light followed by 12 h of dark. After adaptation for 1 day, measures of O2 consumption (VO2), CO2 production (VCO2), respiratory exchange ratio (RER), heat production, and physical activity in the horizontal and vertical planes were collected for 3 consecutive days to determine energy expenditure. These data were obtained for each mouse, grouped according to genotype, and analyzed on an hourly basis or according to light vs. dark portion of the cycle. Respiratory exchange ratios (RERs) were calculated as the ratio of CO2 production to VO2 consumption. Total food and water consumption was measured during the experimental period.2.13Data are presented in the form mean \u00b1 SD and were analyzed using the statistical analysis software GraphPad Prism 9. Parametric, unpaired, two-tailed Student\u2019s t-tests were conducted to compare means between two groups. A p-value of 0.05 or lower was considered to represent statistical significance.33.1Ppar\u03b1 mRNA expression in osteocytes isolated from murine femora was at a level similar to that observed in soleus muscle but much lower than that observed to liver tissue and eWAT  indicated 56 upregulated and 87 downregulated transcripts in PPAR\u03b1-deficient osteocytes (Eno1b) ; sucrose transport solute carrier (Slc45a4) ; Rac family small GTPase 2 (Rac2), which may augment production of reactive oxygen species ; and mitochondria-localized glutamic acid-rich protein (Mgarp), which is involved in maintenance of mitochondrial abundance and morphology . Among the most significantly downregulated and potentially most relevant were transcripts for protein promoting activation of AKT1 (Dynap) ; cell adhesion protein (Megf1) ; and several transcripts coding for proteins involved in neuron development, such as chimerin 1 (Chn1) , semaphorin 3 (Sema3d) , and neurotransmitter receptor for postsynaptic specialization (Rapgef4) . The downregulation of transcripts coding for neuronal proteins is of interest, because osteocytes express large number of such transcripts  were obtained as a result of breeding 10kb Dmp1-Cre and PPAR\u03b1flfl mice, as described in the Materials and Methods section. To determine specificity of deletion, sequences encompassing exon 4 were amplified from genomic DNA collected from bone and other tissues using previously described primers  that, in addition to the abovementioned tissues, Ppar\u03b3 expression is not affected in the cerebellum, small intestine, or kidneys of adult male mice [Ppar\u03b1 expression may also not be significantly affected in these tissues of mature \u03b1OTKO mice.Mice with osteocyte-specific deletion of PPAR\u03b1  staining for lipids indicated increased bone marrow adipose tissue (BMAT) volume in the \u03b1OTKO tibia, specifically in the proximal part of the male tibia  was affected , with a concomitant increase in number of trabeculae (TbN), and a tendency toward increase in trabecular bone volume (BV), but no difference in trabecular bone mass (BV/TV)  but no changes in mineral apposition rate (MAR) or bone formation rate (BFR)  cultures derived from \u03b1KO and \u03b1OTKO animals. As shown in KO BMSCs (in contrast with BMSC cultures from \u03b1OTKO mice) also showed higher number of CFU-OB colonies, which was correlated with increased expression of osteoblast markers, such as Dlx5, Osx, and Ocn in undifferentiated conditions  toward osteoblasts and adipocytes was tested in colony-forming unit assays measuring the number of fibroblast (CFU-F), osteoblast (CFU-OB), and adipocyte (CFU-AD) colonies formed KO mice exhibited 4-fold higher CFU-AD formation as compared to WT BMSCs, while CFU-AD formation in \u03b1OTKO BMSCs was increased only by 40%. In addition, expression of adipocyte-specific gene markers was significantly increased in undifferentiated \u03b1KO BMSCs, whereas expression of these markers did not differ from controls in the case of undifferentiated \u03b1OTKO BMSCs  collected from primary cultures of PPAR\u03b1-deficient osteocytes on differentiation of WT BMSCs. As shown in Ucp1 and Prdm16). This response points to PPAR\u03b1-dependent osteocyte paracrine activities regulating SSC allocation to adipocytes, and correlates with the higher volume of BMAT in the proximal tibia of \u03b1OTKO male mice.Notably, BMSCs derived from \u03b1KO mice . Increased hematopoietic cell commitment, together with increased Rankl expression in \u03b1KO BMSCs, is consistent with the increased number of osteoclasts and enlarged marrow cavities in bone tissue of \u03b1KO mice.Analyses of bone structure and histomorphometry suggested increased bone resorption in \u03b1KO mice Figure\u00a033.4KO and \u03b1OTKO mice was measured by indirect calorimetry using the CLAMS system  and carbon dioxide (CO2) production were increased during the daytime and at night, compared to their respective controls. These shifts resulted in an increase in respiratory exchange ratio (RER), indicating increased use of glucose as fuel, and were accompanied by an increase in heat production  (KD cells. We did not observe differences in extracellular acidification rate (ECAR) between \u03b1A5KD and a scrambled control, indicating no effect on anaerobic glycolysis or lactate production  or unchanged (\u03b1KO) food consumption  repository, accession number GSE225595.The animal study was reviewed and approved by University of Toledo Institutional Animal Care and Utilization Committee.AC and BL-C designed the experiments; AC conducted the experiments and analyzed the data, including transcriptomic analysis; SB, PC, and EC contributed to the experimental design, data analysis, data interpretation, and editing of manuscript; MC and PG contributed to transcriptomic data analysis; and AC and BL-C wrote the manuscript. All authors take responsibility for the integrity of the data analysis. All authors contributed to the article and approved the submitted version."}
