Phytohormones play a critical role in mediating plant stress response. They employ a variety of proteins for coordinating such processes. In Arabidopsis thaliana, some members of a Cys-rich protein family known as C1-clan proteins were involved in stress response, but the actual function of the protein family is largely unknown. We studied At5g17960, a C1-clan protein member that possesses three unique C1 signature domains viz. C1_2, C1_3 and ZZ/PHD type. Additionally, we identified 72 other proteins in A. thaliana that contain all three unique signature domains. Subsequently, the 73 proteins were phylogenetically classified into IX subgroups. Promoter motif analysis of the 73 genes identified the presence of hormone-responsive and stress-responsive putative cis-regulatory elements. Furthermore, we observed that transcript levels of At5g17960 were induced in response to different hormones and stress treatments. At1g35610 and At3g13760, two other members of subgroup IV, also showed upregulation upon GA3, biotic and abiotic stress treatments. Moreover, seedlings of independent transgenic A. thaliana lines ectopically expressing or suppressing At5g17960 also showed differential regulation of several abiotic stress-responsive marker genes. Thus, our data suggest that C1-domain-containing proteins have a role to play in plant hormone-mediated stress responses, thereby assigning a putative function for the C1-clan protein family. Plants demonstrate tolerance or resistance against such adverse conditions by the interplay of several fine-tuned strategies. One such strategy involves phytohormones that serve as key endogenous signals in mediating biotic and abiotic stress responses. The major hormones produced by plants are auxins, gibberellins (GA), cytokinins (Ck), abscisic acid (ABA), ethylene (ET), salicylic acid (SA) and jasmonates (JA). Synergistic and antagonistic interactions between different hormones, also known as hormone crosstalk, provide the plants with a powerful mechanism to regulate their defense response , 2. For ignaling .cis-regulatory elements present in the promoter region of target genes [cis-elements are the functional DNA elements that control transcriptional activity. Analyses of upstream promoter regions of genes for the identification of cis-elements have been instrumental in understanding the major regulatory networks underlining different stress responses. For example, analysis of promoters of A. thaliana genes differentially expressed upon ABA or abiotic stress treatments identified ABA response elements [is-regulatory elements known to control osmotic stress response were used to identify other functionally relevant genes involved in the same process [The maintenance of such an intricate nexus of phytohormone signaling for plant defense response involves signal perception, signal transduction and eventually transcriptional regulation of target genes. Therefore, a better understanding of the stress response cascades necessitates a deeper insight into the gene regulatory networks. Fundamentally, transcriptional regulation requires binding of transcription factors (TFs) to their specific et genes . Thus, celements . Further process .NtDC1A and NtDC1B, two genes encoding divergent C1 (DC1)-domain-containing zinc-finger protein homologs, were identified as early response genes in tobacco BY-2 cells treated with glucan-enriched fraction from Alternaria alternata 102 [A. thaliana [At5g17960 that encodes a C1-domain protein whose role in stress responses may be studied further.Members of specific gene families play key roles in mediating stress responses in plants. For example, nata 102 . Similarnata 102 . Furthernata 102 . In a prthaliana , we idenviz., C1_2, C1_3 and ZZ/PHD type. We also identified 72 other proteins in A. thaliana that contain these three C1 domains and subsequently classified them into nine distinct subgroups based on their phylogenetic relationship. Promoter motif analyses of these 73 genes identified the presence of several hormone-responsive and stress-responsive putative cis-elements. Consequently, At5g17960 was examined for its response to different plant hormones as well as various biotic and abiotic treatments. The gene showed significant upregulation in response to GA, SA, JA, ET and various stress conditions. We generated transgenic lines overexpressing and supressing At5g17960 for studying the early molecular responses to various stress treatments. The transgenic seedlings exhibited differential expression profiles for selected stress-inducible marker genes. Thus, our data suggest that At5g17960 is a hormone-responsive C1-domain-containing protein that mediates abiotic and biotic stress responses in A. thaliana. Based on the data, we discuss the probable mode of action of C1-domain-containing proteins as a link between phytohormone signaling and plant stress responses.In this study, we show that At5g17960 possesses three C1 domains, A. thaliana ecotype Columbia-0 (Col-0) (WT) plants were used in this study. The plants were grown at 22°C with 16 h light/8 h dark conditions at 75% RH. For seedling assays, the seeds were surface-sterilized by rinsing with 70% ethanol for 2 min followed by washing with 15% bleach for 5 min. Subsequently, the seeds were washed four times with sterile water. The surface sterilized, seeds were stratified at 4°C for 3 days in the dark. About 10–15 seeds were transferred to a 50 ml falcon tube with 10 ml Murashige and Skoog (MS) liquid medium (Caisson LABS) containing 1X MS and 2% glucose. The tubes containing the seeds were incubated at 22°C with shaking at 90 rpm under constant white light (~50 µE/m2/s) for growth.35S::At5g17960, full-length coding sequence of At5g17960 was PCR amplified using A15 FP and A15 RP and cloned into pJET vector (Thermo Scientific). Subsequently, the gene was released using XhoI/HindIII and cloned into pGREEN 35S vector digested with the same restriction enzymes. To construct amiR::At5g17960, amiRNA sequence (5’-GCGGGAAGCAAGTATCCACTT-3’) was designed using the amiRNA designer interface WMD [For face WMD , 14. TheA. thaliana plants were generated by Agrobacterium tumefaciens (strain GV3101:pMP90)-mediated floral dip method [Transgenic p method . In the 3 treatment, 12-day-old WT seedlings were incubated in 1/2X liquid MS supplemented with 10 µM GA3 and samples were collected and snap frozen in liquid nitrogen after 30, 60 and 90 min of treatment. For ethylene treatment, 12-day-old seedlings were treated with 10 µM 1-aminocyclopropane-1-carboxylic acid (ACC) for 30 and 60 min. ACC stock was prepared in water. For SA and MeJA treatments also, seedlings were incubated in 1/2X MS liquid supplemented with 10 µM SA and 10 µM MeJA, respectively. However for SA treatment, samples were collected at 30, 60 and 180 min, whereas, MeJA, treatment was only given for 30 and 60 min as specified in the figure legends.For GAFor biotic stress treatment, 12-day-old WT seedlings were treated with chitin at a final concentration of 10 mg/l in 1/2X MS liquid medium for 20, 30 and 60 min. Chitin solution was prepared by grinding shrimp shell chitin (Sigma Aldrich) to fine powder and subsequently, left overnight in sterile water for proper dissolution and finally filtered through muslin cloth.2) for 10 min and then transferred back to liquid MS for revival and collected after 30 and 60 min time points. For cold treatment, 12-day-old seedlings were incubated at 10°C for 60 and 180 min.Among the abiotic stress treatments, salt treatment was given by treating 12-day-old WT and transgenic seedlings with 200 mM NaCl or sterile water (mock treatment) in 1/2X MS solution for 20, 30 and 60 min. Drought treatment was given by leaving the seedlings in the laminar flow hood for 20 min to allow dehydration and subsequently transferred to 1/2X MS liquid medium for revival and samples were collected after 20 and 30 min time points. For UV irradiation treatment, seedlings were exposed to UV  as per the manufacturer’s protocol. About 500 ng of RNA for each sample was reverse transcribed using Maxima First strand cDNA synthesis kit (Thermo Scientific) as per the manufacturer’s protocol. The cDNA samples were diluted 5-fold before use. The reaction mixture for qRT-PCR included 1 µl diluted cDNA, 5 µl 2x KAPA SYBR Master Mix (KAPA Biosystems), 0.2 µl each of the two primers (forward and reverse) and sterile water to a final volume of 10 µl. qRT-PCR was done using StepOne Real-Time PCR Systems  with denaturation at 95°C for 2 s followed by 40 cycles of denaturation at 95°C for 3 s and annealing and extension at 60°C for 30 s. The data analysis was done using StepOne software . For normalization of the data, amplification of ry table  for infot-test (two-tailed) with level of significance P ≤ 0.05.The expression analyses data represent means of three independent biological replicates each with three technical replicates. Statistical analysis was carried out using Student’s http://www.arabidopsis.org/) database. The ClustalW2 program was used for multiple sequence analysis of all the 73 proteins. Phylogenetic tree for the 73 sequences (amino acid) was constructed with MEGA v5.0 using the neighbor-joining (NJ) method and Poisson correction method [Protein domain analysis was performed using Pfam 24.0 . The genn method , 18. TheA. thaliana promoter sequence database.Consensus sequences for the three conserved domains were verified by MEME version 4.9.0. . For knocis-elements were detected by the Dragon Motif Builder program with EM2 option [-3 among the promoters investigated. The biological significance of these motifs was verified by their presence in Transcription Factor Binding databases such as TRANSFAC [Promoter sequence motifs corresponding to previously identified or putative 2 option . Thirty TRANSFAC , PLACE [TRANSFAC  and AGRITRANSFAC . The perA. thaliana genome that encode proteins containing all three unique