Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species  Bombyx mori) and many destructive pests of agriculture and forestry, are among the most diverse and species-rich groups of insects Insects in the order Lepidoptera (moths and butterflies), including the domesticated silkworm  is limited to detect SNPs within restriction endonuclease sites, and technically laborious and expensive as it requires post-PCR processing, such as performing restriction digest reactions, preparing agarose gels, and running electropheresis. As for B. anynana, application of the Illumina Golden Gate platform to SNP genotyping requires multiple preparation steps and is relatively expensive due to the initial high cost of probe production An alternative approach is to develop single nucleotide polymorphisms (SNPs). Owing to their advantages over microsatellites which includes lower error rates and higher abundance in the genome, SNPs have been widely used in human and model species and are becoming the markers of choice for applications in non-model organisms In clinical research and diagnostics, high-resolution DNA melting (HRM) analysis has proved to be a powerful post-PCR technology for SNP genotyping Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae). The O. furnacalis is among the most serious lepidopteran pests of maize (Zea mays) in Asian countries Tpi (triose-phosphate isomerase) previously described The aim of this study is to evaluate the effectiveness of HRM assays for SNP genotyping in the lepidopteran non-model species O. furnacalis obtained from three different populations  were amplified by previously described primers Tpi locus of all the sequenced samples from Of_CU and Of_CC populations, while two single-base mutations were identified in the Of_NC population. One SNP rs84 was a G>A substitution located 84 bp downstream of the reference sequence of tpi (1463 bp in length) reported by rs577 was a C>T substitution located 577 bp downstream of the reference and determined in three other samples. The samples identified by sequencing were considered as known female controls used for genotyping unknown samples in this study.To find SNPs for genotyping, DNA samples from 30 females (10 from each population) of Tpi locus respectively summarized in To produce small amplicons, two primer pairs were designed to bracket the two polymorphic sites within the O. furnacalis. The HRM profiles for the same DNA preparations and the same sequences amplified from different populations were readily clustered together, allowing different genotypes to be consistently distinguished. The accuracy of genotype detection by SA-HRM was compared on the same samples identified using direct bi-directional sequencing in this study.For known female controls, the expected two hemizygous variants at each SNP site were resolved by SA-HRM as distinct normalized melting curves, melting peaks, and difference plots  samples were tested by mixing at the ratio of 1∶1 with the known wild-type female controls. If two hemizygous samples are not identical, mixing will generate a heterozygote that changes the shape of the melting curve rs84  designed to target the SNP SNPs used widely in human and model organisms are becoming the marker of choice for applications in non-model species O. furnacalis. For the demonstration set of 30 known and 90 unknown female samples, both SA-HRM and UP-HRM assays could readily resolve different genotypes of the SNPs identified in O. furnacalis  with that of a known wild-type female, each mixed sample has either homozygous or heterozygous DNA. Genotyping a mixed female sample by HRM assays is analogous to genotype a male sample or an autosomal gene. Compared to SA-HRM (m). This is valuable when there is a small change in Tm between different hemizygotes or homozygotes. Although only the two SNPs available were used for genotyping in this study, either SA-HRM O. furnacalis.HRM analysis was very effective for SNP genotyping in the tested lepidopteran species rnacalis , 3 and 4o SA-HRM , UP-HRM o SA-HRM , primariO. furnacalis, HRM analysis can be readily applied to other non-model species in the order Lepidoptera mainly due to its obvious advantages. The methods described in this study should work on DNA extracted from any other non-model organisms in this order. Since HRM is highly sensitive to detection of single nucleotide differences, all SNPs discovered in the non-model organisms can be genotyped by this technique. One major advantage of the technique is that there is no need for expensive and specific reagents other than a generic saturating DNA dye (e.g. LC Green plus) and the corresponding primer sets, so the reagent cost is very low Although we have applied this technique to However, one limitation of the technique is the scarcity or unavailability of HRM instruments. While currently all real-time PCR instruments are designed to monitor fluorescence during DNA melting, not all of them are able to perform HRM assay O. furnacalis. In our tests of known controls and 90 unknown females of O. furnacalis from diverse geographical sources, the HRM results fully coincided with those obtained by our direct sequencing. Thus, HRM is a reliable, simple and cost-effective post-PCR technique that is suitable for genotyping O. furnacalis populations, and has the potential of wide application for genotyping studies  in any other non-model species of Lepidoptera.In conclusion, we have demonstrated that HRM can readily and inexpensively resolve all the SNPs in O. furnacalis was established from larvae collected in maize fields in central China, as described by Zhou et al. (2003) O. furnacalis (Of_CU) has been maintained at the Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Two additional wild populations of O. furnacalis, Of_CC and Of_NC were sampled in April 2010 from maize fields in central China (CC) and northern China (NC), respectively. Egg masses of O. furnacalis were collected and transferred to State Key Laboratory Breeding Base for Zhejiang Sustainable Plant Pest and Disease Control, Laboratory of Entomo-molecular Biology, Sericulture Research Institute, Zhejiang Academy of Agricultural Sciences, China. The hatched larvae were reared on an artificial diet at 25°C under a 14∶10 light∶dark photoperiod and 70% relatively humidity in the laboratory. After pupation, moths from the cultured and two natural populations were preserved at −80°C until used for DNA extraction.The culture of O. furnacalis was extracted from the adult virgin female moths using a DNeasy Tissue kit (Qiagen) according to manufacturer's instructions. DNA quality and concentration was assessed using a Beckman DU 640 spectrophotometer . DNA solutions were then prepared at a concentration of 10 ng/µl and stored at −20°C until use.Genomic DNA of Tpi was amplified using a pair of primers ECBtpi_for1A (AGATGTCAAAATTCAACTCAG) and ECBtpi_rev5 (ATAGTTTACGAATTACGAGTT), as described by Dopman et al. (2005) et al. (2007) O. furnacalis has a female-heterogametic sex chromosome constitution  O. nubilalisTpi of O. furnacalis was also found to be located on the Z chromosome (unpublicated data). Thus, PCR products from only female moths were purified and directly sequenced using ABI PRISM3730 automatic DNA sequence systems  in order to give unambiguous sequence information. DNA sequences from 10 female moths from each population were edited using DNAstar Seqman software . One sequence (Accession number DQ204987) of Tpi in O. furnacalis reported by Dopman et al. (2005) A DNA fragment of about 1600 bp including the entire coding region and introns of m of between 59–65°C with LightScanner Primer Design software . Pairs of primers were designed to flank the informative SNPs identified newly in this study. Amplicon size was kept below 150 bp, as differences among genotypes are easier to identify when the amplicons are shorter http://www.dna.utah.edu/umelt/umelt.html) a) was optimized in a temperature gradient ranging from 58 to 72°C in order to amplify a specific PCR product. Gradient PCR was carried out using the EDC-810 gradient thermocycler  under the PCR cycling conditions described in the next section. The quality and quantity of PCR products were validated on a 1.5% agarose gel using standard methods. All primer and probe sequences are summarized in Primers and one probe were designed to have a TTpi (Z-linked) analysis, because hemizygous (or homozygous) variants are easier to be detected by mixing known and unknown DNA For the tested female DNA, both mixed and unmixed samples were prepared for Taq polymerase (TransGen Biotech), 250 µg/mL bovine serum albumin (Sigma), and 1 µL LC Green plus . Reaction mixtures were overlaid with 20 µL mineral oil to avoid evaporative losses ensuring melt curves uniformity. Thermal cycling conditions for SA-HRM were 95°C for 2 min, followed by 45 cycles of 94°C for 10 s, Ta for 10 s, and 72°C for 10 s. The final step was set as follows: one cycle of 95°C for 5 s, followed by rapid cooling to 25°C for 30 s, maximizing heteroduplex formation a for 15 s, and the final step was set as above. All reactions were performed in duplicate for every sample, and all samples showing variant melting transitions were amplified for the third time.PCR reactions were performed on the Rapid Cycler 2 instrument  in a total volume of 10 µL containing 10 ng DNA template, 0.2 µM of each primer, 200 µM of each dNTP, 0.5 U of mm, obtained from negative derivative melting curve plots After PCR, glass capillaries carrying samples were transferred to a high-resolution melting instrument  for HRM analysis. A temperature ramp rising at 0.3°C/s was used to acquire fluorescence data during DNA melting from 60°C to 90°C in SA-HRM and from 45°C to 90°C in UP-HRM. HRM curves were analyzed using the Lightscanner software  following the protocols described previously PCR products of all genotypes were purified and cloned into the vector pMD™18-T (TaKaRa). Positive clones were sent to Sangon Biotech Co., Ltd., Shanghai, China, for bi-directional sequencing. Sequences were analyzed as above by using the Lasergene software  and ClustalX 1.83