+{"text": "Nicotinamide phosphoribosyltransferase (NAMPT) plays a central role in mammalian cell metabolism by contributing to nicotinamide adenine dinucleotide biosynthesis. However, NAMPT activity is not limited to the intracellular compartment, as once secreted, the protein accomplishes diverse functions in the extracellular space. Extracellular NAMPT  has been shown to possess adipocytokine, pro-inflammatory, and pro-angiogenic activities. Numerous studies have reported the association between elevated levels of circulating eNAMPT and various inflammatory and metabolic disorders such as obesity, diabetes, atherosclerosis, arthritis, inflammatory bowel disease, lung injury and cancer. In this review, we summarize the current state of knowledge on eNAMPT biology, proposed roles in disease pathogenesis, and its potential as a disease biomarker. We also briefly discuss the emerging therapeutic approaches for eNAMPT inhibition. NMN is onserved \u20138, and o., birds ], but ev., birds  and bact., birds . The NAM., birds , 12. Som., birds \u201315. In a., birds .As mentioned above, NAMPT is not only found inside cells and EVs, but can also be directly secreted into the extracellular space. Historically, diverse names were attributed to extracellular NAMPT (eNAMPT), as its diverse functions were being characterized. eNAMPT was first identified as a cytokine secreted by pre-B cells and named \u201cpre-B-cell colony enhancing factor\u201d (PBEF) due to its ability to synergize with interleukin (IL)-7 and stem cell factor (SCF) in promoting pre-B-cell colony formation . eNAMPT 2As mentioned above, NAMPT is released by a wide range of cell types, including adipocytes, \u03b2-cells, immune cells, neurons, endothelial cells, cardiomyocytes and cancer cells . NAMPT NAMPT gene sequence , but the publication was eventually retracted due to a lack of reproducibility of eNAMPT to IR binding experiments  \u201361, the  in vivo , 66. eNA in vivo , 68. Stu in vivo  , as well as VEGF receptor 2 expression in endothelial and amniotic epithelial cells via the activation of MAPK and phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathways . In 2015, Camp et\u00a0al. revealed that eNAMPT activates the NF-\u03baB pathway by interacting with TLR4 . Consisttivation . Interestivation . Gasparrtivation ). Using tivation , who shotivation .., Il6, Il1b, Cox2, and Tnf), independently of TLR4  , 132, ty5.1.35.1.3.1ApoE knockout atherosclerotic mice  , 151. eN5.2.2Several studies have reported significantly elevated eNAMPT levels in the serum or synovial fluid of OA patients see , 164\u20131675.35.3.1As in OA, elevated eNAMPT levels may contribute to the pathogenesis of RA, an autoimmune disease which is also characterized by joint damage. However, as compared to OA, human RA tissues exhibit higher serum and synovial fluid concentrations of eNAMPT, probably reflecting a higher-grade inflammatory state of the latter , 181. Ac5.3.2A growing body of research suggests that eNAMPT may be a useful biomarker for various autoimmune diseases characterized by chronic inflammation, in particular rheumatoid arthritis (RA) , inflammAs pointed out above, eNAMPT levels are high in RA  and Crohn\u2019s disease (CD) [5.55.5.1In vivo studies show that eNAMPT acts as a leucocyte chemoattractant increasing polymorphonuclear leucocyte (PMN) counts and chemokine levels in bronchoalveolar lavage (BAL) fluid , acute respiratory distress syndrome , and ventilator-induced lung injury (VILI). Neutralization of eNAMPT with an antibody was shown to attenuate inflammatory lung injury in preclinical mouse, rat, and porcine ARDS/VILI models \u2013207 and L) fluid , 208. CoL) fluid . In linefunction . Finallyfunction , 206.5.5.2Circulating levels of eNAMPT are increased in patients with ALI, ARDS [see 5.65.6.1in vitro, e.g., of breast cancer  tin vitro  and cancin vitro .Importantly, eNAMPT may contribute to cancer progression by fostering immunosuppression in the tumor micro-environment. For instance, studies reported that eNAMPT induced the polarization of macrophages towards the pro-tumorigenic M2 phenotype , 71. eNAin vitro studies suggest that eNAMPT could mediate resistance to cancer therapies. A recent study revealed that eNAMPT, by boosting thymidylate synthase expression in cancer cells, reduced the sensitivity of CRC to capecitabine  lesions had significantly higher plasma eNAMPT concentrations than patients with PD-L1\u2212 lesions, hinting at a link between eNAMPT and the tumor-associated inflammatory response  are available , 250. Amin vitro and in vivo studies already explored this therapeutic strategy  . A mecha in vivo , 262. On in vivo . Strikin in vivo . Notwith7Numerous clinical studies, mostly published in the past decade, have reported a correlation between elevated levels of circulating eNAMPT and diverse metabolic and inflammatory disorders, presenting physicians with an opportunity of using eNAMPT levels as a disease