C1 domains . We also scanned the PLAZA database for understanding the divergence of these domains in other plant species. Besides A. thaliana, Oryza sativa and Populus trichocarpa also showed significant divergence for the different C1 domains  . Pfam do domains .2CX12CX2CX8C: 29 amino acids), C1_3 domain (CX2CX11CX2CX7C: 27 amino acids) and ZZ/PHD type domain (CX2CX11CX2CX7CX28CX2C: 59 amino acids)  .A. thaliana chromosomes. The genes were distributed in all five chromosomes of A. thaliana, but compared to other chromosomal arms, the density of distribution of the C1-clan genes was the least along the upper arm of Chromosome 1 -like, Pyrimidine box-like, as-1/ocs-like, JA response element (JAre)-like, Auxin response element (AuxRE)-like, early response to dehydration stress (ERD1)-like, ARABIDOPSIS RESPONSE REGULATOR 10 (ARR10)-B element-like, W box-like, AAAG element-like and others were found to be highly enriched , MYB , bZIP, ERF, ARF1, NAC, ARR-B, WRKY, DOF and others. The total enrichment scores for the different classes of TF are presented in To investigate the possible TFs involved in the transcriptional regulation of these 73 genes, we identified several conserved motifs present in the promoter regions (-1000 to +200 relative to TSS) of these genes. Among the motifs detected, the putative enriched . These pcis-elements, such as GARE-like (gibberellin response element), ARR10-B element (cytokinin response element) [At5g17960 by different phytohormones. GA3 treatment to 12-day-old WT seedlings showed ~19-fold increase in the transcript levels of At5g17960 after 30 min, in comparison to the mock treatment at the same time point  , JAre-lielement) . Hence, me point . ACC treon by ET . Upon SAon by ET . MeJA trr 30 min . These dAt5g17960 in the phylogenetic tree, viz., At1g35610 and At3g13760, for their response to GA3 treatment  and abiotic  stress treatments within 60 min.Among abiotic stress conditions, drought treatment caused ~6-fold upregulation of ery time . Upon come point . Similart3g13760 . AltogetAt5g17960 affects stress response, we generated transgenic lines with 35S::At5g17960  and amiR::At5g17960 (for suppressing At5g17960). For each construct at least 7 independent transgenic lines were obtained (At5g17960 #9–1) and amiR line (amiR::At5g17960 #1–1) were used for functional analyses. Furthermore, to study the impact of overexpression and suppression of At5g17960 on stress response, we selected well-characterized stress-responsive marker genes, one for each of the four abiotic stress conditions, DREB2A , RD29A (drought stress), COR15A (cold stress) and ELIP2 (UV stress) [DREB2A was observed in the amiR line at both 30 and 60 min time points, as compared to the WT  , 28. Upoo the WT . Howevert 20 min . On the f COR15A . Howevern the WT . The afocis-elements  in hormonal crosstalk-mediated plant stress response.Abiotic and biotic stress conditions not only affect plant growth and development adversely, but also affect huge yield losses worldwide . To counAt5g17960, At1g35610 and At3g13760 in response to different stress treatments, because proteins encoded by the three genes are clustered together in the same subgroup IV of the phylogenetic tree  in 35S::At5g17960 #9–1 and amiR::At5g17960 #1–1 transgenic seedlings, upon stress treatments Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 FigArabidopsis thaliana. The gene name is followed by amino acid position, followed by P-value and the amino acids corresponding to C1_2 domain are highlighted in color.The multiple sequence alignment of (A) C1_2 domain, (B) C1_3 domain and (C) ZZ/PHD domain 73 C1-clan proteins of (PDF)Click here for additional data file.S2 Fig(A) The C1_2 consensus sequence is from amino acid 1–29, in total 29aa. (B) The C1_3 the consensus sequence is from amino acid 26–52, in total 27aa. (C) The ZZ/PHD type the consensus sequence is from amino acid 2–60, in total 59aa. The derived consensus sequence is shown below each diagram. X-axis refers to position of amino acid. Y-axis depicts the degree of consensus based on the size of amino acid i.e. the larger the size of amino acid symbol the more conserved it is.(PDF)Click here for additional data file.S3 FigA. thaliana. The green bars represent the A. thaliana chromosomes (numbered one to five at the top of each bar). The 73 genes are represented by their accession numbers. Chromosome mapping was performed using the TAIR online Chromosome map tool.The 73 genes were mapped to the five chromosomes of (PDF)Click here for additional data file.S4 FigAt5g17960 were significantly upregulated in the leaves of 7 selected 35S-At5g17960 independent lines in the T3 generation. (B) Transcript levels of At5g17960 were significantly decreased in the leaves of 8 selected amiRNA-At5g17960 independent lines in the T3 generation. Transcript levels in (A) and (B) were determined by qRT-PCR and are shown relative to TUB2 expression. Values are the mean ± standard deviation of three independent biological replicates each with three technical replicates.(A) Transcript levels of (PDF)Click here for additional data file.


The inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), result from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the genetic heritage.Using a candidate gene approach, 39 mainly functional single nucleotide polymorphisms (SNPs) in 26 genes regulating inflammation were assessed in a clinical homogeneous group of severely diseased patients consisting of 624 patients with CD, 411 patients with UC and 795 controls. The results were analysed using logistic regression.TLR2 (rs1816702), NFKB1 (rs28362491), TNFRSF1A (rs4149570), IL6R (rs4537545), IL23R (rs11209026) and PTPN22 (rs2476601) were associated with risk of CD and the polymorphisms TLR2 (rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs352139), LY96 (rs11465996), NFKBIA (rs696), TNFA (rs1800629), TNFRSF1A (rs4149570), IL10 (rs3024505), IL23R (rs11209026), PTPN22 (rs2476601) and PPARG (rs1801282) were associated with risk of UC. When including all patients (IBD) the polymorphisms TLR2 (rs4696480 and rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs187084), TNFRSF1A (rs4149570), IL6R (rs4537545), IL10 (rs3024505), IL23R (rs11209026) and PTPN22 (rs2476601) were associated with risk. After Bonferroni correction for multiple testing, both the homozygous and the heterozygous variant genotypes of IL23R G>A(rs11209026)  and PTPN22 1858 G>A(rs2476601)  were associated with reduced risk of CD.Sixteen polymorphisms in 13 genes involved in regulation of inflammation were associated with risk of CD and/or UC (p≤0.05). The polymorphisms TLRs suggest that the host microbial composition or environmental factors in the gut are involved in risk of IBD in genetically susceptible individuals.The biological effects of the studied polymorphisms suggest that genetically determined high inflammatory response was associated with increased risk of CD. The many SNPs found in  Chronic inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis (UC), are complex diseases that result from the interaction of numerous genetic and environmental factors Genetic association studies have identified innate immunity as a critical component in the development of IBD. Until now, more than 163 IBD susceptibility polymorphisms have been confirmed, most of which are associated with both CD and UC, by candidate and genome wide association studies (GWAS) Functional polymorphisms in genes in the inflammatory pathways may explain some of the genetic heritage involved in IBD. The transcription factor NFκB is a central regulator of inflammation. NFκB can be activated by Toll like receptors (TLRs). TLRs recognize pathogen-associated molecular patterns (PAMPs) that are broadly shared by pathogens but distinguishable from host molecules such as bacterial or viral DNA, flagellin or lipopolysaccharide (LPS). The TLRs initiates a kinase cascade that ultimately activates the IKK-complex, which phosphorylates and degrades the NFκB inhibitor IκBα. NFκB is shuttled from the cytosol to the nucleus where it initiates expression of pro- and anti-inflammatory cytokines including TNF-α, IL-6 and IL-10 To identify susceptibility loci we assessed 39 mainly functional polymorphisms in genes involved in inflammation, particular in the NFκB pathway, in a homogeneous Danish cohort of 624 patients with severe CD, 411 patients with severe UC and 795 healthy controls. The candidate gene approach using functional polymorphisms allows interpretation of the underlying biological mechanisms based on increased or decreased gene expression or protein activity.TLR2, TLR4, TLR5, TLR9, LY96, CD14, MAP3K14, SUMO4, NFKBIA and NFKB1), TNF-α signaling , cytokines regulated by NFκB  and other genes involved in regulation of inflammation  were studied, as genetically determined variation in the inflammatory pathways may be associated with severe disease among patients with CD and UC.Functional polymorphisms in genes in the inflammatory pathways involved in regulation of the NFκB pathway  and the Department of Respiratory Diseases B or the Department Clinical Microbiology, Aarhus University Hospital  were collected from 01.09.2009 to 30.03.2011 (9217 patients). Patients with intestinal diseases (ICD-10 code K50–K63) were identified by linking the unique personal identification number of Danish citizens (CPR-number) from each blood sample with the National Patient Registry (2659 patients). Patient records from 18 medical departments were examined (1378 patients) and identified 1035 ethnic Danish patients with IBD where blood and clinical data were available. The patients either received or were considered candidates to anti-tumor necrosis factor-α (TNF-α) therapy . The control group consisted of 795 healthy blood donors recruited from Viborg, Denmark A prior anti-TNF naïve Danish cohort of patients with IBD was established. In short, blood samples