In contrast to other similar tools  RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions.The RNA Bricks database ( Folded RNA molecules exhibit hierarchical organization. They are composed of modular units, in particular regularly shaped double-stranded helices formed by ribonucleotide residues paired in the Watson–Crick (WC) sense, and irregularly shaped motifs formed by residues engaged in various non-WC interactions. Examples of structural motifs include kink-turn , sarcin–Experimental structure determination for a growing number and type of RNAs revealed that structural motifs are often conserved in homologous (evolutionarily related) molecules, but they may also appear in different structural and functional contexts in non-homologous molecules. Consequently, structural motifs are not necessarily accompanied by a conserved RNA sequence or secondary structure, hence their discovery and comparison is not trivial .Currently, several databases that classify the RNA structural motifs exist. Some of them provide also information about tertiary interactions in RNA molecules. SCOR  is a manSupplementary Example S2 in Supplementary Data 1 for details). Another issue is the common practice of using the maximum resolution of diffraction data as a measure of structure quality. It is well known, however, that crystal structure model quality is a local property and must be locally validated , models are split into several PDB files that are not necessarily independent. For example the asymmetric unit of one of the crystal structures of the 70S ribosome is composed of four PDB files  , but onlalidated .We developed a database named RNA Bricks to resolve the above-mentioned issues. Our database provides information about local environments of the collected motifs, including contacts with other RNA motifs, proteins, metal ions, water molecules or small molecule ligands. Furthermore, RNA Bricks stores data on contacts between symmetry mates in crystals and between molecules from split PDB entries . A unique feature of RNA Bricks is the availability of three structure-quality scores with single-nucleotide resolution. These may be used to select most reliable subsets of stored RNA structures and motifs. We also implemented an algorithm for PDB-wide structure-based searches. The algorithm has similar capabilities to the above-mentioned WebFR3D, but additionally enables making PDB-wide queries.The term ‘RNA (3D) motif’ has many meanings ,24. In thttp://djangoproject.com). The visualization of reduced RNA graphs is based on a JavaScript InfoVis Toolkit (http://thejit.org). Secondary structure representations of RNA motifs were rendered using an in-house modified version of Varna (http://www.jmol.org/).Utility programs were implemented in Python 2.7 with the extensive use of routines from the Computational Crystallography Toolbox (CCTBX) . The datof Varna , which eModels of macromolecular structures containing RNA molecules (with or without proteins), excluding DNA/RNA hybrids, were downloaded from the PDB . StructuFor each RNA chain found in a PDB model, secondary structure was annotated with MC-Annotate . After rq and a set of RNA 3D motifs M = {m1, m2, m3, … mn}. The task is to find a set of rigid transformations that superposes M members onto q with lowest RMSD over backbone atoms. An additional restriction is that all mi nucleotides must have their counterpart in the query structure q. A symmetric version of the problem, where we want to find all matches of q within M members, is handled in the analogous way.The input to the tertiary structure-based search algorithm are a query RNA structure M that cannot match q. First, we compare distances between selected backbone atoms within motifs from M and the structure q. At this step the carbon C3′ atoms are used by default. The user, however, may request the use of any other backbone atom type. All motifs from M that have distances not observed in q are rejected. Next, for structure q and remaining motifs from M we analyse all possible triplets of the selected backbone atoms. Again, we reject all motifs that do not contain at least one triplet with similar edge distances to a triplet from q. Finally, for each of the remaining motifs we pick a triplet of the selected backbone atoms, and try to superimpose it onto all similar triplets from the structure q. The obtained transformation is applied to the whole motif and quality of the match is scored with the RMSD of the closest pairs of the selected backbone atoms from q and mi after superposition. If the RMSD is below a user-provided threshold, the superposition is further refined with the use of all backbone atoms.The algorithm starts with a two-step procedure of filtering motifs from Putative hydrogen bonds were detected using our own utility scripts developed with the CCTBX library. We used our own implementation of the algorithm from the HB-PLUS program . MissingThe detection of putative interactions of RNA with molecules other than RNA and proteins, i.e. with water, ions and small molecules, was based on a simple distance-based criterion. Two neighbors were classified as being in contact if the smallest distance between their non-hydrogen atoms was below 3.9 Å.http://iimcb.genesilico.pl/clarna/).Base pairs were annotated using MC-Annotate , RNAViewAll nucleotide residues in RNA bricks are annotated with structure-quality scores that describe real-space correlation coefficients (RSCCs) with electron density data , backbone geometry and possible steric conflicts. These scores can be used to identify poorly modeled residues and to assist in the selection of well-modeled motifs. The measure of steric clashes, i.e. a number of non-H-bond overlaps (0.4 Å or greater) per 1000 atoms, was determined with the use of the Probe program  from theThe RNA Bricks web interface provides intuitive access to the data on RNA 3D motifs, i.e. ‘RNA bricks’ . The use3D structure of RNA bricks and their complete local environment (including symmetry mates and neighboring molecules from split PDB entries) can be visualized with the Jmol applet. Users may toggle visibility of selected types of neighboring molecules . Contact with protein and RNA molecules are presented down to the level of single hydrogen bonds .case sensitive option to search for modified residues that are represented by a lower-case symbol of a related residue . Additionally the query results that are within 1.0 Å backbone RMSD distance may be aggregated. This option may be useful if a number of results is very large.The database enables searching for RNA motifs based on sequence and secondary structure similarities. Queries must be in the FASTA format, and define sequences of continuous RNA chain fragments that form a motif. The search engine supports regular expression queries and accounts for cyclic permutations in the order of segments. By default, the sequence-based search is case-insensitive. The users, however, may select query-in-motif searches for instances of the query structure within RNA motifs, whereas motif-in-query attempts to cover the query structure with motifs from the database. Because of the complexity of the structure-matching algorithm, the exhaustive search involving all the available motifs is computationally prohibitive. Therefore a single query is limited to representatives of the RNA 3D motif clusters . These can be simply medoids (default), motifs derived from a set of non-redundant RNA structures , and 220 460 RNA 3D motifs. A majority of these are loops (51.8%). In particular terminal loops constitute 15.9%, internal loops 28.2%, three-way-junctions (3wj) 4.2% and loops composed of more than 3 strands 3.5% of all the RNA 3D motifs. Canonical helices (double stranded) and single-stranded terminal fragments comprise 40.6% and 7.6% of all RNA 3D motifs, respectively. After applying the clustering procedure , we obtained 16 089 motifs that represent clusters of RNA 3D motifs with the RMSD of backbone atoms ≤ 1.0 Å. Fraction of representative stem motifs is 13.4%, which is significantly smaller than overall fraction of stems in the database. All types of loops constitute 63.2% of representatives. Majority of these are internal loops . Internal loops, 3wj and loops composed of more than three strands constitute 20.5%, 5.7% and 4.6% of all representatives, respectively. Relatively large fraction of the representatives (23.4%) are single-stranded terminal fragments.To assess the mutual dependence of the quality scores used in RNA Bricks we calculated the Spearman's rank correlation coefficient for all pairs of score values. For the calculations we used only RNA motifs derived from crystallographic structures that have experimental data deposited in the PDB.The correlation coefficients for all three pairs of scores calculated for complete RNA structures are 0.42 for poor electron density and clash-score, 0.38 for poor electron density and suite-score and 0.44 for clash-score and suite-score, respectively. These correspond to relatively low, but statistically significant correlations. Analogous correlation coefficients determined for RNA motifs are 0.35, 0.28 and 0.31, which again corresponds to weak, but statistically significant correlation. These results suggest that the three quality scores should be used together, as they contribute complementary information, but they should not be treated as completely independent from each other.The RNA Bricks was developed to enable studies of RNA tertiary interactions. To demonstrate its capabilities we analyzed contacts formed by the conserved T-loop motif. The T-loop  is a recquery-in-motif mode and RNA motifs derived from a non-redundant set of X-ray structures solved at 3.0 Å resolution or better. The results were manually curated with the use of RNA Bricks web interface to remove hits that matched the query with low RMSD, but were not T-loops (e.g. had fifth and sixth nucleotide stacked with each other). Families of structures not covered by the current release of Rfam database were assigned using Infernal  from the first match. Among 20 motifs, 16 were derived from RNA structures annotated as tRNA, 3 as tRNASec and 1 as group-II-D1D4-3 (PDB: 4FAW). In both tRNA and tRNASec families, the third uridine residue and the apex cytosine residue of the motif interact with conserved guanine residues . RNA Bricks provides also three structure-quality scores with a single-nucleotide resolution. These may be used to select the most reliable subsets of stored RNA structures and motifs. We also implemented an algorithm for making PDB-wide structure-based queries. In contrast to other similar tools RNA Bricks accepts user-provided queries in PDB format. Besides, the search utility enables searches for ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions.To address the problem of secondary structure visualization of large RNA molecules (e.g. ribosomes) we developed the reduced graph representation. The graphs make it possible to display complex interaction networks  and provide efficient mapping between secondary structure elements and the RNA 3D motifs .RNA Bricks database provides a simple clustering method that groups RNA motifs with the same secondary structure and number of ribonucleotide residues. Therefore in some cases two motifs that have similar tertiary structures are classified to separate clusters due to differences in secondary structure annotation. Furthermore, very similar 3D motifs with variable loops (e.g. T-loop described above) do