activity indicator, prognostic biomarker, or even as a patient stratification tool. There is also mounting, well-documented, preclinical evidence suggesting that elevated eNAMPT levels may contribute to disease pathophysiology by exerting pleiotropic and systemic effects on metabolism and immunity via DAMP/adipocytokine/pro-angiogenic factor activities. Consequently, therapeutic approaches for eNAMPT inhibition, principally with mAbs, are now being developed and tested in various disease models. The leading experimental drug, mAb ALT-100, is in early clinical development, so we may soon learn more about the potential of eNAMPT as therapeutic target.All that said, while eNAMPT may be a pleiotropic factor implicated in disease pathophysiology, it is also ubiquitously secreted and present at low physiological levels in healthy individuals, and the benefits of physiological eNAMPT release are not fully appreciated, aside from specific examples, such as maintaining pancreatic beta cell function. Likewise, little is known about how eNAMPT physiological levels in tissues and in the circulation are affected by diverse environmental and physiological cues, by age, activity, etc. Other aspects of eNAMPT biology remain unclear as well, in particular the exact modalities of eNAMPT interaction with target cells and its purported cell surface receptors. We hope that future research will help shed light on the currently unknown aspects of this fascinating protein.ES: Conceptualization, Investigation, Writing \u2013 original draft, Writing \u2013 review & editing, Visualization. AN: Writing \u2013 review & editing, Visualization. KM: Conceptualization, Project administration, Supervision, Visualization, Writing - review & editing."}
+{"text": "We also observed an altered, opposite impact of Huntington\u2019s and Parkinson\u2019s disease on functional connectivity within the sensorimotor cortex, once again with relevant associations with clinical symptoms. The high resolution offered by the 7T scanner has thus made it possible to explore the complex interplay between the disease effects and their contribution on the subthalamic nucleus, and sensorimotor cortex. Taken altogether, these findings reveal for the first time non-invasively in humans a differential, clinically meaningful impact of the pathophysiological process of these two movement disorders on the overall sensorimotor functional connection of the subthalamic nucleus and sensorimotor cortex.Huntington\u2019s and Parkinson\u2019s disease are two movement disorders representing mainly opposite states of the basal ganglia inhibitory function. Despite being an integral part of the cortico-subcortico-cortical circuitry, the subthalamic nucleus function has been studied at the level of detail required to isolate its signal only through invasive studies in Huntington\u2019s and Parkinson\u2019s disease. Here, we tested whether the subthalamic nucleus exhibited opposite functional signatures in early Huntington\u2019s and Parkinson\u2019s disease. We included both movement disorders in the same whole-brain imaging study, and leveraged ultra-high-field 7T MRI to achieve the very fine resolution needed to investigate the smallest of the basal ganglia nuclei. Eleven of the 12 Huntington\u2019s disease carriers were recruited at a premanifest stage, while 16 of the 18 Parkinson\u2019s disease patients only exhibited unilateral motor symptoms (15 were at Stage I of Hoehn and Yahr off medication). Our group comparison interaction analyses, including 24 healthy controls, revealed a differential effect of Huntington\u2019s and Parkinson\u2019s disease on the functional connectivity at rest of the subthalamic nucleus within the sensorimotor network, i.e. an opposite effect compared with their respective age-matched healthy control groups. This differential impact in the subthalamic nucleus included an area precisely corresponding to the deep brain stimulation \u2018sweet spot\u2019\u2014the area with maximum overall efficacy\u2014in Parkinson\u2019s disease. Importantly, the severity of deviation away from controls\u2019 resting-state values in the subthalamic nucleus was associated with the severity of motor and cognitive symptoms in both diseases, despite functional connectivity going in  et al. report that participants with early Huntington\u2019s and Parkinson\u2019s disease show opposite pattern of resting-state functional connectivity in the subthalamic nucleus and sensorimotor cortex. Despite going in opposite direction, functional connectivity values in each disorder showed pathological associations with motor and cognitive symptoms.Using MRI at 7T, Evangelisti   In particular, motor connections follow two competing pathways linking cortex and thalamus, the indirect and the direct pathways.6 PD, on the other hand, is a progressive neurological disorder characterised by a degeneration of dopaminergic neurons in the substantia nigra (SN) pars compacta that manifests itself with various motor symptoms, one of the most characteristic of which is bradykinesia, a decremental slowness of movement.7HD is a fatal, autosomal dominant neurodegenerative disease that initially predominantly affects the GABAergic medium-size spiny neurons of the striatum, leading to choreic movements.8 Conversely, while degeneration of the STN in HD has been reported more than four decades ago,9 evidence for its functional involvement in HD is