retrieved as part of the routine screening for latent TLR2 and TLR4 were included A candidate gene approach was used with focus on polymorphisms in the TNF-α and NFκB pathways. In addition, polymorphisms in genes which have been shown to be associated with CD and/or UC, polymorphisms in inflammatory cytokines and polymorphisms in Functional polymorphisms in relevant genes were found by searching pubmed with “polymorphism AND gene-name AND (reporter gene OR luciferase OR ELISA OR enzyme-linked immunosorbent assay OR RT-PCR OR reverse transcriptase PCR OR EMSA OR electrophoretic mobility shift assay OR flow cytometry)”.http://www.lgcgenomics.com/). The SNPs studied were TLR2 , TLR4 , TLR5 (rs5744168), TLR9 , LY96 (MD-2) (rs11465996), CD14 (rs2569190), MAP3K14 (NIK) (rs7222094), SUMO4 (rs237025), NFKBIA (IκBα) , NFKB1 (NFκB1) (rs28362491), TNFA (TNF-α) , TNFRSF1A (TNFR1) (rs4149570), TNFAIP3 (A20) (rs6927172), IL1B (IL-1β) , IL-1RN (IL-1RA) (rs4251961), IL4R (rs1805010), IL6 (rs10499563), IL6R (rs4537545), IL10 , IL17A (rs2275913), IL23R (rs11209026), IFNG (IFN-γ) (rs2430561), TGFB1 (TGF-β1) (rs1800469), PTPN22 (rs2476601), PPARG (PPAR-γ) (rs1801282) and NLRP3 (rs4612666).For patients with IBD the DNA was extracted from cryopreserved blood clots by using the Maxwell 16 Blood purification kit (Promega) according to the manufacturers' instructions with a median yield of 4.90 µg (range 0.8–25 µg) pr 300 µl total blood TNFA (TNF-α) −857 C>T (rs1799724) and −863 C>A (rs1800630) failed due to their close proximity to each other. All genotyping of −857 C>T (rs1799724) either failed or were erroneously genotyped as homozygous wild type when the patients were carriers of the AA genotype of −863 C>A (rs1800630) due to genotyping bias.Genotyping of The 39 genotypes were replicated in 94 randomly selected samples and yielded >99% identical genotypes.Logistic regression was used to compare genotype distributions among patients with CD, UC and IBD versus healthy controls  and S2. Statistical analyses were performed using STATA version 11 .The study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committees of Central (M20100153) and Southern (S-20120113) Denmark and the Danish Data Protection Agency of Central (RM: J. 2010-41-4719) and Southern (RSD: 2008-58-035) Denmark. The Ethics Committees gave suspension for obtaining written informed consent.Characteristics of the Danish patients with CD, UC and healthy controls are shown in TLR2 (−16934 A>T (rs4696480)) (p = 0.02), TLR4 (rs1554973 T>C) (p = 0.03), TLR9 (1174 G>A (rs352139)) (p = 0.02), and TGFB1 (−509 C>T (rs1800469)) (p = 0.02). None of the deviations remained statistically significant after correction for multiple testing.The genotype distributions among the healthy controls deviated from Hardy-Weinberg equilibrium for TLR2 C>T (rs1816702) , TNFRSF1A −609 G>T (rs4149570)  and IL6R C>T (rs4537545)  were associated with increased risk of CD. Both the homozygous and the heterozygous variant genotypes of NFKB1 −94ins/del (rs28362491) , IL23R G>A (rs11209026)  and PTPN22 1858 G>A (rs2476601)  were associated with reduced risk of CD (The homozygous variant genotype of sk of CD  and S2.IL23R G>A (rs11209026)  and PTPN22 1858 G>A (rs2476601)  were associated with reduced risk of CD.After Bonferroni correction for multiple testing both the homozygous and the heterozygous variant genotypes of TLR2 C>T (rs1816702)), increase TNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)), increase IL-6r and IL-6 levels (IL6R C>T (rs4537545)), decrease NF-κB p50 subunit expression (NFKB1 −94ins/del (rs28362491)), decrease IL-17 serum levels (IL23R G>A (rs11209026)) and decrease TNF-α serum levels (PTPN22 1858 G>A (rs2476601)), respectively ), increased TNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)) and higher IL-6r and IL-6 levels (IL6R C>T (rs4537545)) were associated with increased risk of CD. In addition, decreased IL-17 serum levels (IL23R G>A (rs11209026)) and decreased TNF-α serum levels (PTPN22 1858 G>A (rs2476601)) were associated with reduced risk of CD. The NF-κB p50 subunit can act both pro-inflammatory as part of the p50/p65 complex or anti-inflammatory as p50 homodimer (NFKB1 −94ins/del (rs28362491)) could be a differential lowering of the anti-inflammatory response which was associated with reduced risk of CD.Thus, polymorphisms associated with higher TLR2 levels (omodimer . A lowerTNFRSF1A −609 G>T (rs4149570)  and PPARG C>G (rs1801282)  and both the homozygous and the heterozygous variant genotypes of TLR2 C>T (rs1816702) , TLR4 T>C (rs12377632) , NFKBIA 2758 A>G (rs696)  and IL10 C>T (rs3024505)  were associated with increased risk of UC.The homozygous variant genotype of TLR9 1174 G>A (rs352139)  and the heterozygous genotype of LY96 −1625 C>G (rs11465996)  were associated with reduced risk of UC. In addition, both the homozygous and the heterozygous variant genotypes of TLR4 T>C (rs1554973) , TNFA −308 G>A (rs1800629) , IL23R G>A (rs11209026)  and PTPN22 1858 G>A (rs2476601)  were associated with reduced risk of UC (The homozygous variant genotype of sk of UC  and S2. TNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)), increase TLRs and TNF-α mRNA levels (PPARG C>G (rs1801282)), increase TLR2 levels (TLR2 C>T (rs1816702)), increase NFKBIA expression (NFKBIA 2758 A>G (rs696)), increase MD-2 (LY96) and TNF-α levels (LY96 −1625 C>G (rs11465996)), increases TNFA expression (TNFA −308 G>A (rs1800629)), decrease IL-17 serum levels (IL23R G>A (rs11209026)) and decrease TNF-α serum levels (PTPN22 1858 G>A (rs2476601)), respectively (TLR4 T>C (rs12377632), TLR4 T>C (rs1554973), TLR9 1174 G>A (rs352139) and IL10 C>T (rs3024505) are unknown.The variant allele of the polymorphisms have been shown to increase ectively . The bioTNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)), increased TLRs and TNF-α mRNA levels (PPARG C>G (rs1801282)) and increased TLR2 levels (TLR2 C>T (rs1816702)) were associated with increased risk of UC. Furthermore, polymorphisms associated with decreased IL-17 serum levels (IL23R G>A (rs11209026)) and decreased TNF-α serum levels (PTPN22 1858 G>A (rs2476601)) were associated with reduced risk of UC. In contrary, lower activity of the NFκB pathway through increased NFKBIA (IκBα) expression (NFKBIA 2758 A>G (rs696)), an inhibitor of the NFκB pathway, was associated with increased risk of UC. In addition, polymorphisms associated with increased MD-2 (LY96 −1625 C>G (rs11465996)) and TNF-α levels (TNFA −308 G>A (rs1800629)) were associated with reduced risk of UC.Thus, polymorphisms associated with increased PPARG (rs1801282)   and S2.TLR2 A>T (rs4696480)  and IL6R C>T (rs4537545)  and both the homozygous and the heterozygous variant genotypes of TLR2 C>T (rs1816702) , TLR4 T>C (rs12377632) , TLR9 −1486 T>C (rs187084) , TNFRSF1A −609 G>T (rs4149570)  and IL10 C>T (rs3024505)  were associated with increased risk of IBD. The homozygous variant genotype of TLR4 T>C (rs1554973)  and both the homozygous and the heterozygous variant genotypes of IL23R G>A (rs11209026)  and PTPN22 1858 G>A (rs2476601)  were associated with reduced risk of IBD , the homozygous variant genotype of k of IBD  and S2.IL23R G>A (rs11209026)  and PTPN22 1858 G>A (rs2476601)  were associated with reduced risk of IBD.After Bonferroni correction for multiple testing both the homozygous and the heterozygous variant genotypes of IL6R C>T (rs4537545)), increase TLR2 levels (TLR2 C>T (rs1816702)), increase TNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)), decrease IL-17 serum levels (IL23R G>A (rs11209026)) and decrease TNF-α serum levels (PTPN22 1858 G>A (rs2476601)), respectively (TLR2 A>T (rs4696480), TLR4 T>C (rs12377632), TLR9 −1486 T>C (rs187084), TLR4 T>C (rs1554973) and IL10 C>T (rs3024505) are unknown.The variant allele of the polymorphisms have been shown to increase IL-6r and IL-6 levels (ectively . The bioIL6R C>T (rs4537545)), higher TLR2 levels (TLR2 C>T (rs1816702)) and increased TNFRSF1A expression (TNFRSF1A −609 G>T (rs4149570)) were associated with increased risk of IBD. In addition, polymorphisms associated with decreased IL-17 serum levels (IL23R G>A (rs11209026)) and decreased TNF-α serum levels (PTPN22 1858 G>A (rs2476601)) were associated with reduced risk of IBD.Thus, polymorphisms associated with higher IL-6r and IL-6 levels  was associated with reduced risk of CD  and combined CD and UC .The TLR4.No associations were found for TLR9 haplotype combination 11 (rs187084CC and rs352139GG) was associated with increased risk of UC . The TLR9 haplotype combination 12 was associated with increased risk of IBD . Indirectly, these results support the analysis of the individual SNPs, where the variant allele of rs352139 (included in haplotype combination 22 which was used as reference) was fond to be associated with lowered risk of UC.The PTPN22 (1858 G>A) IL10 (rs3024505 C>T) PPARG (rs1801282 C>G) IL23R (rs11209026 G>A) IL6R (rs4537545 C>T)), UC (TLR4 (rs1554973 T>C and rs12377632 T>C), TLR9 (1174 G>A), LY96 (−1625 C>G), NFKBIA (2758 A>G) and PTPN22 (1858 G>A)) or CD and UC (TLR2 (−16934 A>T and rs1816702 C>T), TLR9 (−1486 T>C) and TNFRSF1A (−609 G>T)) were identified. Other cohort studies of patients with CD and UC have found that other polymorphisms in the TLRs (toll like receptors) TNFRSFs (TNF receptors) In this study of severely ill patients, 16 functional polymorphisms in 13 genes involved in regulation of inflammation were found to be associated with CD, UC or CD and UC combined (IBD) . As showTLR2 (rs1816702 C>T), TNFRSF1A (−609 G>T) and IL6R (rs4537545 C>T) was associated with increased risk of CD and lower activity of the inflammatory genes NFKB1 (NFκB) (−94ins/del ATTG), IL23R (rs11209026 G>A) and PTPN22 (1858 G>A) was associated with reduced risk of CD (TNFRSF1A (−609 G>T), PPARG (rs1801282 C>G) and TLR2 (rs1816702 C>T) was associated with increased risk of UC and lower activity of the