not belong to the same clusters. Being aware of these limitations we implemented a structure search algorithm that allows for comparison of RNA structure fragments according to the mutual position of individual atoms and regardless of the secondary structure and the molecule size. Users should utilize this tool to query the database for all RNA 3D motifs that share common substructures with a motif of interest.We compared the structure-based search tool implemented in RNA Bricks to other publicly available web servers that enable PDB-wide searches using coordinates of small, user-defined RNA fragments. To the best of our knowledge among currently available methods only R3D-Blast , WebRF3DOur solution of the 3D motif search problem is similar to the algorithm implemented in ARTS  for the Supplementary Example S1 in Supplementary Data 1 for details). All occurrences of a sarcin–ricin motif loop, including non-local ones, were found within Haloarcula marismortui 50S ribosomal subunit (PDB: 1S72). In addition, we were able to carry out the search for all known RNA structures, which was not possible using the web version of FR3D , the presence of severe steric clashes (clashscore) and low RSCCs for experimental diffraction data . In this work we showed that although the three scores are not independent, the correlation between them is relatively weak. Notably, RNA motifs that correspond to well-resolved electron density maps do not necessarily have favorable RNA backbone conformers (data not shown). Therefore we suggest that users should always take all the available quality scores into consideration. RNA Bricks provides the RSCCs determined for all structures with experimental data deposited in the PDB, including those, for which refinement parameters reported by the authors cannot be reproduced. It means that in some cases the overall low correlation coefficient may reflect gross errors in the diffraction data deposited by their authors, rather than the actual quality of a structure. In borderline cases users should refer to specialized tools like the Electron Density Server  or PDB_RSupplementary Data are available at NAR Online.Polish Ministry of Science [Iuventus Plus0066/IP1/2011/71 to G.Ch.]; European Research Council (ERC) [StG grant RNA+P=123D to J.M.B.]; the development of the substructure search tool by T.W. has been supported by the National Science Centre (NCN) [2011/01/D/NZ1/00212 to G.Ch.]. Funding for open access charge: Polish Ministry of Science [Iuventus Plus 0066/IP1/2011/71].Conflict of interest statement. None declared.


Inhibiting sodium-dependent glucose transporters (SGLTs) has been proposed as a new therapy for the treatment of diabetes . SGLT2 aConsequently, any drug discovery project aiming for promising SGLT2 inhibitors has to take into account significant selectivity towards SGLT1. In order to describe the selectivity on a structural basis we extensively used molecular modelling techniques since no x-ray structural data is available for neither SGLT1 nor SGLT2. For both transporters homology models were generated using the published x-ray structure from vSGLT (PDB-code 3DH4) [


Self-rated health (SRH) has been reported as a predictor of mortality in previous studies. This study aimed to examine whether SRH is independently associated with hypertension and if there is a gender difference in this association.16,956 community dwelling adults aged 20 and over within a defined geographic area participated in this study. Data on SRH, socio-demographic factors  and health behaviors  were collected. Body mass index and blood pressure were measured. Logistic regression models were used to determine a relationship between SRH and hypertension.p < 0.001), and the older age groups rated their SRH more negatively in both men and women (p < 0.001). While the multivariate-adjusted odds ratio  of participants rating their SRH as very poor for hypertension in men was OR 1.70 (1.13-2.58), that in women was OR 2.83 (1.80-4.44). Interaction between SRH and gender was significant (p < 0.001).32.5% of the participants were found to have hypertension. Women were more likely than men to rate their SRH as poor (SRH was independently associated with hypertension in a Korean adult population. This association was modified by gender. Self-rated health (SRH) is based on a simple question where people are asked to rate their own overall health. It is a subjective consciousness about personal health rather than an objective measure of health. However, SRH has been reported as a predictor of morbidity and mortality even after controlling for other related factors -8. FurthGender differences in health vary according to differential vulnerabilities in men and women. Previous studies on gender difference in the relationship between SRH and mortality implied a different process by which men and women assess their general health state ,7,14-18.The purpose of this study was to examine whether SRH is independently associated with hypertension and whether the association is modified by gender in a Korean adult population.The study population was community-dwelling men and women aged 20 and over within a defined geographical area of Jeollanamdo Province, South Korea in 2006. A total of 1,485,843 community residents  were identified from the national registration records and 41,250 candidates were selected by random sampling stratified by cities and counties to be a representative sample of Jeollanamdo Province. In total, 16,956 residents provided informed consent and participated in the Jeollanamdo Community Survey, which collected information on the health behavior of community-dwelling adults. The response rate was 41.1%.This study relied on secondary data from a Jeollanamdo Community Survey. The data set included the socio-demographic information , health behaviors , body mass index, blood pressure and SRH. Only non-identifiable aggregate results were released and they were openly available. No institutional review board approval was sought for this reason.Well-trained Health Center staffs visited randomly selected residents. Socio-demographic information  and health behaviors  were collected from the participants using structured questionnaires. Smoking status was categorized as current smoker, former smoker, and non-smoker. Alcohol consumption and physical activity were identified as yes or no.SRH was based on the answer to the question, "in general, how would you describe your current health?" The responses were categorized into five levels: excellent, good, fair, poor or very poor.Weight and height were measured and body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Blood pressure was measured by a mercury sphygmomanometer in the sitting position. The average of two consecutive readings of systolic and diastolic blood pressure was used in the analysis. Participants were considered to have hypertension if the average systolic blood pressure exceeded 140 mmHg and/or their diastolic pressure exceeded 90 mmHg, or they were taking antihypertensive drugs.T-test and chi-square tests were used to examine the distribution of covariates for subjects by hypertension status, and a trend test for age by SRH and chi-square test for gender by SRH were performed. The prevalence of hypertension with 95% confidence intervals was presented by SRH, and a trend test for hypertension by SRH was done. Logistic regression models were used to adjust for other covariates. SRH was entered as a categorical variable, and excellent SRH was taken as a reference group. After finding significantly related factors with hypertension in univariate analysis, age, marital status, education, smoking status, alcohol consumption, physical activity and BMI were entered as covariates in the models. Because of gender differences in the association between SRH and hypertension, regression models were stratified and interaction terms were tested. IBM SPSS statistics 19 software was used for the analysis. A p-value of less than 0.05 was considered significant.t-test or chi-square test  of the participants were found to have hypertension. Age, gender, marital status, education, smoking status, alcohol consumption, physical activity, BMI and SRH were identified as the related factors of hypertension by st Table .p < 0.001).The prevalence of hypertension for each level of SRH was 21.1% (excellent), 24.5% (good), 30.9% (fair), 50.9% (poor) and 55.7% (very poor) , and women were more likely to rate their health status poorer than men. Both men and women who were older tended to rate their SRH as poorer than the younger subjects .Results of logistic regression analyses are summarized in Table In this study of a community dwelling Korean adult population, SRH was independently associated with hypertension after controlling for other related factors such as age, gender, marital status, education, smoking status, alcohol consumption, physical activity and BMI. Further, there were significant gender differences in the association between SRH and hypertension. SRH was more strongly associated with hypertension in women, compared to men.Our findings are similar to the results from previous studies suggesting an association between SRH and morbidity or mortality -8. AccorAlthough several studies revealed that the effect of SRH on the prediction of mortality seems to be more apparent for men than women, little is known about gender differences in the association between SRH and hypertension ,14,15. WThis study has several limitations. First, the study was a population-based cross-sectional survey. With this kind of study design, it is often not possible to establish a temporal relationship. To confirm this association, a prospective study needs to be done. Second, there is a possibility of reverse causality; SRH could be not the cause but the outcome for hypertension, because hypertension labeling may adversely affect SRH. Hypertension is an asymptomatic condition that affects 30% or more adults  and mostIn conclusion, SRH was found to be independently associated with hypertension in men  and women  after adjustment for other related factors. Women were more likely to rate their health status poorer than men, and SRH was more strongly associated with hypertension in women than in men. Further research is needed to confirm the association in a prospective manner.The authors declare that they have no competing interests.HYS and MHS designed the study and performed the statistical analyses. HYS, MHS, and JAR interpreted the results. HYS and MHS drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/135/prepub


OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops , whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the  Flaviviridae family. This family includes known pathogenic viruses that cause Yellow fever, Dengue fever, Japanese encephalitis, and Tick-borne encephalitis Flaviviridae family members despite the lack of extensive sequence homology West Nile virus (WNV) is an 11 kb positive-sense, single-stranded RNA virus that belongs to the Flaviviridae family members represents a highly structured and important regulatory region.Two long distance interactions mediated by complementary base pairing between the 5′-UTR/initial coding region and 3′-UTR are thought to be required for WNV genome cyclization, creating a panhandle structure. Both the 5′-UAR  and 5′-CS  to achieve cyclization. Panhandle formation enables recruitment of factors, including the viral RNA dependent RNA polymerase The innate immune system is the first line of defense against viral infection, and susceptibility of IFN α/β receptor deficient mice to WNV infection points towards the importance of antiviral immunity conferred by the Type 1 IFN et al. OAS1 gene acts as a host genetic risk factor for humans in WNV infection FlaviviridaeSeveral lines of evidence suggest that OAS enzymes play a key role in the IFN response to WNV infection. The importance of OAS enzymes in the antiviral response against WNV can be inferred from the finding that RNase L limits WNV spread in mouse models Flaviviridae family form conserved dsRNA stem loops that are necessary for the regulation of viral genome replication in vitro activation of OAS1 by the SLI/II/III of WNV. Small-angle X-ray scattering experiments demonstrated that the WNV SLI/II/III adopts multiple conformations, including a subset that supports the predicted secondary structure. Truncations to the SLI/II/III were used to narrow the minimal region required for OAS1 activation, and mutations in the RNA binding site of OAS1 confirmed the specificity of the interaction To date, no specific OAS1 recognition sites have been identified within the WNV RNA genome. Given the importance of OAS enzymes to restrict viral propagation via dsRNA binding, we sought to identify and analyze specific dsRNA regions of the WNV genome responsible for OAS1 activation. dsRNAs with stable secondary structure are ideal activators of OAS1. Regions including the 5′-UTR/initial coding regions and the 3′-UTR from members of the GB1, Nus A, Streptavidin, 6xHis and TEV protease site) was performed as described previously Expression of recombinant human OAS1 p42 isoform (transcript variant 2) as a fusion protein in BL21 DE3-RIL cells  and subsequent purification of the free protein from the cleaved N-terminal affinity tag (GNSHT: in vitro transcription from a linearized plasmid using a non-denaturing column chromatography approach TCTCAAAGAAACACGTGCCGCTTACGCCCACAGTGTTCT) was transcribed/purified in an identical manner to serve as a negative control and to ensure that no unintended by-products of the transcription reaction were leading to activation.The SLI/II/III encompassing the 5′-UTR and initial coding region of WNV  was generated by 20,bs) which were then corrected to standard solvent conditions  using the algorithm SEDNTERP 20,ws (S) values for individual concentrations were then extrapolated to infinite dilution to obtain 020,ws (S).The sedimentation velocity (SV) experiment for OAS1 was performed using an Optima XL-I analytical ultracentrifuge with an An60-Ti rotor at 20.0 °C as described previously Hr in place of the translational diffusion coefficient:Dynamic light scattering data were collected using a Zetasizer Nano S system  as described previously oη is the solvent viscosity, oρ is the solvent density and N is the Avogadro's number.where 2 at pH 7.0) were also collected at multiple concentrations . Primary data analysis was performed using the program PRIMUS Gr) and the maximum particle dimension (maxD) using the program GNOM Ab initio shape reconstruction of OAS1 WT was performed using the program DAMMIF, that utilizes simulated annealing protocol ab initio shape determination, high-resolution structure of human OAS1 (PDB code: 4IG8 SAXS data for proteins (wild type and mutants) and for the WNV SLI/II/III was collected using an in-house Rigaku instrument as described previously RNA (100 nM) was titrated with increasing concentration of OAS1, in 50 mM Tris, 100 mM NaCl (pH 7.5) buffer for EMSA experiments. The binding reaction was allowed to proceed for 10 minutes at room temperature (∼20.0°C) and the mixed with native loading buffer  was added. Protein-RNA complex formation was analyzed on a Tris borate-EDTA poly-acrylamide gel (8%), and electrophoresis was performed at 65 V at ∼4.0°C in 0.5X TBE running buffer. Sybr gold  was used to visualize the RNA-containing species on the gel.V) were calculated by linear regression within the linear range of time course in at least triplicate. The apparent dissociation constant (appK) and the maximum reaction velocity (maxV) were determined using the following equation: V = maxV/(1+(appK/[RNA])) OAS1 activity was measured using an established colorimetric assay that quantifies the amount of pyrophosphate (PPi) produced as a by-product of 2′ 5′-oligoadenylates formation by the active enzyme −13 sec) suggesting that the protein is homogenous in mass and conformation (Hr) of 3.0±0.3 nm for OAS1  of 7.1 nm and a radius of gyration (Gr) of 2.28±0.02 nm were obtained for OAS1 from the pair distribution function analysis  of ∼0.9 was obtained for each individual model, signifying excellent agreement between the experimental scattering data and the calculated scattering data. The superimposed ab initio models provided an averaged model that was highly similar to each individual model in terms of shape as evidenced by normalized spatial discrepancy (NSD) parameter of 0.52±0.02  for the purpose of determining the solution structure by SAXS. Denaturing gel electrophoresis demonstrated a single band of appropriate size (data not shown), and DLS analysis confirmed that the sample was monodisperse with a rH of 5.1±0.2 nm  of the SLI/II/III were determined based on the ab initio models, and excellent agreement was found with the experimentally determined hydrodynamic properties  displays heterogeneity under native conditions consistent with the subspecies observed by SAXS. This heterogeneity is not observed under denaturing conditions (data not shown).The SLI/II/III and initial coding region of the WNV genome is comprised of three double-stranded stem loops. We sought to determine whether this region of the genome could bind to and activate OAS1 man OAS1 Fig. 4A.To identify the specific region(s) within the SLI/II/III that interacts with OAS1, we generated five different RNA molecules in addition to the SLI/II/III region that represent either two stem loops in combination  or individual stem loops . We believe this truncation approach is feasible based on the predicted secondary structure and our observed solution conformation of the SLI/II/III. Complex formation with increasing OAS1 concentration was then performed for each RNA molecule in order to determine which secondary structural elements mediated the interaction. We observed significant complex formation that appeared as a high molecular weight species in the EMSA of SLI/II with OAS1 Fig. 4B.in vitro, we were further interested to investigate whether this interaction leads to activation of OAS1 catalytic activity. A colorimetric assay was performed which correlates the detection of pyrophosphate (PPi) with the production of 2′-5′(A) chains. We prepared buffered reactions containing OAS1, ATP, and Mg2+ in the presence of SLI/II/III, polyinosinic-polycytidylic acid , or a single-stranded RNA (ssRNA) negative control. The experiments were performed over a 120-minute period, followed by progressive measurement of PPi production  and ssRNA (negative control). Remarkably, of all the SLI/II/III truncations, only SLII/III is capable of achieving activation level comparable to the full length SLI/II/III Fig. 5A.appK), a measure of affinity, and the maximum reaction velocity (maxV), a measure of catalysis. fitR). The SLI/II/III and SLII/III demonstrate potent stimulation of OAS1 activity (∼200-fold enhancement relative to the ssRNA negative control for both) despite the 4-fold higher affinity for OAS1 shown by SLI/II/III compared to the SLII/III. Despite having a maxV value approaching that of the negative control, the SLI/II RNA demonstrated a nearly 3-fold increase in affinity relative to the SLI/II/III. None of the individual stem loop structures reveals appreciable stimulation of OAS1 catalytic activity, and only SLI demonstrates detectable affinity (4-fold lower affinity than the SLI/II/III). Together, the kinetic analysis supports a model where SLII/III is the minimal construct capable of OAS1 activation despite having a weak binding affinity for the enzyme.Due to the observed discrepancy between binding and activation, detailed dose-response experiments in which initial reaction velocities of OAS1-catalyzed PPi production were performed under increasing RNA concentrations for each of the SLI/II/III constructs Fig. 5B.Gr and maxD values were within error of the wild type results (data not shown). Therefore, we conclude that these mutations do not affect the solution conformation of OAS1.To confirm that OAS1 activation is an RNA-mediated effect, the binding affinity and catalytic activity of two point mutants of human OAS1 (R195E and K199E) were investigated. Based on the human OAS1 structure, these mutations are in the positively charged dsRNA-binding groove on the enzyme face distal to the active site As expected, higher molecular weight RNA-protein complexes were not observed in EMSAs of SLI/II/III with increasing concentrations of R195E or K199E OAS1 Fig. 6B.The S162G mutation has been reported in a previous study as a very common single nucleotide polymorphism (SNP) in the OAS1 gene, and is more prevalent in WNV-susceptible individuals Flaviviridae family members. We conclude that a direct interaction between the SLI/II/III of the WNV and OAS1 occurs in vitro, and therefore warrants further investigation in a cellular context.OAS1 and other OAS isoforms play an important role in the recognition of viral dsRNA and subsequent amplification of the initial interferon-mediated innate immune response in vitro. The affinity of OAS1 for the SLI/II/III (appK of 1453±199 nM) is consistent with affinities for other short viral RNAs maxV of 21±2 μM/min) is within error of the positive control (synthetic poly I:C)  Table 2.We studied two point mutations in OAS1 within the RNA binding domain on the basic tract opposite the active site Genome cyclization has been established as necessary for WNV genomic RNA replication, and involves a panhandle structure comprising long-range base pairing interactions between nucleotides in the SLI/II/III , whereas the remaining models lack domain resolution Fig. 3C.