scarcer in humans. Studies on different HD animal models have, however, recently demonstrated an early impairment of spontaneous STN activity.10-13To date, only invasive studies have been able to functionally investigate the STN in HD and PD at the level of detail required to identify this subcortical structure and, crucially, to distinguish it from the adjacent SN. The exact functional role of the STN in PD, explored in numerous studies of deep brain stimulation (DBS) in patients with advanced disease, remains unclear, however. Inhibition, decorrelation and even elevation of its activity all improve motor symptoms, suggesting it is an abnormal pattern of activity that lies at the heart of this movement disorder.14-16 and to study its spontaneous neuronal fluctuations  using resting-state functional MRI (rs-fMRI).17Conventional non-invasive measures in human, such as MRI at 1.5 or 3T, usually lack the resolution and contrast to achieve such a key distinction, typically with voxels inferior to 1.5\u2005mm isotropic in size. Improvements gained by using state-of-the-art 7\u2005T provide a unique opportunity to isolate the signal from the STNboth HD and PD at high resolution using 7T MRI. Including the two movement disorders in the same study makes it possible for the first time to directly compare in humans the three states of basal ganglia inhibition: decreased (HD), increased (PD) and normal  at an unprecedented level of detail. The main aims of our study were 2-fold: first, to assess whether we could detect in the STN, and the sensorimotor cortex, the theoretical opposite states in basal ganglia dysfunction for HD and PD; second, whether these differential functional signatures could be related to clinical symptoms in HD and PD.Here, we investigated the functional connectivity of the STN and sensorimotor cortex in The study was approved by the local Research Ethics Committee  and written consent was obtained from each participant (recruited 2014\u201320).18We aimed to include participants early in the course of the disease to limit confounding effects, and to keep the groups as homogenous as possible to maximize the detection of differences. We primarily included in this study participants with premanifest HD on the one hand, and unilateral PD participants at Hoehn and Yahr\u2014H&Y\u2014Stage I off medication on the other hand.Exclusion criteria pertained to the safety risks specific to the 7T scanner, such as dental implants, head/neck/shoulder tattoos, or any surgery where vascular clips could have been used .19 and the Hopkins verbal learning test (HLVT).20 Following amended criteria described in the TRACK-HD study,22 the HD carriers were classified based on a combination of total motor score (TMS), total functional capacity (TFC) and diagnostic confidence score, which are subscales of the UHDRS. Eleven participants were at a premanifest stage, and one participant showed manifest HD; Twelve HD carriers were recruited through the HD clinic at the Oxford University Hospitals (OUH). To characterize motor and cognitive symptoms, a trained neurologist administered the Unified Huntington\u2019s Disease Rating Scale (UHDRS)23\u00a024 While 2 participants showed bilateral motor symptoms, the vast majority\u201416 of the PD patients\u2014were unilateral .Eighteen early-stage PD participants were recruited through the Oxford Parkinson\u2019s Disease Centre (OPDC) and the Parkinson\u2019s clinic at the OUH. A trained neurologist administered the Movement Disorders Society-Unified Parkinson\u2019s Disease Rating Scale (MDS-UPDRS Parts I\u2013IV) to quantify motor and non-motor symptoms.P = 0.0013 uncorrected), constipation (P = 0.048 uncorrected) and light headedness on standing (P = 0.041 uncorrected), as well as neck rigidity (P = 0.0013 uncorrected) and right hand movements (P = 0.00074 uncorrected)  and (ii) postural tremor of the left hand .Comparing the 3 unmedicated PD participants with the 15 medicated ones, other than differences inherent to their medication status, there was no significant difference between the two groups (under heteroscedastic assumptions) except, at an uncorrected level, in: (i) daytime sleepiness  centre, part of the Wellcome Centre for Integrative Neuroimaging, using a 7T Magnetom syngo B17 scanner with a 32-channel head coil .The MRI protocol included, amongst other sequences:Two whole-brain high-resolution volumetric structural scans:3 resolution, inversion time (TI) of 1050\u2005ms, echo time (TE) of 2.84\u2005ms and repetition time (TR) of 2200\u2005ms, flip angle of 7\u00b0, bandwidth of 240\u2005Hz/Px, generalized autocalibrating partial parallel acquisition (GRAPPA) acceleration with factor 2;(i) a T1-weighted image, acquired using a magnetization-prepared rapid gradient echo (MPRAGE) sequence: sagittal orientation, matrix 256 \u00d7 256, field of view (FOV) of 240\u2005mm, 176 slices per slab, 0.94 \u00d7 0.94 \u00d7 0.94\u2005mm3 resolution, TE of 2.84\u2005ms and TR of 1240\u2005ms, flip angle of 7\u00b0, bandwidth of 240\u2005Hz/Px, GRAPPA acceleration with factor 2.