inflammatory genes IL23R (rs11209026 G>A) and PTPN22 (1858 G>A) was associated with reduced risk of UC. In contrast, a genetically determined higher activity of the NFκB inhibitor IκBα (NFKBIA 2758 A>G) and lower activity of TLR9 (haplotype 11 (−1486CC and 1174GG)) were associated with increased risk of UC. Furthermore, a genetically determined higher activity of LY96 (−1625 C>G) and TNFA (−308 G>A) was associated with reduced risk of UC. However, the risk of CD and UC seem to have shared mechanisms through the TLR2 (rs1816702 C>T), TNFRSF1A (−609 G>T), IL23R (rs11209026 G>A) and PTPN22 (1858 G>A) polymorphisms confirming that the inflammatory pathways are involved in risk of both CD and UC The biological interpretation indicates that a genetically determined higher activity of the inflammatory genes sk of CD . The picIL10 (rs3024505 C>T) and increased risk of CD CD14 (−159 G>A) and increased risk of CD, UC and IBD IL10 (rs3024505 C>T), as a GWA study with more than six thousands CD cases and fifteen thousands control found that the varaint allele was associated with increased risk of CD but with an odds ratio of 1.12 IL10 has previously been found to be associated with risk of CD and UC in the Danish population CD14 (−159 G>A) may be due to genetic differences between the Korean and Danish population.This study was unable to confirm the associations between the variant allele of NFKBIA (IκBα) (2758 A>G) TNFA (−308 G>A) were associated with higher and lower risk of UC, respectively, and the deletion polymorphism in NFKB1 (NFκB) (−94ins/del ATTG) was associated with lower risk of CD in our cohort study. However, there seems to be no consensus regarding these polymorphisms in other cohort studies The variant allele in IL1B (−31 T>C) IL-10 (−592 C>A) PPARG (rs1801282 C>G) MAP3K14 (rs7222094 T>C), SUMO4 (163 T>C), TNFAIP3 (rs6927172 C>G), IL1RN (rs4251961 T>C), IL4R (rs1805010 A>G), IL6 (−6331 T>C), IL17A (197G>A), IFNG (874 T>A), TGFB1 (−509 C>T) and NLRP3 (rs4612666 C>T) were not statistically associated with CD, UC or IBD in our cohort. None of the polymorphisms studied in these genes have been reported to be associated with CD or UC. We can not exclude that we did not find associations between these polymorphisms and risk of disease due to lack of power in this cohort.No associations were found for TLR2 (rs4696480 A>T), TLR4 (rs1554973 T>C), TLR9 (1174 G>A) and TGFB1 (−509 C>T) were not in Hardy-Weinberg equilibrium among the healthy controls which is probable due to chance because of the number of polymorphisms analyzed. When corrected adequately for multiple testing they did not deviate from Hardy-Weinberg equilibrium. In the light of the obtained P-values and the number of statistical tests performed, we cannot exclude that some of our positive findings may be due to chance. If the results were corrected for multiple testing only the well known susceptibility polymorphisms in IL23R (rs11209026 G>A) and PTPN22 (1858 G>A) were associated with reduced risk of both CD and IBD. We successfully tested 37 polymorphisms and, assuming a 5% acceptance level, two would be expected to be associated with susceptibility by pure chance. In this study 16 polymorphisms were found to be associated with susceptibility and the found associations were biologically plausible. A major strength was that this clinically homogeneous and well-characterised cohort was rather large including 1035 patients with IBD and 795 healthy controls. All the patients were considered for anti-TNF treatment and were therefore considered to have a severe disease course. Genetic determinants may be expected to be strong among severely ill cases The results in this study should be interpreted with care. TLRs associated with risk of CD or UC support that the host microbial composition, diet or environmental molecules in the gut are important factors driving the inflammatory response in genetically susceptible individuals.In conclusion, 16 functional SNPs in 13 genes involved in regulation of inflammation were found to be associated with susceptibility of severe CD, UC or IBD. Eleven of the SNPs have not previously been reported as susceptibility polymorphisms of CD, UC or IBD  althoughTable S1Odds ratios (OR) (unadjusted) for genotypes studied among healthy controls and patients with Crohn's disease (CD), ulcerative colitis (UC) and combined inflammatory bowel disease (IBD).(DOC)Click here for additional data file.Table S2Odds ratios (OR)  for genotypes studied among healthy controls and patients with Crohn's disease (CD), ulcerative colitis (UC) and combined inflammatory bowel disease (IBD).(DOC)Click here for additional data file.Table S3Association of the TLR2 haplotype combinations and risk of Crohn's disease (CD), ulcerative colitis (UC) and all inflammatory bowel disease (IBD).(DOC)Click here for additional data file.Table S4Association between TLR4 haplotype combinations and risk of Crohn's disease (CD), ulcerative colitis (UC) and all inflammatory bowel disease (IBD).(DOC)Click here for additional data file.Table S5Association between TLR9 haplotype combinations and risk of Crohn's disease (CD), ulcerative colitis (UC) and all inflammatory bowel disease (IBD).(DOC)Click here for additional data file.


During the 2014–15 influenza season in the United States, influenza activityDuring September 28, 2014–May 23, 2015, World Health Organization (WHO) and National Respiratory and Enteric Virus Surveillance System collaborating laboratories in the United States tested 691,952 specimens for influenza viruses; 125,462 (18.1%) were positive . Of the Through the peak of the 2014–15 season, H3N2 viruses predominated nationally, with lesser numbers of influenza B viruses and influenza A (H1N1)pdm09 viruses also identified. Based on the percentage of specimens testing positive for influenza to determine the peak of influenza activity, the peak occurred during week 52  nationally; however, differences among U.S. Department of Health and Human Services regionsAlthough H3N2 activity peaked between late December and early January, substantial influenza B activity occurred late in the season. Influenza A viruses predominated until late February, with influenza B viruses predominating from the week ending February 28, 2015 (week 8) through the week ending May 23, 2015 (week 20). The highest proportion of influenza B viruses was observed in Region 4 (19.8%), and the lowest proportion of influenza B viruses was detected in Region 10 (11.1%).During the 2014–15 influenza season, three cases of human infection with novel influenza A viruses have been reported. One infection with an influenza A (H3N2) variant virus occurred during the week ending October 18, 2014 (week 42) in Wisconsin, and one infection with an influenza A (H1N1) variant (H1N1v) virus was reported to CDC during the week ending January 24, 2015 (week 3) from Minnesota. Both patients had illness onset in October 2014 and reported contact with swine in the week preceding illness. Both patients fully recovered, and no further cases were identified in contacts of either patient. The third case, a fatal infection with an H1N1v virus was reported from Ohio during the week ending April 2, 2015 (week 17). The patient worked at a livestock facility that housed swine, but no direct contact with swine in the week before illness onset was reported. The patient died from complications of the infection, and no ongoing human-to-human transmission was identified.WHO collaborating laboratories in the United States are requested to submit a subset of their influenza-positive respiratory specimens to CDC for further virus characterization. CDC has antigenically and/or genetically characterizedA total of 246 (18.6%) of the 1,324 H3N2 viruses tested have been characterized as A/Texas/50/2012-like, the influenza A (H3N2) component of the 2014–15 Northern Hemisphere influenza vaccine. A total of 1,078 (81.4%) of the 1,324 viruses tested showed either reduced titers with antiserum produced against A/Texas/50/2012 or belonged to a genetic group that typically shows reduced titers to A/Texas/50/2012. The viruses that showed reduced titers to A/Texas/50/2012 belonged to multiple genetic groups; most but not all were antigenically similar to the influenza A (H3N2) virus selected in September 2014 for the 2015 Southern Hemisphere and in February 2015 for the 2015–16 Northern Hemisphere influenza vaccines, A/Switzerland/9715293/2013. A total of 948 of the 1,324 A (H3N2) viruses were further characterized; 889 (93.7%) were antigenically similar to A/Switzerland/9715293/2013, and fifty-nine (6.2%) showed reduced titers with antiserum produced against A/Switzerland/9715293/2013 virus.Of the 810 influenza B viruses tested, 582 (71.9%) belonged to the B/Yamagata lineage, and the remaining 228 (28.1%) influenza B viruses tested belonged to the B/Victoria/02/87 lineage. A total of 571 (98.1%) of the 582 B/Yamagata-lineage viruses were characterized as B/Massachusetts/2/2012-like, which was included as an influenza B component of the 2014–15 Northern Hemisphere trivalent and quadrivalent influenza vaccines. Eleven (1.9%) of the B/Yamagata-lineage viruses tested showed reduced titers to B/Massachusetts/2/2012. Among the 582 B/Yamagata lineage viruses characterized, 576 (98.9%) viruses were antigenically similar to B/Phuket/3073/2013 virus, the B/Yamagata lineage virus selected for the 2015 Southern Hemisphere influenza vaccine and 2015–16 Northern Hemisphere influenza vaccine. Six (1.0%) showed reduced titers with antiserum produced against B/Phuket/3073/2013 virus. A total of 223 (97.8%) of the 228 B/Victoria-lineage viruses were characterized as B/Brisbane/60/2008-like, the virus that is included as an influenza B component of the 2014–15 Northern Hemisphere quadrivalent influenza vaccine. Five (2.2%) of the B/Victoria-lineage viruses tested showed reduced titers to B/Brisbane/60/2008.Since October 