Transcriptomic studies in clinical research are essential tools for deciphering the functional elements of the genome and unraveling underlying disease mechanisms. Various technologies have been developed to deduce and quantify the transcriptome including hybridization and sequencing-based approaches. Recently, high density exon microarrays have been successfully employed for detecting differentially expressed genes and alternative splicing events for biomarker discovery and disease diagnostics. The field of transcriptomics is currently being revolutionized by high throughput DNA sequencing methodologies to map, characterize, and quantify the transcriptome.In an effort to understand the merits and limitations of each of these tools, we undertook a study of the transcriptome in sickle cell disease, a monogenic disease comparing the Affymetrix Human Exon 1.0 ST microarray (Exon array) and Illumina’s deep sequencing technology (RNA-seq) on whole blood clinical specimens..Analysis indicated a strong concordance (R = 0.64) between Exon array and RNA-seq data at both gene level and exon level transcript expression. The magnitude of differential expression was found to be generally higher in RNA-seq than in the Exon microarrays. We also demonstrate for the first time the ability of RNA-seq technology to discover novel transcript variants and differential expression in previously unannotated genomic regions in sickle cell disease. In addition to detecting expression level changes, RNA-seq technology was also able to identify sequence variation in the expressed transcriptsOur findings suggest that microarrays remain useful and accurate for transcriptomic analysis of clinical samples with low input requirements, while RNA-seq technology complements and extends microarray measurements for novel discoveries. With the completion of the human genome project, the monitoring of changes in the entire human transcriptome is an increasingly attractive method for dissecting the molecular basis of disease processes ,2. In thUntil recently, high throughput microarray technologies have been the method of choice in clinical studies with limited amounts of RNA from blood samples, biopsy specimens, or enriched cell populations, to obtain gene expression profiles. The high density Affymetrix Human Exon 1.0 ST array (Exon array) with 1.2 million probesets targeting every known and predicted exon in the entire genome has been successfully employed in clinical investigations for obtaining gene expression profiles and associated alternative splicing events in disease processes -9. DespiWith the advent of next-generation sequencing technologies, RNA-seq has emerged as a powerful tool for transcriptome analysis -12. By mConsidering the merits and limitations of each of the technologies, we undertook the current study using complex whole blood specimens from patients with sickle cell disease (SCD) and matched healthy controls to address the following major questions:(1). How do the technologies compare with regard to sensitivity, specificity, and variability of gene expression data? (2). Do the differentially expressed transcripts and alternatively spliced exons in SCD correlate well between RNA-seq and Exon arrays? (3). Does the abundant expression of globin transcripts in SCD interfere with RNA-seq analysis? (4). Are we able to discover novel differentially expressed transcripts in SCD using RNA-seq? (5). Can we detect sequence variants in the expressed transcripts?Although previous comparative studies ,16 have The study was approved by the National Institute of Diabetes and Digestive and Kidney Diseases institutional review board (NIH protocol 03-CC-0015) and written informed consents were obtained from study participants. Patients selected for this study included patients (n = 6) with sickle cell disease of mean age 41.6 ± 10.1 and the control group (n = 4) of self-identified African American subjects of mean age 42.2 ± 8.9, without sickle cell disease. The biosamples were collected in steady state condition and none of the individuals was receiving anti-platelet medication.Blood specimens (2.5 ml) collected in PAXgene™ tubes from each subject were incubated at room temperature for 4 h for RNA stabilization and then stored at − 80 °C. RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit following the manufacturer's guidelines. Briefly, samples were removed from −80 °C and incubated at room temperature for 2 hours to ensure complete lysis. Following lysis, the tubes were centrifuged for 10 min at 5,000 × g the supernatant was discarded and 500 μL of RNase-free water added to the pellet. The tube was vortexed thoroughly to re-suspend the pellet, centrifuged for 10 min at 5000 × g and the entire supernatant was discarded. The pellet was re-suspended in 360 μL of buffer BR1 by vortexing and further purification of RNA was done following the manufacturer's protocol with on-column DNase digestion. Quality of the purified RNA from was verified on an Agilent® 2100 Bioanalyzer ; RNA concentrations were determined using a NanoDrop® ND-1000 spectrophotometer .Total RNA from six SCD patients and four healthy controls were used for detailed analysis on RNA-seq and exon array platforms. Data analysis was carried out on 6 SCD and 3 healthy controls after removing a control sample which was identified to be an outlier based on principal component analysis (PCA) of the transformed data from RNA-seq.Globin mRNA was depleted from a portion of each total RNA sample isolated from PAXgene tubes using the GLOBINclear™-Human kit . In brief 2 μg of total RNA from human whole blood was mixed with a biotinylated Capture Oligo Mix in hybridization buffer. The mixture was incubated for 15 minutes to allow the biotinylated oligonucleotides to hybridize with the globin mRNA species. Streptavidin magnetic beads were then added, to capture the globin mRNA and the magnetic beads were then pulled to the side of the tube with a magnet and the RNA, depleted of the globin mRNA, was transferred to a fresh tube. The RNA was further purified using a rapid magnetic bead-based purification method .50 ng RNA samples were amplified using the WT-Ovation™ Pico RNA Amplification System  in a 3 step process as recommended by the manufacturer. All processes were performed in an automated manner using the genechip array station (GCAS). In brief, first strand cDNA was prepared using a unique first strand DNA/RNA chimeric primer mix and reverse transcriptase. In the second step, DNA/RNA Heteroduplex Double Stranded cDNA was generated which serves as the substrate for SPIA amplification - a linear isothermal DNA amplification process developed by NuGEN. RNase H degrades the RNA in the DNA/RNA heteroduplex at the 5´ end of the first cDNA strand exposing part of the unique priming site for the initiation of the next round of cDNA synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of SPIA cDNA. Following amplification and purification, 3 μg of the amplified cDNA were processed with the WT-Ovation Exon Module to produce sense strand ST-cDNA. Following purification and quantitation, 5 μg ST-cDNA was fragmented and labeled with the FL-Ovation™ cDNA Biotin Module using a proprietary two-step fragmentation and labeling process. The first step is a combined chemical and enzymatic fragmentation process that yields single-stranded cDNA products in the 50 to 100 base range. In the second step, this fragmented product is labeled via enzymatic attachment of a biotin-labeled nucleotide to the 3-hydroxyl end of the fragmented cDNA generated in the first step. Hybridization, washing and laser scanning of Affymetrix Human Exon 1.0 ST microarrays were performed according to the manufacturer’s protocol . Hybridization was performed at 45 °C overnight, followed by washing and staining using FS450 fluidics station. Scanning was carried out using the 7 G GCS3000 scanner.http://www.netaffx.com, HuEx-1_0-st-v2.na29.hg18.probeset.csv).Exon-level core RMA-sketch intensity values for each of the chips were collected using Affymetrix Expression Console (EC) Software . The 284,258 core probesets were annotated using the Affymetrix annotation file from Netaffx (t-test (SCD N1 = 6 and control N2 = 3) was computed on 9 samples in order to determine differential gene expression between SCD and controls. Microarray RMA values for each transcript cluster for each of 9 samples were submitted to a Principal Component Analysis in order to detect possible outliers. Alternative splicing analysis was computed using the ExonANOVA available from software developed by two of the authors, J.B and P.J.M (http://affylims.cit.nih.gov/MSCLtoolbox). The ExonANOVA model fits the following formulaGene level intensity values were obtained by taking the average RMA values over probesets for each transcript cluster. A two sample to the data.yijk is the log 2 expression intensity, i is treatment, j is replicate within treatment and k indexes exons. The μ is the mean and the two fixed factors are the treatment effect (A) and exon effect (C). The random factor (β) is the sample within treatment effect and (ϵ) is the error. The fixed interaction between treatment and exon (AC) models the alternative splicing event. In this study, the treatment effect is sickle cell or control. The significance of a detected alternatively spliced event is denoted p-AC. Alternatively spliced events are declared if p-AC < = 10^-8 and the maximum absolute interaction effect (maxik|ACik|) is greater than or equal to 2. A p-AC < = 10^-8 corresponds to less than 