(ii) a PD-weighted image, also acquired with an MPRAGE sequence: sagittal orientation, matrix 256 \u00d7 256, FOV of 240\u2005mm, 176 slices per slab, 0.94 \u00d7 0.94 \u00d7 0.94\u2005mm3 resolution, TE of 68.2\u2005ms, TR of 5382\u2005ms, flip angle of 90\u00b0, bandwidth of 1488\u2005Hz/Px, GRAPPA acceleration with factor 2, multi-band acceleration factor 2, 64 isotropically distributed diffusion gradient directions, b-value of 1000\u2005s/mm2. Two non-diffusion-weighted imaging scans were acquired with opposite phase encoding directions (A/P and P/A).Whole-brain diffusion-weighted high-resolution imaging, which was performed using an echo planar imaging (EPI) sequence: axial orientation, matrix 160 \u00d7 160, FOV of 192\u2005mm, 104 slices, 1.2 \u00d7 1.2 \u00d7 1.2\u2005mm3 resolution, TE of 25\u2005ms, TR of 1853\u2005ms, flip angle of 40\u00b0, bandwidth of 1644\u2005Hz/Px, GRAPPA acceleration with factor 2, multi-band acceleration factor 4, 220 volumes. Subjects were instructed to keep their eyes open but no fixation cross was shown.A 7-minute high-resolution rs-fMRI scan acquired with a gradient EPI sequence: axial orientation, A/P phase encoding direction, matrix 160 \u00d7 160, FOV of 192\u2005mm, 104 slices, 1.2 \u00d7 1.2 \u00d7 1.2\u2005mm25-27; https://fsl.fmrib.ox.ac.uk/fsl/fslwiki).Image preprocessing was performed using tools from the FMRIB software library (FSL28 accounting for high-intensity outlier voxels (around vessels) with a combination of upper thresholding and the use of an additional tissue class.To improve contrast-to-noise ratio and image inhomogeneities, T1 images were divided by PD images within subject. For this, we registered PD to the corresponding T1 scan with a rigid registration (6 degrees of freedom). The resultant T1/PD image was used as the main structural image for processing, and as the \u2018native\u2019 space for each subject\u2019s other imaging modalities. It was brain extracted, and then segmented into grey matter (GM),30 Rs-fMRI was motion-corrected,31 then unwarped using the diffusion-derived fieldmap. There was no large head motion, with a maximum mean relative displacement of 0.5\u2005mm, and no difference between the groups . Temporal filtering was performed with a high-pass filter (cut-off at 100\u2005s). These functional images were then brain extracted. Denoising of rs-fMRI was performed with single-subject independent component analysis (ICA)32: we performed a manual classification of components into \u2018signal\u2019, \u2018unknown\u2019 and \u2018noise\u2019, and noise components were regressed out.33-35 Finally, functional images were linearly registered to the corresponding T1/PD \u2018native\u2019 space using boundary-based registration approach.36First, we needed to create a map to unwarp the rs-fMRI scans. In lieu of a fieldmap, we estimated the displacement field from blip-up-blip-down pairs of diffusion images to correct for rs-fMRI distortions.37 GM maps of a subset of participants  were linearly registered to a GM prior template in MNI space. By taking the mean of these images and their flipped counterparts along the x-axis, an initial symmetrical template in MNI space was created. The same steps were repeated with non-linear registration to the initial template to create an improved symmetrical, study-specific and unbiased template. Last, all subjects\u2019 GM images were non-linearly registered to the study-specific template to create structural-to-MNI warpfields. Exclusion masks for the signal dropout region in structural images due to inhomogeneities in the B1 transmit field of the coil (mainly around a right temporo-cerebellar area) were created specifically for each participant and used in both linear and non-linear registrations to improve the alignment.Optimized registration to the Montreal Neurological Institute (MNI) standard space was carried out with a multi-step approach optimizing the alignment across subjects based on GM information. First, registration from T1/PD native space to MNI was performed using FSL-VBM.de facto in the same study-specific standard space.Boundary-based registration matrices from functional to native structural space, and warpfields from structural to study-specific standard space were then combined to transform the resting-state images with a single interpolation. Therefore, GM from structural T1/PD and rs-fMRI was n = 220, to increase signal-to-noise ratio [SNR]), after unwarping and motion-correction (http://www.thehumanbrain.info/brain/sections.php) was used as anatomical reference.Both for visualization, and to create probabilistic regions-of-interest (ROIs), individual, subject-specific masks were manually drawn for the STN. Thanks to the sufficient contrast and high resolution of the rs-fMRI data, STN and SN could be clearly distinguished directly on each subject\u2019s average functional image  as additional, informative ROIs of the basal ganglia circuitry that can be difficult to isolate from one another. For the manual segmentation of the GPe and GPi, we used each subject\u2019s structural image, as the medial medullary lamina\u2014separating GPi from GPe\u2014was clearly visible on the T1/PD images . The GPeAll the manually drawn ROIs were registered to the study-specific standard space, applying corresponding spatial transformations described above, and specific thresholding after interpolation . The ROIs were then averaged to get probabilistic group reference masks for each region.38 for the post-central and pre-central gyri (at a 5% probability threshold) and the supplementary motor area (at a 20% probability threshold).Finally, a large mask of the sensorimotor cortex was created by combining probabilistic masks from the Harvard Oxford atlasData analyses were performed using