1, 2014, a total of 4,192 influenza virus specimens have been tested for resistance to influenza antiviral medications. All 896 influenza B viruses and 3,232 influenza A (H3N2) viruses tested were sensitive to oseltamivir and zanamivir. All 896 influenza B viruses and 1,723 influenza A (H3N2) viruses tested were sensitive to peramivir. Among 64 pH1N1 viruses tested for resistance, one (1.6%) was found to be resistant to oseltamivir and one (1.6%) to peramivir. All 58 influenza A (H1N1)pdm09 viruses tested for resistance to zanamivir were sensitive. High levels of resistance to the adamantanes (amantadine and rimantadine) persist among influenza A viruses currently circulating globally (the adamantanes are not effective against influenza B viruses).The Food and Drug Administration’s Vaccines and Related Biological Products Advisory Committee has recommended that the 2015–16 influenza trivalent vaccines used in the United States contain an A/California/7/2009 (H1N1)pdm09-like virus, an A/Switzerland/9715293/2013 (H3N2)-like virus, and a B/Phuket/3073/2013-like (B/Yamagata lineage) virus. It is recommended that quadrivalent vaccines, which have two influenza B viruses, contain the viruses recommended for the trivalent vaccines, as well as a B/Brisbane/60/2008-like (B/Victoria lineage) virus ILINet data are used to produce a weekly jurisdiction-level measure of ILI activityState and territorial epidemiologists report the geographic distribution of influenza in their jurisdictions through a weekly influenza activity code.CDC monitors hospitalizations associated with laboratory-confirmed influenza virus infections using the FluSurv-NETAs of April 30, 2015, among the FluSurv-NET adult patients for whom medical chart data were available, the most frequent underlying conditions were cardiovascular disease (51.0%), metabolic disorders (45.8%) and obesity (33.1%). Among children hospitalized with laboratory-confirmed influenza and for whom medical chart data were available, 43.3% did not have any recorded underlying conditions, and 26.4% had underlying asthma or reactive airway disease. Among the 626 hospitalized women of childbearing age (15–44 years), 200 (31.9%) were pregnant.During the 2014–15 influenza season, the percentage of deaths attributed to pneumonia and influenza (P&I) exceeded the epidemic thresholdFor the 2014–15 influenza season, as of May 23, 2015, a total of 141 laboratory-confirmed, influenza-associated pediatric deaths had been reported from 40 states and New York City. The deaths occurred in 14 children aged <6 months, 23 aged 6–23 months, 22 aged 2–4 years, 45 aged 5–11 years, and 37 aged 12–17 years; mean and median ages were 7.2 years and 5.9 years, respectively. Among the 141 deaths, 109 were associated with an influenza A virus, 29 were associated with an influenza B virus, two were associated with an influenza virus for which the type was not determined, and one was associated with an influenza A and influenza B virus coinfection.Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the total number of influenza-associated pediatric deaths had previously ranged from 34 to 171 per season; this excludes the 2009 pandemic, when 358 pediatric deaths were reported to CDC during April 15, 2009–October 2, 2010.The 2014–15 influenza season was moderately severe overall and especially severe in adults aged ≥65 years, with predominant circulation of antigenically and genetically drifted influenza A (H3N2) viruses. Influenza activity peaked during late December, with influenza A (H3N2) viruses predominant early in the season through the week ending February 21, 2015 (week 7). Influenza B became the predominant virus starting week 8 . The majority of influenza A (H3N2) viruses sent to CDC for antigenic and/or genetic characterization were different from the influenza A (H3N2) component of the 2014–15 Northern Hemisphere seasonal vaccines (A/Texas/50/2012).Previous influenza A H3N2)–predominant seasons have been associated with increased hospitalizations and deaths compared to seasons that were not influenza A (H3N2)–predominant, especially among children aged <5 years and adults aged ≥65 years , compared with previous seasons when most circulating and vaccine strain viruses were well-matched. Data collected during November 10, 2014–January 30, 2015, indicated that the influenza vaccine was 19%  effective in preventing medical visits against all influenza across all age groups, and was 18% CI = 6%–29%) and 45% (CI = 14%–65%) effective in preventing medical visits associated with influenza A (H3N2) and influenza B (Yamagata lineage), respectively –predominant and moderately severe season overall, but was especially severe for adults aged ≥65 years. This age group had the highest laboratory-confirmed influenza hospitalization rates and also accounted for the majority of pneumonia and influenza deaths. Antigenic and genetic characterization showed that most of the circulating influenza A (H3N2) viruses were different from the influenza A (H3N2) component of the 2014–15 Northern Hemisphere vaccines, resulting in reduced vaccine effectiveness.What are the implications for public health practice?Influenza vaccination remains the most effective way to prevent influenza illness and its associated complications. Although vaccine effectiveness was reduced this season because of antigenic drift in H3N2 viruses, vaccination was still protective against vaccine-like influenza A (H3N2) viruses and influenza B viruses. Timely influenza surveillance informs vaccine strain selection; the influenza A (H3) and influenza B components of the subsequent 2015–16 season vaccine have been changed to more optimally match circulating viruses. As an adjunct to vaccination, timely empiric antiviral treatment is also recommended for all patients with severe, complicated, or progressive influenza illness and those at higher risk for influenza-associated complications, including adults aged ≥65 years.http://www.cdc.gov/flu/weekly. Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is available at http://www.cdc.gov/flu.Influenza surveillance reports for the United States are posted online weekly and are available at


The cytochrome P450 (CYP450) 2C19 681 genotypes affect the antiplatelet activity of clopidogrel. We investigated the correlation of CYP 2C19 681G > A mutation with clopidogrel resistance (CR). Additionally, we studied the effect of CR on clinical prognosis of patients with acute coronary syndrome (ACS).One hundred ten ACS patients undergoing percutaneous coronary intervention, who were followed-up for 1 year, were included in the study. The patients were co-administered aspirin 100 mg/d and clopidogrel 75mg/d following a loading dose of 300 mg. CR was assessed on the basis of polymorphism observed in the CYP2C19 subgroup.p < 0.01). CYP2C19 681GG genotype group was associated with lower CR than CYP2C19 681A allele (GA + AA) group (9/59 vs. (12+5)/51; p = 0.009). Over a follow-up of 12 months, the incidence of recurrent angina, acute myocardial infarction, and intra-stent thrombosis in CYP2C19 681 GG carriers was significantly lower than that in CYP2C19 681A allele (GA + AA) group .Patients in GG genotype group exhibited greater inhibition of platelet aggregation than patients in GA and AA genotype groups (16.2 ± 10.1%; 10.2 ± 9.9%; 8.0 ± 5.9%, respectively, CYP 2C19*2 is associated with reduced clopidogrel antiplatelet activity and might be an important marker for poor prognosis of ACS. Clopidogrel, which irreversibly inhibits the adenosine diphosphate (ADP) platelet receptor P2Y12, has been widely used in preventing thromboembolic events in patients with acute coronary syndrome (ACS). However, a great number of patients under the treatment of clopidogrel still experience cardiovascular events. Various studies have demonstrated that the rate of clopidogrel resistance (CR) ranges 4.2–27.8% . The CYPIn this study, we investigated whether CYP2C19 681G > A mutation is correlated with low inhibition of platelet aggregation (IPA) in ACS patients after administration of clopidogrel. Furthermore, we assessed the correlation between CR at pre-discharge and 12-month incidence of adverse cardiovascular events.This study was a prospective, single center research. Enrollment began in January 2010, which lasted for two years. Clinical follow-up was completed in January 2013. In this study ACS patients (18–80 years), who underwent percutaneous coronary intervention (PCI), were enrolled. The clinical diagnosis of ACS was based on the 2007 ACC/AHA guideline for the diagnosis and treatment of ACS, including unstable angina (UA), acute ST-segment elevation myocardial infarction (STEMI), and acute non-ST-segment elevation myocardial infarction (NSTEMI). Exclusion criteria included allergic or intolerance to aspirin or clopidogrel, bleeding disorder or tendency, chronic oral anticoagulation drugs (such as warfarin), exposure to clopidogrel in the last 2 weeks, contraindication to antiplatelet therapy, surgery within the preceding 8 weeks, severe anemia (hemoglobin < 60 g/L), renal impairment (serum creatinine > 220 μmol/L), NYHA function class IV, and tumor or severe immune system disorder. This study was approved by the review board of The First Affiliated Hospital of Wannan Medical College. Written informed consent was obtained from all the patients.The patients were pre-treated with a loading dose of 300 mg clopidogrel and 300 mg aspirin before undergoing coronary angiography, which was followed by the daily administration of 75 mg clopidogrel and 100 mg aspirin. Tirofiban, a glycoprotein IIb/IIIa inhibitor, was administered only when the coronary flow to Thrombolysis In Myocardial Infarction (TIMI) flow grade was < 2.Blood specimens were collected for platelet function assays using test tubes containing 3.8% sodium citrate before and 24 h after administration of clopidogrel. Samples were processed