1% false discovery rate (FDR) using the method of Benjamini and Hochberg [In the above formula, Hochberg .http://www.illumina.com). Briefly, mRNAs were purified using poly-T oligo-attached magnetic beads and then fragmented. The first and the second strand cDNAs were synthesized and end repaired. Adaptors were ligated after adenylation at the 3'-ends. After gel purification, cDNA templates were enriched by PCR. cDNA libraries were validated using a High Sensitivity Chip on the Agilent2100 Bioanalyzer™ . The samples were clustered on a flow cell using the cBOT. After clustering, the samples were loaded on the Illumina GA-II machine. The samples were sequenced using a single lane with 36 cycles. Initial base calling and quality filtering of the Illumina GA-II image data were performed using the default parameters of the Illumina GA Pipeline GERALD stage (http://www.illumina.com).High quality total RNA at 1.5 μg was used for analysis on the Illumina GAII analyzer on six SCD samples and four healthy controls. cDNA library preparation and sequencing reactions were carried out using Illumina library prep, clustering and sequencing reagents following the manufacturer's recommendations (.The raw data Fastq sequence files obtained from GAII were mapped to the human genome (build HG18) to get genomic addresses using Bowtie/Tophat  allowingt-test  was used on the normalized, transformed data to test for differential expression. Alternative splicing analysis was computed using the ExonANOVA as with the microarrays data. A conservative and reasonable background limit at 4.5 RMA units was applied for Exon arrays and 1.0 in RPKM units as recommended by Mortazavi et al. [Transcript cluster level reads were counted per probeset within each transcript cluster. Very low count transcript clusters (fewer than 6 samples with 6 or more counts) were ignored. This filtered out a total of 7,146 transcript clusters leaving 11,562 for further statistical analysis. In order to normalize the data, transcript cluster counts were divided by the median transcript cluster count for that sample, and logarithm base 10 transformed, yielding transformed, normalized counts. After principal component analysis (PCA) of the transformed data, one outlier, a control sample was detected and disregarded from further analysis leaving 9 samples. A two sample i et al.  was applIn order to identify novel transcription events, we counted reads mapping to each 200 base pair region of the genome. Only populated bins (5 or more samples had 6 or more reads) bins were considered further. This filter retained 187,764 bins for analysis. We disregarded bins that fell within annotated RefSeq exons. The remaining 48,462 bins, describe novel, unannotated transcription events. To normalize these data, counts were divided by the 90th percentile of counts for that sample and base 10 logarithm transformed. p-values were required to be 0.0005 or less (corresponding to FDR < 0.15), and the fold-change was required to be 2-fold or greater. If differential expression was found, it was classified as a novel transcript.First-strand cDNA was synthesized using 500 ng of RNA and random primers in a 20 μl reverse transcriptase reaction mixture using Invitrogen’s Superscript cDNA synthesis kit  following the manufacturer’s directions. Quantitative real-time PCR assays were carried out on 11 differentially expressed genes from both the platforms with the use of gene-specific double fluorescently labeled probes in a 7900 Sequence Detector . Probes and primers were obtained from Applied Biosystems. In brief, PCR amplification was performed in a 384 well plate with a 20-μl reaction mixture containing 300 nm of each primer, 200 nm probe, 200 nm dNTP in 1x real time PCR buffer and passive reference (ROX) fluorochrome. The thermal cycling conditions were 2 min at 50° C and 10 min at 95° C, followed by 40 cycles of 15 sec denaturation at 95° C and 1 min annealing and extension at 60° C. Samples were analyzed in duplicate and the Ct values obtained were normalized to the housekeeping gene ß actin. The comparative CT (ΔΔ CT) method  which coPrincipal component analysis was first used to identify outliers within the SCD and healthy controls groups. Figure To assess the dynamic range  of each platform, a scatter plot Figure  was consThe technical reproducibility and coefficient of variation of the array and RNA-seq platforms at both the gene and exon levels was examined using the mean expression levels over all samples. The pooled coefficient of variation calculated over all samples were broken up into 4 bins  to identify such novel transcripts. We identified 86 novel regions that manifest a significant , greater than 2-fold change between sickle cell disease and control groups Table .One novel region was found within the differentially expressed ALAS2 gene. Figure https://dnanexus.com/) for SCD and control subjects in the neighborhood of the known single mutation driving sickle cell disease. In SCD, glutamic acid (coded by CTC) is replaced by Valine (coded by CAC). Figure RNA-seq is an attractive method that enables the identification of sequence variations in expressed transcripts. To illustrate the feasibility of identifying sequence variation in expressed genes, we visualized the sequence of reads from the beta globin gene  is a powerful transcriptional profiling technology using next generation sequencing platforms ,23-25 anAlthough attractive, clinical application of RNA-seq is feasible only if the tool can demonstrate high specificity, sensitivity and reproducibility with limited amount of starting material. Although current technology for transcriptome sequencing requires at least 100 ng total RNA , along with additional enrichment steps to select for poly(A) + RNA and/or to reduce the content of ribosomal RNA (rRNA) prior to NGS library construction, to minimize the loss of input material researchers tend to start with a minimum of 1 microgram total RNA. The utility of RNA-seq data generated is believed to be sensitive to read length, mapping and assembly of reads and statistical and computational challenges. However, with the current availability of substantially improved mapping software, these challenges are expected to be well tackled. Microarrays, on the other hand, are known to suffer from reduced dynamic range of signals due to saturation biases and high background, and non-specific or cross-hybridization resulting in false-positive signals, especially for transcripts that have low expression levels.Considering these challenges inherent to each of the high throughput platforms, we undertook this comparative study in an attempt to better understand the relative merits of high density exon microarrays and RNA-seq for biomarker discovery in the clinical setting. We chose the sickle cell disease because strong differential expression has been previously observed, and the phenotype of the disease is in manifested in blood, an accessible tissue for study. Sampling whole blood, a globin abundant tissue, allowed us to examine the potential interference of high abundant globin transcripts during sequencing and also to potentially discover novel genes that are associated with sickle cell disease from cell types such as nucleated red cells in addition to the conventional peripheral blood mononuclear cells. We believe that this is the first study that has compared the 2 platforms on a monogenic human disease model using easily accessible whole blood clinical specimens mimicking a large scale clinical research project.In this study we used 50 ng of total RNA on exon arrays without any globin reduction or poly(A) + enrichment to identify differentially expressed genes and alternative splicing events in sickle cell disease. The same samples were also analyzed by RNA-seq using 1.5 micrograms of total RNA. RNA-seq analysis generated an average of 83% mappable reads from the whole blood samples after poly(A) + enrichment. The globin reduction process had insignificant effect on the mappable read count, even for sickle cells samples which have high levels of globin RNA suggetsing that it is not necessary to reduce the globins while analyzing whole blood samples by RNA-seq.As expected, comparison of the dynamic range of the two platforms confirmed that RNA-seq has a dramatically larger range varying from 4 to 16 fold. This enticing feature of RNA-seq effectively removes the saturation biases inherent to the array platform. Examination of the technical reproducibility and coefficient of variation of the array and RNA-seq platforms at the gene and exon levels, as a function of the mean expression level, indicated that the coefficient of variation for microarray is much lower than that for RNA-seq and is also independent of the expression level for each transcript, suggesting that technical variability within group is higher in the RNA-seq platform than the arrays. A similar observation has been reported by Marioni et al. . They obOur comparative analysis of detection sensitivity with material from clinical samples revealed that even with the usage of 30 times less starting material (50 ng vs 1.5 micrograms) Exon arrays could detect as many transcripts above background as in RNA-seq. It should be noted that the sequencing depth in this study (~10 million reads) is comparable with most published RNA-seq studies. Xu and others  from theWe found 331 transcripts with differentially expressed transcripts in SCD. These included genes involved in pathways related to sickle cell disease such as inflammatory