FSL, R and SPSS.32 The same number of participants from each group was included to ensure an unbiased decomposition. Following a dual-regression approach,39 all 30 ICs\u201410 of which were identified as resting-state networks, RSNs\u2014were first regressed into rs-fMRI data to obtain a participant-specific timecourse for each IC above and beyond all other ICs. Second, timecourses were regressed into the imaging data to obtain subject-specific spatial maps, which could then be compared between groups. This dual-regression approach inherently accounts for potential differences in baseline signal that might exist across subjects.Group-ICA was run on rs-fMRI data to create 30 independent components (ICs).15\u2014output timecourses from the first regression were regressed into unsmoothed data to retain as much of the initial spatial resolution as possible.While the 30 group-ICs were created using the data smoothed with a Gaussian kernel of 3\u2005mm full width at half maximum (FWHM)\u2014below the recommended maximum 4\u2005mm FWHM smoothing that guarantees that the majority of the signal in the centre of the STN originates from the STN itself, as opposed to from the SNAll subsequent analyses, i.e. group comparisons and correlations, focus on the primary sensorimotor network component that was identified amongst the 10 RSNs .P = 0.003, Tukey), HC participants were manually split into two groups so that they were age-matched to each HD and PD group: HCHD and HCPD. There were subsequently no significant differences in age between HD and HCHD , or PD and HCPD . No significant differences in sex existed before (\u03c72(2) = 2.9, P = 0.24) or after (\u03c72(3) = 3.5, P = 0.32) splitting the HC group.Due to inherent baseline differences in age between our HD carriers and PD patients groups \u2212(PD\u2212HCPD)(HD\u2212HCHD)+(PD\u2212HCPD)\u2212 was performed on the dual-regression spatial maps using the above contrasts, as well as a mean contrast for each group individually.P-values generated by the step described above. For completeness and future replicability,41 we also reported results and effect sizes for uncorrected P < 0.001 in our additional basal ganglia ROIs.In particular, STN clusters were considered significant using false discovery rate (FDR) after correction for multiple comparisons across the voxels making up the probability group mask (created by thresholding at 20%), using the uncorrected 42 in the sensorimotor cortical ROI, as this method intrinsically favours larger ROIs. Results were considered significant for TFCE-corrected P < 0.05.Separately, multiple comparison correction was performed using threshold-free cluster enhancement (TFCE) as test statistic within the non-parametric permutation toolP)-value maps to get parameter estimates in supra-threshold clusters of interest within our ROIs of the STN  and of the sensorimotor cortex. The presence of potential outliers  was verified in the weighted averages of parameter estimates extracted from both full GM  and the full group-level ICA map of our entire sensorimotor network, controlling for age in the regression. One HC and one PD were excluded from further imaging analyses following these procedures, without any issue to the matching .In particular, for HD participants, and excluding those scores that were constant for all participants or non-null only for the one manifest HD patient, these were:CAG number of repeats 43)Disease burden UHDRS\u2014Functional capacity: all sub-scoresUHDRS\u2014Functional assessment scaleUHDRS\u2014Independence scaleUHDRS\u2014Cognitive assessment: all sub-scoresUHDRS\u2014Behaviour assessment: all sub-scoresHVLT Separately, for PD participants, excluding the scores that were null for all patients, these were:Disease durationUPDRS Part I: all sub-scoresUPDRS Part II: all sub-scoresUPDRS Part III: all sub-scoresUPDRS Part IV: all sub-scoresComposite scores:For tremor: sum of scores for II-tremor, III-postural tremor of hands, III-kinetic tremor of hands, III-rest tremor amplitude, III-constancy of rest tremor.For posture and gait: sum of scores for II-walking and balance, III-freezing, III-gait, III-freezing of gait, III-postural stability.For rigidity: sum of scores for III-rigidity of neck, right and left upper and lower extremities.For right and left motor symptoms, separately: sum of scores for III-rigidity of upper and lower extremities, finger tapping, hand movements, pronation/supination movements of hands, toe tapping, leg agility, postural tremor of hands, kinetic tremor of hands, rest tremor amplitude of upper and lower extremities.For axial motor symptoms: sum of scores for III-speech, facial expression, rigidity neck, arising from chair, gait, freezing of gait, postural stability, posture, body bradykinesia, rest tremor amplitude of lip/jaw.R . We applied an FDR correction, which holds under dependency, to these P-values. Considering the small sample sizes in both HD and PD groups, results were considered significant at a threshold of 10% FDR, corrected across all clinical and behavioural measures. For completeness, all correlations with uncorrected-P < 0.05 are also reported.Rho correlations were evaluated with a two-sided Spearman\u2019s rank test, and their significance was determined in Using state-of-the-art 7T, we were able to gather data at a very fine resolution crucial to study small structures that play a key role in both movement disorders and cannot be easily disentangled from one another at lower resolution . 