within 3 hours after blood collection.9 thrombocytes/l). Platelet aggregation was measured using turbidimetric aggregometry by applying a 4-channel LBY-NJ4 aggregometer . ADP  was used to decrease the aggregation. Light transmission in PRP was measured 5 min after the addition of ADP at a final concentration of 5 μmol/L. Results were expressed as percentage of maximal light transmission using PPP from the same patient as reference (100% aggregation).Platelet-rich plasma (PRP) was prepared by centrifugation of citrated venous blood at 1000 rpm for 10 min. Residual blood samples, after removing PRP, were centrifuged at 3000 rpm for 5 min and were called as platelet-poor plasma  at pre-and-post treatment was determined, and IPA, which was referred to as ΔA (ΔA = pre-treatment PAR-post-treatment PAR), was calculated. ΔA < 10%  is referred to as the presence of CR . AccordiGenomic DNA from whole-blood samples was extracted using a purifier  according to the reagent kit instructions.CYP2C19*2 genetic polymorphism was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). The sequences were amplified using PCR in a Gradient PCR Thermal Cycler  with conditions listed below: pre-denaturation at 94°C for 5 min, 35 cycles with denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 20 sec, and final extension at 72°C for 5 min.To detect the CYP2C19*2 polymorphism, 10 μL of the PCR products (334 bp) were digested with SmaI restriction enzyme  and the products were transferred to a water bath (30°C) for 6 h. Agarose gels electrophoresis was performed for the digested fragments  Figs.Study visits were conducted for 12 months. The main endpoint was the cumulative incidence of recurrent angina, nonfatal MI, cerebral stroke, stent thrombosis, and sudden cardiac death (SCD). The patients were either directly consulted or contacted by telephone by two referring physicians.t-test and one way analysis of variance (ANOVA). Chi square analysis was performed to evaluate the categorical data. Statistical analysis was performed using STATA software version 11.0 (StataCorp.). A p value of < 0.05 was considered as statistically significant.Hardy-Weinberg genetic equilibrium test was used to test the population representativeness. Continuous data was described as mean±SD, whereas the categorical data was represented as counts and percentage. The comparison between the groups was made by One hundred eighteen patients (8 patients lost to follow-up) were enrolled for the study . Baselinp < 0.01). After 24 h of clopidogrel administration, the patients in GG group exhibited significantly lower laboratory CR than patients with CYP2C19 681 allele group (9/59 vs. (12+5)/51, p = 0.009)  enrolled patients, 59 patients (53.6%) were CYP2C19 681 wild-type homozygotes (GG), 43 were (39.1%) CYP2C19 681 mutant heterozygotes (GA), and 8 were (7.3%) mutant homozygotes (AA). The IPA was significantly higher in GG genotype group than GA genotype and AA genotype groups  . Since tp < 0.05). However, the incidence of cerebral stroke and SCD was similar in both the groups (p > 0.05)  during the follow-up. Recurrent angina, AMI, and intra-stent thrombosis occurred less often in CYP2C19 681 GG patients than CYP2C19 681A allele (GA+AA) patients  . The cliCytochrome P450 (CYP450) enzymes belong to a superfamily of heme-containing monooxygenases . The funOur data suggested that the presence of CYP2C19*2 polymorphism is associated with higher laboratory CR in ACS patients . We found that the carriers of CYP2C19 681 allele are associated with 3-fold increased risk for adverse cardiovascular events when treated with clopidogrel. From these findings, it was inferred that that patients with CYP2C19*2 allele treated with clopidogrel post-PCI should be warned about the risk of these clinical events. It is necessary to recognize the CYP2C19*2 loss of function in patients with ACS undergoing PCI, including relevant clinical features of weakened functional reaction to clopidogrel. Increasing the dose of clopidogrel may be one of the options to overcome CR in ACS patients with low- or non-response to clopidogrel [CYP2C19 681 G > A mutation, associated with reduced clopidogrel antiplatelet activity, is an important marker for poor prognosis of ACS patients receiving clopidogrel treatment. Genotyping of CYP2C19*2 has the potential to guide the use of antiplatelet treatment.The sample size was relatively small, and the results could not be extrapolated to all the ACS population. In order to validate the results further, the clinical trials involving much larger patient cohorts are required.S1 Fig(TIF)Click here for additional data file.S2 FigA: GG genotype; B: GA genotype; B: AA genotype.(TIF)Click here for additional data file.


Different flavonoids are known to interfere with influenza A virus replication. Recently, we showed that the structurally similar flavonoids baicalein and biochanin A inhibit highly pathogenic avian H5N1 influenza A virus replication by different mechanisms in A549 lung cells. Here, we investigated the effects of both compounds on H5N1-induced reactive oxygen species (ROS) formation and the role of ROS formation during H5N1 replication.Baicalein and biochanin A enhanced H5N1-induced ROS formation in A549 cells and primary human monocyte-derived macrophages. Suppression of ROS formation induced by baicalein and biochanin A using the antioxidant N-acetyl-L-cysteine strongly increased the anti-H5N1 activity of both compounds in A549 cells but not in macrophages.These findings emphasise that flavonoids induce complex pharmacological actions some of which may interfere with H5N1 replication while others may support H5N1 replication. A more detailed understanding of these actions and the underlying structure-activity relationships is needed to design agents with optimised anti-H5N1 activity. Complication rates are much higher in H5N1 patients than in seasonal influenza or pandemic H1N1/09 patients . As of 2During an initial pandemic phase, matched vaccines will be restricted and antiviral drugs will be critical. The efficacy of the approved anti-influenza drugs  is limited, resistant strains emerge, and H5N1 strains appear to be less sensitive to the established anti-influenza drugs than seasonal influenza strains -13. Henchttp://www.who.int). Flavonoids are known to exert multiple pharmacological effects including anti-inflammatory and anti-viral activities including inhibition of seasonal influenza A (H1N1) viruses [In 2009, the “WHO public health research agenda for Influenza” expressed a need for additional drugs including those that exert immunomodulatory effects and recommended to investigate natural products for anti-influenza activity . They may display anti- or pro-oxidative effects . Influen+ monocytes were differentiated into MDMs as described previously [50/mL) as described previously [t-test, more groups by ANOVA followed by subsequent stepwise multiple comparison procedure using the Student-Newman-Keuls method.A549 cells  and Vero cells  were cultivated as described previously . Human meviously . Cells weviously . FlavonoH5N1 infection of A549 cells at a multiplicity of infection (MOI) 0.01 resulted in enhanced ROS formation compared to control 24 h after infection Figure . Human mIn conclusion, we show that two flavonoids that interfere with H5N1 replication by different mechanisms of action exert similar effects at the level of ROS induction. Baicalein interferes with the H5N1 neuraminidase activity but biochanin does not. Biochanin A  inhibits the activation of signalling molecules involved in H5N1-induced signalling including AKT, ERK 1/2, and NFκB . DespiteNAC: N-acetyl-L-cysteine; ROS: Reactive oxygen species.The authors declare that they have no competing interests.MM and JC designed the study, analysed the data, and wrote the manuscript. PS performed experiments and analysed data. All authors read and approved the final manuscript.


Cardiac MR (CMR) is the reference standard for assessing macroscopic myocardial scar. Determination of the extracellular volume fraction (ECV) by T1 estimation using the modified Look-Locker inversion recovery (MOLLI) correction enables quantitation of diffuse fibrosis. The purpose of this study is to evaluate the feasibility and reproducibility of an optimized high-resolution MOLLI (HR-MOLLI) technique at 3T and 1.5T for RV ECV calculation in healthy volunteers.25 healthy volunteers  were scanned at 3T  and 15  were scanned at 1.5T (MAGNETOM Aera). T1 mapping was performed in the axial orientation using an investigational HR-MOLLI technique with a 1 × 1 mm 2 in-plane resolution that applies motion correction with synthetic image estimation. Motion corrected images were used to generate parametric maps with (T1) and without (T1*) the MOLLI correction. The MOLLI sequence uses a 5 heart-beat (HB) acquisition, 3 HB recovery, 3 HB acquisition scheme with a single shot bSSFP diastolic readout. Images were acquired pre- and 10-25 minutes post- administration of 0.2 mmol/kg gadobenate dimeglumine . T1 and T1* parametric maps were used to quantify the T1 of tissue and blood, respectively. Two reviewers quantified basal and mid RV free wall, interventricular septal, and lateral LV wall T1 values on T1 parametric maps. RV and LV ECV ranges were calculated assuming normal hematocrit values . Global ventricular ECV values were compared using the student's t-test. Intra and inter-observer variance was measured by the intraclass correlation coefficient (ICC).Representative T1 and T1* parametric maps are shown in Figure This feasibility study demonstrates that HR-MOLLI can quantitate the global RV ECV fraction at both 1.5 and 3T with good intra- and interobserver variance. Our results demonstrated a field-strength influence on RV and LV ECV values, and showed that ECV calculations using blood pool extimates without a look-locker correction are necessary at 3T.Funding pending.