response, oxidoreductase pathways, stress response, cell signaling and apoptosis. Of these 331 transcripts which showed a high degree of correlation (R = 0.64), 96 genes were identified by both the technologies. A similar observation has also been reported by correlating gene expression arrays and RNA-seq on their study on differentially expressed liver and kidney tissues . Only 11Gene ontology analysis of these genes helped to classify their molecular functions into ten highly significant functional pathway such as cellular cycle regulators, apoptosis, oxidative stress response, inflammation and immune response, free radical scavenging, protein modification, and hematopoiesis. Examination of these pathways suggests that differentially regulation may be in response to oxidant and hemolytic stress, vascular injury and participation in repair and homeostasis ,30-34. IWe also observed upregulated expression of several reticulocyte specific genes as expected in SCD where a higher proportion of reticulocytes are observed. This finding validates the performance of both technologies in identifying alterations relevant to sickle cell disease. From a biological perspective, the whole blood expression profile provided a window into real time erythrocyte expression profiles. Insights into the transcription profile of these red blood cells may contribute greatly to our understanding of mechanism of disease, prognosis, and responses to therapeutics.RHCE and RHD are components of the important Rh antigen system on red cells. However the potential implications of altered Rh splicing in SCD is still unclear. Deficiency of UNC13D is known to result in defective exocytosis of cytolytic granules of cytotoxic T lymphocytes and natural killer cells, causing immune dysregulation [Using ExonAnova analysis on RNA-seq data, we identified 16 alternatively spliced genes. While further validation of these splice variants is needed, it is interesting to note that both gulation . WhetherALAS2 gene with SCD patients showing at least six times higher expression levels compared to the control subjects (p = 0.003). This could suggest alternative splicing in SCD which might serve as an ALAS2 transcription regulator. Follow up of this suggestion would require a functional analysis of this newly identified region of ALAS2 but is beyond the scope of the current study but is planned for the future. ALAS2 gene expression is restricted to the erythroid lineage [To illustrate the power of RNA-seq in detecting differential transcription not associated with known genes, we scanned the entire genome for novel differential expression focusing only on unannotated genomic regions. By doing so, we found an interesting region to include an apparently novel, minor exon between exons 4 and 5 in the  lineage  and play lineage . This abAnother key advantage of RNA-seq over existing technologies for transcriptomic studies is its ability to identify sequence variations in expressed transcripts. To illustrate the feasibility of identifying sequence variation in expressed genes, we focused on the known single nucleotide mutation in SCD in which glutamic acid-6 is replaced by valine (GAG replaced by GTG). We were able to successfully detect this mutation in all the sickle cell patients. Interestingly, we were also able to identify, that same mutation in heterozygous combination with a hemoglobin C beta globin variant having glutamic acid replaced by lysine (GAG replaced by AAG) in one compound heterozygous sickle cell patient, thereby demonstrating the ability of RNA-seq to reliably identify heterozygous single base mutations in the expressed transcripts.In conclusion, our study clearly illustrates a high level of concordance between the array platform and the RNA-seq technology, and suggests that the high density Exon array still remains a powerful tool to generate meaningful data when the amount of material is limited. Although RNA-seq is still in the early stages of use in clinical studies, it has clear advantages over the array based transcriptomic methods, based on its ability to discover novel transcripts, identify sequence variants, and increased dynamic range of signals leading to increase fold change in measured expression levels. With the rapid evolution of NGS instruments and library preparation methods with multiplexing barcodes, longer read lengths and large number of paired end reads associated with reduced cost per lane is highly feasible in the near future. The use of picogram to few nanogram amounts to total RNA for RNAseq still needs to be optimized in order to capture low abundance transcripts.We believe that the results from this study provide guidelines on the choice of tools in the form of arrays or RNA-seq for clinical transcriptomic studies using limited amount of starting material. We believe that the selection of an appropriate tool for clinical genomic studies is mostly driven by the biological question underlying the study: whether a formal hypothesis is being tested or is the study intended to better describe the complete transcriptome and discover novel transcripts. An emerging approach is to apply both RNA-seq and arrays in combination, in large scale clinical studies, where RNA-seq is used first to define the transcriptome elements associated with the disease in question, followed by high throughput and reliable screening of these elements on thousands of patient samples using the arrays. Integrating data from both microarray and RNA-seq experiments may open up new possibilities for creating meaningful informational networks which will aid our understanding of disease pathology and development of novel therapeutics.The authors declare that they have no competing interests.NR designed the study, wrote the manuscript, NR, PL, KW, performed the experiments. YY helped in mapping the NGS data. JB, PJM performed data analysis and edited the manuscript. GK initiated the NIH protocol for patient recruitment, sample collection and edited the manuscript. DL and CJO funded the study and edited the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/5/28/prepubFigure S1. Gene Ontology analysis on the differentially expressed genes. The top 13 highly significant classification/functions of genes are shown in the figure.Click here for fileFigure S2. Effect of Globin Reduction on RNA-seq expression. Y-axis: Read counts per transcript, normalized by median for globin reduced sample; X-axis: Read counts normalized by median for same sample using standard preparation. Correlation coefficient is 0.93. Click here for fileTable S2. Validation of few differentially expressed genes by QPCR.Click here for fileFigure S3. Putative Exon 4A . UCSC Genome Browser view of the BAM files for each sample showing genomic region chromosome X: 55067500–5506725. The first 3 aligned tracks show the control samples, the following 5 tracks show the sickle cell disease samples. Aligned reads are Red if to the negative strands and blue if to the positive strands. The total reads per sample is: C3: 15,715,705, C4: 15,131,360, C5: 15,730,372, S6: 16,570,843, S1: 13,481,528, S2: 16,707,788, S3: 14,650,161, S4: 18,580,778 and S5: 16,460,443. S6: 16570843, S1.Click here for fileTable S1. Complete List of differentially expressed genes (n = 331). Click here for file


For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry.Functional Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites  was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay.Both asynchronous and tightly synchronized asexual blood stage cultures of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry.Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of in vitro assays such as ADCI assay.A flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional  The antibody-dependent cellular inhibition (ADCI) assay may provide one such tool [Plasmodium falciparum-specific antibodies can co-operate with human blood monocytes to control parasite multiplication in vivo and have accordingly developed the in vitro correlate of this immune effector mechanism – the antibody-dependent cellular inhibition (ADCI) assay [in vivo relevance of a monocyte-dependent, antibody-mediated mechanism by showing a correlation between the acquisition of clinical immunity and levels of cytophilic IgG subclasses (IgG1 and IgG3) [in vitro ADCI assay [in vitro ADCI assays [Development of vaccines for malaria remains a high priority in the effort to control malaria worldwide. Blood-stage vaccines are important components of these efforts and functional uch tool . DruilheI) assay . Immune-nd IgG3) . Cytophind IgG3) ,6. Upon nd IgG3) ,7. AmongCI assay ,9. When I assays ,11. ThesI assays -15. A roP. falciparum growth in vitro has been monitored by microscopy, radioactive hypoxanthine uptake [Historically, e uptake , and by e uptake . Radioace uptake . These se uptake ,20. Thuse uptake , SYBR Gre uptake -24, thiae uptake , acridine uptake , ethidiue uptake , hydroete uptake ,29, SYTOe uptake , or prope uptake  have beee uptake . Dyes whe uptake . Howevere uptake . Moreovee uptake ,36 and de uptake . CMXRos e uptake . Of the e uptake  and rhode uptake  have preP. falciparum strain NF54 was cultured as previously described [+ve, human erythrocytes  in parasite growth medium (PGM) consisting of RPMI 1640  supplemented with 25 mM HEPES, 2 mM L-glutamine, 24 mM NaHCO3, 25 μg/ml gentamicin and 10% (v/v) heat-inactivated human AB serum in an atmosphere containing 5% O2, 5% CO2 and 90% N2 at 37 °C. Parasites were synchronized for ring stages by repeated sorbitol