44 The average functional connectivity of the sensorimotor network within each of the four groups  is shown in The main sensorimotor network that resulted from group-ICA comprised the upper limbs (arm/elbow/wrist/fist/fingers) and lower limbs (thighs/feet/toes) regions of the primary cortex, medial and posterior areas of the premotor cortex, and primary and secondary somatosensory cortex .44 The aopposite differences between HD and PD compared with their respective HC group in the STN  and GPe .In addition, formal comparison between differences in left and right hemisphere for the same contrast revealed a trend for the effects being stronger in the left sensorimotor cortex than the right  and within the STN clusters .The two HC groups, by construction, differ in age by 20 years on average to match the two patient groups. We formally tested For the HD group, correlations between functional connectivity values in the clusters of interest and clinical scores consistently revealed associations related to the pathological effect of the disease, i.e. worse symptoms were associated with functional connectivity values furthest away from the values of HC , Fig. 4.In particular, abnormal functional connectivity in the STN showed pathological association with delayed recall and verbal fluency, and with finger tapping for the posterior STN cluster. We also found numerous correlations within the HD group between the functional connectivity values in the sensorimotor cortex cluster and several motor, cognitive and functional items. The strongest pathological associations for motor scores were with the Luria test, and gait, chorea and postural instability symptoms. For the cognitive domain, the strongest correlations were found with immediate recall, verbal fluency and word naming, and there were moderate associations with functional scores. In addition, both CAG and/or disease burden were correlated with functional connectivity in the anterior STN, and in the sensorimotor cortex . Of note, the association between CAG and functional connectivity values in the anterior STN was the only correlation within the HD group compensatory in nature .In the PD patients, functional connectivity in the posterior STN cluster revealed consistent pathological association, most strongly with a composite measure of postural instability and gait, with dysarthria and dysphagia, as well as apathy, and whether the patients were taking medication , Fig. 4.For both HD and PD groups, additional correlations with the functional connectivity values in SN (likely in the pars reticulata) and GPe are reported in Our study has revealed that HD and PD exhibit opposite pattern of functional connectivity of the STN and the sensorimotor cortex. Such an altered resting state of the STN is clinically meaningful, with the severity of deviation from controls largely associated with worsening motor and cognitive symptoms, despite functional connectivity going in opposite directions in each disorder. Similarly, we have also found an opposite pattern in the resting state of the sensorimotor cortex itself between HD and PD. These results take full advantage of the high resolution afforded by using a high-field 7T scanner to investigate the functional resting state of a small structure such as the STN, and from the fact that both participants with HD and PD were involved for the first time in the same MRI study.3\u2014twice as small as the highest resolution achieved so far to investigate the human STN function using whole-brain fMRI17\u2014thus offering 70+ voxels to cover the entire \u223c130\u2005mm3 volume of the STN.15 This allowed us to distinguish STN from SN directly in the imaging modality of interest at the individual level (P = 0.439 for HD versus HCHD and P = 0.937 for PD versus HCPD). We deliberately used ICA to ensure that the signal we observed was specifically that of the functional connectivity of the STN within the sensorimotor network above and beyond any other resting-state contributions of the STN. While functional images needed to be smoothed to create a group-ICA resting-state template, we made sure to use a relatively small Gaussian kernel of 3\u2005mm\u2014which guarantees that 75% of the signal in the STN originates from this structure itself, and not from other surrounding tissue, esp. that of the SN.15 Crucially, once we obtained our sensorimotor resting-state network template, we regressed it into the unsmoothed data.State-of-the-art 7\u2005T scanner and EPI sequence, using both GRAPPA and multi-band acceleration approaches, made it possible to achieve an isotropic resolution of 1.2\u2005mm. In other words, each voxel represented a volume of 1.7\u2005mmal level , but als44 Our findings reflecting a differential effect of HD and PD on the functional connectivity within this network recapitulated all of its cortical regions and markedly so in the left hemisphere, in premotor and primary sensorimotor areas corresponding to the right hand  precisely corresponded to those of the DBS \u2018sweet spot\u2019 .50 This sweet spot was identified as the cluster of maximum overall efficacy for improvement in rigidity, bradykinesia and tremor in advanced PD patients. These differences in the overall synchronization of the spontaneous oscillations of the STN within the sensorimotor network\u2014likely the sum of