The correct legend is:Figure S1 is labeled incorrectly. The second column should be labeled “E. coli OP50” and the third column should be labeled “E.c. OP50 Amp(10)”. The correct legend is:S1 Figfer-1(b232ts) L1 animals were exposed to S. enterica—GFP for 72 hours and transferred to the indicated bacteria-antibiotic plates for 48 hours. Overall GFP intensity in the intestinal lumen was determined using an MZFLIII Leica stereomicroscope. Three levels of colonization were determined as heavy, weak, or none as described in Materials and Methods. The mean of 2 plates is shown. For each condition, we assayed 20–40 animals.(TIF)Click here for additional data file.The legend for Figure S3 is incorrect. The correct legend is:S3 Fig(A-B) Transcript levels of 4 selected down-regulated genes (A) and 5 selected up-regulated genes (B) as determined using qRT-PCR. Black striped bars represent gene expression changes in L1 animals grown on E. coli for 72 hours and then treated with Kanamycin for 24 hours relative to L1 animals grown on E. coli for 96 hours. Gray striped bars represent gene expression changes in L1 animals grown on S. enterica for 72 hours and then treated with Kanamycin for 24 hours relative to animals grown on S. enterica for 96 hours. (C-D) Comparison of gene expression changes in 4 selected down-regulated genes (C) and 5 selected up-regulated genes (D) during recovery with Tetracycline or Kanamycin. Gray bars represent gene expression changes in L1 animals grown on S. enterica for 72 hours and then treated with Tetracycline for 24 hours relative to animals grown on S. enterica for 96 hours. Gray striped bars represent gene expression changes in L1 animals grown on S. enterica for 72 hours and then treated with Kanamycin for 24 hours relative to animals grown on S. enterica for 96 hours. qRT-PCR studies were performed in duplicate. SEM is shown.(TIF)Click here for additional data file.


Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T  in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks.Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine  viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys The influenza A/California/07/2009 H1N1) virus emerged in Mexico, and was identified in the United States at the beginning of 2009. It caused outbreaks of respiratory illness in Mexico and in the United States, N1 virus , 4.The quick emergence and spread of A/California/07/2009 (H1N1) virus has been of great concern globally. Therefore, the development of A /California/07/2009 vaccine was an essential component of the global preventive strategy for the pandemic preparedness and response. WHO recommended that pandemic influenza A (H1N1) vaccines must contain A/California/7/2009 (H1N1)-like virus .http://www.ifpma.org/resources/influenza-vaccines/influenza-vaccines/vaccine-manufacture-virus-processing.html#sthash.5GUYGsGs.dpuf.To date, several vaccines have been developed against A/California/07/2009 (H1N1) virus. Some vaccine viruses were prepared by classical reassortment (e.g. X-179A and X-181); others by reverse genetics (e.g. 121XP). They were manufactured as seasonal influenza vaccines manufactured i.e. after virus cultivation; the allantoic fluid of the egg is harvested and is then usually clarified by centrifugation to remove cellular debris. Then whole virus subjected to purification, concentration, and inactivation steps.—See more detail at: Orthomyxoviridae family [The A/California/07/2009 (H1N1) strain is an influenza A virus, a single-stranded RNA virus from e family . The famSpontaneous mutations easily emerge in RNA viruses, and the consequences of their accumulation must be identified to ensure the safety and potency of vaccines. The genetic diversity within a viral population is important, as RNA viruses in particular have high mutation rates, can evolve rapidly, and may exhibit adaptation to specific hosts and/or tissues . BecauseMost mutants are present in viral populations at relatively low levels, making them difficult to detect using conventional sequencing methods. A highly sensitive mutant analysis by PCR and restriction enzyme cleavage (MAPREC) , 14 and Other approaches based on analysis of electrophoretic mobility in gels , 17 do nRecently, several versions of high-throughput sequencing technology, known also as deep or massively parallel sequencing (MPS), enabled rapid generation of large amounts of sequence information . These nViruses and reference sequences4 pfu at 33°C. Allantoic fluid was harvested at 72 hours post infection and clarified by spinning at 4000 rpm for 10 min at 4°C. The clarified viruses were saved at -80°C.Viruses X-179A, X-181 and 121XP were passaged in eggs under similar conditions to those of the manufacturers , 26, andTotal RNA was isolated from 140 μl of virus suspension using QIAamp Viral RNA Mini Kit  according to the manufacturer’s protocol. The RNA was eluted in a final volume of 60 μl of sterile RNase-free water. cDNA was synthesized using Superscript III Reverse Transcriptase  with Universal_F primer: 5’ GACTAATACGACTCACTATAGGGAGCAAAAGCAGG 3’. cDNA synthesis was performed in a reaction containing 10 μl of isolated viral RNA, 1.6 μM of Universal_F primer, 600 units of SSIII, 0.5 mM dNTPs, 50 mM DTT, and first strand buffer in a total volume of 50 μl. The reaction mixture was incubated at 55°C for two hours, followed by SSIII inactivation at 70°C for 20 min.Two different PCR protocols were used to simultaneously amplify all genome segments of influenza virus.2, and 1 unit of Phusion DNA polymerase . PCR cycling conditions were as follows: an initial denaturation at 98°C for 30 sec followed by 35 cycles of 98°C for 10 sec, 55°C for 30 sec and 72°C for 5 min, and the final elongation at 72°C for 10 min.Phusion protocol: Four microliters of viral cDNA was used as a template for PCR amplification in a total reaction volume of 50 μl, containing 0.2 μM of each universal primers: Universal_F (see above) and Universal_R: 5’GACATTTAGGTGACACTATAGAAGTAGAAACAAGG 3’, 200 μM of dNTP, 3 mM MgClExtensor protocol: Four microliters of viral cDNA was used as a template for PCR amplification in a total reaction volume of 50 μl containing 0.4 μM of each universal primer , 500 μM of dNTP, 5 μl of 10x Extensor Buffer 2, and 2.5 units of Extensor PCR Enzyme Mix (5U/μl) . PCR cycling conditions were as follows: an initial denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 10 sec, 60°C for 30 sec and 68°C for 5 min, and the final elongation at 68°C for 10 min.The resulting eight DNA amplicons representing all segments of viral genome were separated by electrophoresis in 1% agarose gels with ethidium bromide  and visualized using the Kodak Gel Logic 200 Imaging System and Kodak Molecular Imaging Software .The PCR product was purified by QIAquick PCR Purification Kit  and fragmented by focused ultrasonicator (Covaris) to generate the optimal fragment sizes of 350–400 bp suitable for Illumina sequencing.® DNA Sample Prep Reagent Set 1 . The primers containing multiplexing index adapters (barcodes) to identify sequencing reads belonging to each sample were synthesized according to the specifications provided by Illumina Inc. for multiplex pair-end sequencing. Deep sequencing was performed at Macrogen  using HiSeq2000 (Illumina) producing 101 bp-long paired-end reads, or at our laboratory using MiSeq (Illumina) producing 100 to 300 bp-long paired-end reads.The DNA fragments were analyzed for DNA size by the 2100 Bioanalyzer system using a high-sensitivity kit . Next, the fragmented DNAs were used for library preparation with NEBNextNEBNext mRNA Sample Prep Master Mix Set 1  was used for preparation of Illumina sequencing libraries from total RNA contained in the sample (whole-RNA library). Briefly, 0.5 μg total RNA (extracted as mentioned above) was used for fragmentation by focused ultrasonicator (Covaris) to generate the optimal fragment sizes of 200–300 bp suitable for RNA Illumina sequencing. The fragmented RNA was used for cDNA synthesis using ProtoScript II Reverse Transcriptase and random primers. The cDNA was further converted into double stranded cDNA with end-repair procedure , and ligated to Illumina paired end (PE) adaptors. Size selection was performed using 2% agarose gel electrophoresis, generating cDNA libraries ranging from 275 to 325 bp in size. Finally, the libraries were expanded using 15 cycles of PCR with multiplex indexed primers and purified with magnetic beads using Agencourt Ampure Beads (Beckman Coulter).Reference RNA sequences of X-179A, X-181, 121XP, A/California/07/2009 (H1N1), and A/PR/08/34 viruses were downloaded from GenBank. Accession numbers for segments 1 to 8 of X-179A were CY058516 to CY058523 respectively. The same sequence references were used for X-181 except for segments 2, 4, and 6 were replaced by GQ906800—GQ906802 respectively. For 121XP virus, the reference sequences for segments 1 to 8 were GQ454863, GQ454864, GQ454865, GQ280121, GQ454866, GQ280122, GQ454867, and GQ454868 respectively. As references for A/PR/8/34 viruses, we used the sequences downloaded from GenBank; for segments 1–7 the accession numbers were EF467818- EF467824 respectively and EF467817 for segment 8. For A/California/07/2009 (H1N1) the following NCBI sequences were used for segments 1 to 8: CY121687, CY121686, CY121685, CY121680, CY121683, CY121682, CY121681, and CY121684 respectively.https://hive.biochemistry.gwu.edu/dna.cgi?cmd=main).The RNA sequences were used as references for alignment of the viral sequence reads generated in this study. First, sequencing reads with low quality (phred) scores were removed from the data set. The remaining sequences aligned with reference influenza virus sequences using custom software that is implemented in a highly integrated virtual environment (HIVE) computer cluster  profiles, read