treatments [The laboratory-adapted escribed  with slieatments  and schieatments . The preParasitaemia was determined by microscopy of standard blood smear. In brief, thin blood smear were prepared on glass slides and fixed with 100% methanol. Smears were stained with filtered 1/10 dilution of Giemsa  solution in phosphate buffered saline (PBS), incubated for 20 min at room temperature, washed with distilled water, dried, and observed under oil immersion lens (100x). Parasitaemia was determined by counting approximately 5,000 erythrocytes.The CMXRos dye (8-(4’-chloromethyl) phenyl-2,3,5,6,11,12, 14,15-octahydro-1 H,4 H,10 H,13 H-diquinolizino-8 H-xanthylium chloride) was prepared as per the manufacturer’s recommendation . CMXRos dye was dissolved in DMSO at a concentration of 1 mM and stored at -20 °C until use. A 5 μM working solution in pre-warmed PGM was prepared directly from the stock solution just prior to staining cultured parasites.6 cells were harvested by centrifugation in a 96-well, round-bottom plate  at 2,000 rpm for 5 min, re-suspended in 100 μl of 5 μM CMXRos or 0.5 μM coriphosphine O  in warm PGM, and incubated at 37 °C for 30 min in the dark. Hydroethidine was used for staining as described earlier [4 events or more than 300 infected erythrocytes were recorded on a Beckman Coulter (cytomics FC500 MPL) and plotted in a two-dimensional scattergram of FL3 and FSC or SSC in the logarithmic display. Bound CMXRos dye was visualized by excitation at 488 nm by an argon ion laser [For enumeration of parasitaemia by flow cytometry, approximately 10on laser .P. falciparum NF54 at the mid-trophozoite stage (~4% parasitaemia) was treated with a mixture of atovaquone-proguanil (150 μg/ml) for 30 min. For starvation, a highly synchronized culture of P. falciparum NF54 at the mid-trophozoite stage was subjected to temperature changes with minimal quantities of PGM. Briefly, the culture at an initial parasitaemia of 3% was kept at 4 °C for 24 hours, incubated overnight at 37 °C, and transferred again to 4 °C for eight hours. Growth medium was not replenished during this treatment. Both conditions included a control culture, which was not subjected to any of the treatments. Parasitized erythrocytes were enumerated by flow cytometry after staining with CMXRos, coriphosphine O, or hydroethidine. Parasites were also visualized by microscopy after staining with Giemsa before and after the treatment. CMXRos and DAPI pictures were taken immediately after mounting a drop of the stained parasite suspension on a slide.A highly synchronized culture of Total IgG from clinically immune Liberian adults and Danish blood donors (NIG) never exposed to malaria was purified as previously described . Five Li2 for adherence of monocytes. After incubation, wells were washed three times with 150 μl PGM thereby separating non-adherent mononuclear cells from attached monocytes (~2 × 105 monocytes per well). It has previously been observed that monocytes from individual donors may either enhance or inhibit P. falciparum growth [P. falciparum growth in vitro. Of these, monocytes from six donors neither enhanced nor inhibited parasite growth in the absence of IgG. Monocytes purified from either fresh or frozen PBMCs from two of these donors, repeatedly gave similar SGI values (~50%) in presence of HIG and were therefore used throughout this study. Adhered monocytes were co-cultured with highly synchronized mature P. falciparum NF54 schizonts at a 0.5% parasitaemia and a 2.5% haematocrit. Purified IgG dialyzed against RPMI medium was added to wells at the indicated concentration. Final volume in each well was adjusted to 200 μl with PGM. In addition to test IgGs, the following controls were run simultaneously in every experiment on each plate: (i) parasite culture without monocytes, (ii) culture with monocytes and without IgG, (iii) culture with NIG, (iv) culture with monocytes and NIG, (v) culture with HIG, (vi) culture with monocytes and HIG. At 48 hours and 72 hours an additional 50 μl of PGM was added to each well. At the end of the assay (96 hours), parasitaemia was determined in each well by flow cytometry after staining with CMXRos dye and by Giemsa microscopy. In ADCI, detached monocytes could be distinguished from parasitized erythrocytes after plotting acquired data in a two-dimensional scattergram of FL3 and SSC since SSC separate cell populations according to their granularity. The specific growth inhibitory index (SGI) which estimates the parasite growth inhibition due to the effect of test antibodies (Abs) co-operating with monocytes (MN) was calculated as follows: SGI = 100 × (1 − (% parasitaemia with MN and test Abs/% parasitaemia test Abs)/(% parasitaemia with MN and NIG/% parasitaemia NIG)).Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy Danish blood donors never exposed to malaria by LymphoPrep  and used as a source of monocytes for ADCI. One million PBMCs were added to 96-well, flat-bottom culture plates  in monocyte medium  and incubated for two hours at 37 °C and 5% COm growth ,47. MonoBland-Altman test was used for assessing agreement between two methods of estimating parasitaemia. Differences in parasitaemia estimated by each dye before and after drug treatment were analysed using two-way ANOVA for overall comparison, followed by the Bonferroni test for pairwise comparisons. All statistical analysis was carried out using GraphPad Prism software version 4.0 .P. falciparum NF54 was stained with CMXRos dye. The staining of the cells were observed under fluorescence microscopy and evaluated through flow cytometry. The uninfected erythrocytes did not show any detectable staining when observed through microscopy and appeared as a tightly clustered population in flow cytometry with minimal fluorescence  difference in parasitaemia measured by the three dyes. This difference was due to a significant  reduction in parasitaemia measured by CMXRos staining but not by coriphosphine O and hydroethidine staining Figure A. This io Figure B. These In order to determine the ADCI effect mediated by antibodies through co-operation with human monocytes and for more accurate calculation of SGI values, parasitaemia was determined by flow cytometry after CMXRos staining, with minimum 30,000 events recorded for each culture sample. This method allowed evaluation of a larger and more homogenous proportion of live, parasitized cells within a culture population without subjective errors associated with classical microscopy counts. The reproducibility of SGI values from 12 independent experiments of purified IgG from hyper-immune Liberians (HIG) and Danish blood donors (NIG) never exposed to malaria were evaluated for ADCI effect Figure A. SGI vaOptimized ADCI assay, using Mitotracker Red CMXRos staining and flow cytometry as readout to replace the labour-intensive microscopy was proposed, thus allowing for high throughput and reproducible operator-independent readout method. The main advantage of Mitotracker Red CMXRos dye is that it specifically stains live parasites and binds irreversibly to the polarized mitochondrial membrane, thereby potentially increasing the accuracy of the readout in the ADCI assay. Using monocytes from two pre-selected blood donors and the CMXRos-based flow cytometry readout for obtaining live parasite counts, it has been demonstrated that HIG (1 mg/ml) in 12 independent experiments gives highly reproducible SGI values of approximately 40% with an inter-assay coefficient of variation (CV) of 18.22%. In contrast, IgG from a Danish blood donor never exposed to malaria gave SGI values of approximately 0%. Since the two IgG preparations contain similar IgG subclass profiles, it was concluded that the differences in ADCI activity were due to parasite-specific IgG antibodies in the HIG. In some experiments NIG gave a SGI value below zero. A negative SGI value can be attributed to enhancement of parasite growth, which has also been observed in other ADCI studies . SGI valP. falciparum mitochondria. Accordingly, JC-1 has also been used at a similar high concentration for the staining of the P. falciparum parasites [In ADCI, cross-linking of Fcγ receptors is thought to activate human monocytes to secrete inflammatory cytokines, such as TNF , known tarasites .To verify the specificity of the flow cytometry method, a parasite culture was treated with the combination drug, atovaquone-proguanil. Atovaquone causes depolarization of mitochondrial membrane potential  leading A new method for enumeration of parasitaemia based on flow cytometry was developed after Mitotracker Red CMXROS staining. Using this method as readout, the ADCI assay is able to differentiate between IgG samples from hyper-immune and malaria-naïve individuals in a highly reproducible manner with an inter-assay coefficient of variation below 20% for the highest concentration of test IgGs. Moreover, the SGI value obtained is a reflection of the level of parasite-specific antibodies in the test sample. This method is suitable for high-throughput analysis of samples from clinical trials and possible for future down-selection of blood-stage vaccine candidates.CMXRos, Mitotracker Red CMXRos; HIG, pool of hyper immunoglobulins; NIG, pool of normal immunoglobulins; GLURP, glutamate-rich protein.The authors declare that they have no competing interests.PSJ, SS and MT conceived the study. PSJ, SKS, MC and MD performed the laboratory work and the statistical analysis. PSJ, SKS, SS and MT wrote the manuscript. All authors have read the manuscript and agree with its contents.