the contributions from the hyperdirect and indirect pathways\u2014were clinically meaningful, and associated with worsening of the symptoms in both PD and HD. While the PD population included here presented with very early disease  unlike the DBS study of Akram and colleagues,50 we nonetheless observed a trend between STN functional connectivity and rigidity of the upper extremities . This area of the STN also encompasses associative connections,51 likely explaining the correlations observed between two cognitive tests  and STN functional connectivity in the HD carriers .55 One possibility is that the changes in functional connectivity observed here in PD and HD reflect an opposite shift in the synchronization between sensorimotor cortex and STN signals,54 as a result of the pathological alteration in the influence of indirect and hyperdirect pathways. Importantly, it also has been reported recently that an imbalance in excitation:inhibition (E:I) ratio, such as precisely witnessed in PD and HD, could be related to functional connectivity in the same regions.56 Indeed, an increase in the E:I ratio appears to cause an increase in neuronal firing and relative blood flow, but remarkably a reduction in local and long-range functional connectivity in the same brain regions.56 Our results showing a decrease in functional connectivity in the sensorimotor cortex are thus in line with the increase of excitation that is known to happen in this region in HD; and vice versa in PD. Similarly, the findings of a reduced functional connectivity in the STN of PD patients tie in with the known overall increase in the E:I balance caused by a decrease of the inhibitory input onto the STN; and vice versa in HD. More specifically, in HD, pathological changes resulting in a vastly decreased inhibitory indirect pathway (possibly together with a modestly increased excitatory hyperdirect pathway), lead overall to the reduced excitation in the net output of the STN, and so a decrease in the E:I balance.57 Relatively recent animal models studies of HD seem to indeed confirm a reduced activity of the STN.11 For the same reasons, the fact that the STN is at the centre of convergence between an excitatory and an inhibitory pathway also probably explains why, on balance, there is on average no functional connectivity at rest of the STN within the sensorimotor network in HC, contrary to what we can be observed separately in the HD and the PD groups . However, this can also be considered as a strength in that what we observe here, non-invasively and in participants with very early disease, is the reflection of the overall effect of each of the two movement disorders on the functional connections of the sensorimotor cortex and the STN. Another strength of rs-fMRI is that, while task-activation studies have a poor signal-to-noise ratio because the signal (task-related modulation) is often small relative to the ongoing \u2018noise\u2019 and accounts for at best about 20% of the blood oxygenation level dependent (BOLD) variance, most of this \u2018noise\u2019 is actually made of spontaneous activity which is precisely the focus of rs-fMRI and can account for up to 80% of the variance, making rs-fMRI a potentially richer and more sensitive source of disease-related signal changes.62 Movement in the scanner can also be a possible confounding factor.63 We motion-corrected our imaging data,31 but also took great care in denoising every single rs-fMRI timeseries using single-subject ICA, in particular by regressing out from the data motion-related components.33-35 In addition, while the vast majority of our participants were either at a premanifest stage of HD, or at a very early unilateral stage of PD, we made sure that the one manifest HD carrier, and the three PD participants with H&Y two off medication\u2014of which two were identified as having bilateral symptoms\u2014did not drive our results (A clear limitation of this study is that resting-state functional connectivity does not allow us to distinguish between the contribution of the indirect and the hyperdirect pathways to the STN, unlike effective functional connectivity using DBS recordings results . Similar results .64 More importantly, this MRI study is unique in that it not only combines for the first time HD and PD participants\u2014both at a very early stage\u2014but in that it leverages the high signal-to-noise offered by the 7T scanner to achieve the highest resolution of any resting-state study in these populations and of the STN. This made it possible to explore the complex interplay between the disease effects and their overall contribution on the STN, demonstrating for the first time in vivo in humans a differential, clinically meaningful effect of each movement disorder on the sensorimotor cortex and STN.This work\u2019s most obvious limitation resides in the inherently low number of participants included, particularly for the two clinical populations HD and PD. We note however that, as long as valid statistical testing is used\u2014in our case, using non-parametric testing and focusing on supra-threshold clusters fully corrected for multiple comparisons over space\u2014the actual effect observed has to be larger for a small group to reach significance than it would have needed to be for a larger group.fcad282_Supplementary_DataClick here for additional data file."
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