depth and the entropy for the entire viral genomes.To amplify all segments in one reaction, the reverse transcriptase Superscript III was used for cDNA synthesis, and two different PCR amplification protocols were used for DNA amplification.The agarose gel electrophoresis results showed that all eight segments were amplified efficiently from passaged A/California/07/2009 (H1N1) pandemic vaccine viruses  stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount. Sinai School of Medicine and was passaged in CBER to produce CBER stock, this virus was sequenced five times by MiSeq and HiSeq using whole-RNA library and DNA library approaches. The results of sequencing the RNA library showed high levels of reproducibility. There was a slight difference between MiSeq and HiSeq results only for mutations present at low levels  in the NP gene which was present in about 20% of the RNA sequences in all three A/PR/8/34 viruses.Another A/PR/8/34 virus stock was provided by National Institute for Biological Standards and Control (NIBSC) and was propagated 13 times in eggs and then was grown (passage 14) separately in two different eggs, and the resulting stocks were designated as A/PR/8/34-1 and A/PR/8/34-3 . Both of57G (Glu18Gly) in the PA segment. No mutations were observed in the NA segment. Three low-level non-synonymous mutations were found in HA: A456G , A736G  and G739A . The Asn146Asp mutation is located in the Sa antigenic site.A/California/07/2009 (H1N1) strain was sequenced by HiSeq using the whole-RNA library approach, and the summary of the results is presented in The 121XP virus was generated by the reverse genetics method followed by serial passages in chicken eggs; this virus contains the HA and NA genes from A/California/07/2009 (H1N1) and all other genes from A/PR/8/34 (H1N1) .16Ser and Lys226Glu that were present at low levels . The latter mutation is located in the Ca antigenic site in HA. Mutation Lys136Asn, located close to the antigenic site Sa, emerged and accumulated to about 78% of the population.To evaluate its genetic stability, the 121XP virus was passaged ten times in eggs under manufacture conditions . MutatioTen mutations were found in the NA gene, and eight of them led to amino acid changes. Interestingly, three of them were found in codon 88. Sequencing of other genomic segments revealed several heterogeneities in most of them, with some mutations leading to amino acid changes. There was a good agreement between results obtained by sequencing of the whole-RNA library by MiSeq and HiSeq, as well as the DNA library by HiSeq. For more details, see 314Gln (17%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was present as low level mutation in A/California/07/2009 (H1N1). The confirmation of the presence of this mutation by Sanger sequencing method is presented on 328Thr mutation.The X-179A virus is a reassortant prepared by New York Medical College (NYMC) using classical reassortant methodology from A/California/7/2009 (H1N1) virus and NYMC X-157 virus, with the HA, NA and PB1 genes originating from A/California/7/2009 (H1N1), and the other internal genes from A/PR/8/34 (H1N1) . This vi95Ile, was present at a low level (7%) in NA of sample X179A-M3.One non-synonymous mutation, SerIn general, samples derived from X-179A virus were heterogeneous and contained several complete substitutions compared to its published sequence.HiSeq sequencing of DNA libraries prepared from X-179A viruses revealed more heterogeneity than HiSeq/MiSeq sequencing of RNA libraries prepared from the same samples. Mutations found in other genomic segments are presented in Similar to X-179A virus, X‐181 virus was developed by New York Medical College using conventional reassortment technology. It has a 5:3 gene constellation with three genes  coming from A/California/07/2009 (H1N1) and the remaining five genes from A/PR/8/34 . The A/P1398G  in the X-181-M3 virus and G756T  in the X-181-M4 virus. The latter mutation is located in the conserved region of the antigenic site Ca. One non-synonymous mutation, Thr466Ala, was present at a low level (8%) in the NA gene of X-181-M2. Interestingly, the mutation Asn146Asp is located in the Sa antigenic site and exists as a complete substitution in X-181 virus which is derived from X-179A virus. As mentioned above, this mutation was observed in X-179A-M5 at a high level (78%) and at a low level (11%) in the wild-type A/California/07/2009 (H1N1) virus.Viruses derived by passaging of the X-181 virus in chicken eggs under manufacture conditions  were anaViruses derived by passaging of X-181 in chicken eggs were heterogeneous and contained several complete mutations in PB2, NP and NS segments  Tables.As for X-179A viruses, HiSeq sequencing of DNA libraries prepared from X-181 viruses revealed more heterogeneity than HiSeq/MiSeq sequencing of whole-RNA libraries prepared from the same samples. Mutations found in other genomic segments are presented in Rapid emergence of mutations is an inherent property of RNA viruses, leading to genetic heterogeneity of viral populations . This alThe larger number of eggs used during manufacture process would decrease the level (percentage) of each individual mutant, but could nevertheless increase the overall heterogeneity of virus population. Cumulatively, they could lead to a similar loss of antigenic properties as one mutation present at a higher level. Therefore, it may be important to monitor the changes that take place in major protective epitopes of the seed virus stocks to avoid losses in protective efficacy of the vaccine. Not all mutations that emerge in viruses are harmful to vaccine properties. Mutations that consistently accumulate have higher fitness and could increase virus yields and vaccine potency. Therefore, studying mutations emerging in vaccine strains could be used for both quality control, and for optimization of the genetic composition of vaccine strains.Seed viruses used in influenza vaccine manufacture are produced either by reassortment technology that uses co-cultivation of a high-growth reference strain with the current virus isolate leading to exchange of genomic segments , or by rTo reveal sequence heterogeneity in viral stocks we have used a deep sequencing approach that was recently shown to be an effective tool for quantifying all mutants in viral populations . MutatioThe deep sequencing results revealed several heterogeneities in most genomic segments, and several mutations led to amino acid changes. No big differences were observed between the results obtained by sequencing of whole-RNA libraries using MiSeq and HiSeq, and HiSeq sequencing of DNA libraries  Tables. Deep sequencing of several stocks of high-growth reference strain A/PR/8/34 revealed also some mutations and sequence heterogeneities. The CBER master stock of this strain contained several complete mutations in comparison to the published sequence, and most of these mutations were silent. There were also low levels of additional mutations. This indicates that this A/PR/8/34 virus is genetically stable, and the use of this virus as backbone for development of influenza vaccine may improve the vaccine production.Another A/PR/8/34 virus stock was passaged 13 times on eggs and was grown (passage 14) separately in two different embryonated chicken eggs, the harvested 2 viruses (designated as A/PR/8/34-1 and A/PR/8/34-3) were deep-sequenced using HiSeq protocol. These two viruses shared most sequence heterogeneities, but the percentages of mutants were slightly different . Therefo18Gly) of the viral population, this mutation is very rare and nothing known about its biological function.The parental wild type A/California/07/2009 (H1N1) virus was sequenced by HiSeq using the whole RNA library . Its con314Gln present at 17% and Asn146Asp present at 78% of the population) were identified in X-179A-M5 virus. Interestingly, the latter is located in the antigenic site Sa; it was detected at a low level (11%) in A/California/07/2009 (H1N1) strain and was present as a complete substitution in the X-181 virus stock derived from the X-179A virus (328Thr mutation at a low level (9%). Viruses derived from X-179A were heterogeneous and contained some complete nucleotide substitutions in comparison to their published sequences in PB2, PB1, NP, and in NS segments  mutation emerged in HA of the X181-M4 virus, and it is located in the conserved region of the antigenic site Ca.The X-181 virus was developed from the X-179A seed lot by another round of reassortment, and also is subjected to several passages in eggs . Deep se16Ser and Lys226Glu amino acid substitutions emerged at relatively low levels  in HA. The latter mutation was located in the Ca antigenic site in HA, next to codon 225 that was shown to participate in the modulation of HA receptor avidity/specificity [136Asn was present at a high level (78%). It is located close to the antigenic site Sa in close proximity within the sialic acid-binding pocket, suggesting its functional importance [Unlike the two previous viruses, 121XP was developed by reverse genetics . Deep secificity . In addicificity , 41. Mutportance . InteresIn conclusion, our deep sequencing approach based on RNA library preparation was shown to be effective and reproducible for detection of low quantities of mutants in the entire genome of influenza viruses. It revealed that the viruses derived from the three of pandemic A/Ca/07/2009 (H1N1) vaccine viruses have varying levels of sequence heterogeneities. It can be used for assessment of the quality of seed viruses, and for ensuring that they do not contain mutations that could affect immunogenic properties of the vaccine.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLS)Click here for additional data file.S10 Table(XLS)Click here for additional data file.