To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i) genes with low CpG density and H3K9me3 in the gene body or (ii) genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF) genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively. Methylation of cytosines at promoters is an epigenetic mark, which is linked to gene silencing. The main regulatory function of DNA methylation has been associated with CpG-rich regions, CpG islands, which are frequently co-localized with gene promoters. It is well known that DNA methylation prevents binding of a number of transcription factors and attracts methylated-CpG binding domain proteins (MBDs) together with chromatin modifiers, which are responsible for the repressive chromatin structure Arabidopsis thaliana, in Neurospora crassa and in mammalian heterochromatin repeats de novo DNA methyltransferase, DNMT3B de novo DNA methyltransferase, DNMT3A Connections between DNA methylation and chromatin modification processes have recently become apparent. Aberrant CpG island DNA methylation in cancer has been linked to the Polycomb-associated repressive histone mark, H3K27me3 Arabidopsis thalianaRecently, a number of publications reported on the presence of extensive DNA methylation in gene bodies, downstream of transcription start sites (intragenic methylation). It was noticed that actively transcribed genes are associated with increased DNA methylation levels in To more precisely understand the crosstalk between histone modifications, DNA methylation and transcription in mammalian cells, we profiled DNA methylation and chromatin modifications on human chromosome 19 in human bronchial epithelial cells (HBEC). To understand the impact of DNA methylation on chromatin and transcription, we performed a similar analysis in the HCT116 colon cancer cell line with double knockout of DNMT1 and DNMT3B, a cell line characterized by loss of 95% of DNA methylation To understand the interdependence between DNA methylation and histone modifications, we profiled DNA methylation patterns and several histone modifications along human chromosome 19 (chr19) on NimbleGen tiling arrays in human bronchial epithelial cells immortalized with hTERT and CDK4 LSM4 gene  the H3K36me3 and DNA CpG methylation patterns in gene bodies are not directly dependent on each other, and (ii) from their CpG density-dependent gene-specific localization patterns, we suggest that there is a similar functional role for intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively. Future studies will determine whether the role of these two intragenic repressive marks indeed lies in suppression of spurious intragenic gene transcription as is assumed by some current models or whether they have other functional roles that are currently not anticipated.HCT116-WT and HCT116-DKO (DKO1 line) cells were generous gifts from B. Vogelstein (Johns Hopkins University) SETD2 gene, we used siRNA D-012448-03, HYPB (Dharmacon). For non-targeting control, siRNA D-001810-01-05 was used (Dharmacon).For siRNA treatment, cells were transfected twice, 96 hours apart, each transfection using Lipofectamine 2000 (Invitrogen) and OptiMEM® I medium (Invitrogen) according to the manufacturers' instructions. For the SETD2 siRNA experiments were used. Samples were denatured in Laemmli sample buffer  and resolved in pre-cast gels (BioRad). After semi-wet transfer, membranes were blocked with 5% milk (BioRad) in PBS with 0.1% Triton X-100 and further incubated with specific antibodies. We used the following antibodies for Western blotting: H3K36me3 (Abcam and SA Bioscience), histone H3 (Abcam), and beta-actin (Sigma). Detection was achieved using HRP-conjugated secondary antibodies (BioRad) and ECL Western Blotting System .For protein analysis, the same number of cells for control and http://www.mged.org/Workgroups/MIAME/miame.html. Expression data were verified by real-time RT-PCR with cDNA obtained with iScript cDNA Synthesis Kit (BioRad).Total RNA was isolated with the RNAeasy kit . For RNA analysis in HBEC, Affymetrix human gene 1.0-ST arrays were used. For expression profiles of HCT116-WT, HCT116-DKO, and HBEC non-targeting siRNA and HBEC SETD2 siRNA experiments, Affymetrix human exon 1.0-ST microarrays were applied. Microarrays were processed at the City of Hope microarray core facility. Arrays were scanned at 5 µm resolution using the Affymetrix GCS 3000 7G scanner and GeneChip Operating Software v. 1.4 to produce. CEL intensity files. Raw intensity measurements of all probe sets were background-corrected, normalized and converted into transcript or exon level expression measurements using the Affymetrix Expression Console v1.1.1. All data is MIAME compliant. The raw microarray data have been deposited in the Gene Expression Omnibus repository (accession number GSE26020), which is a MIAME compliant database, as detailed on the MGED Society website DNA isolation was done with the DNAeasy kit according to the manufacturer's instructions (Qiagen). Bisulfite DNA conversion was done with the EZ DNA Methylation-Gold™ Kit . For DNA methylation analysis, bisulfite-modified DNA was amplified with FastTaq polymerase (Qiagen) and gene-specific primers (primer sequences are available on request). The amplicon was digested dependent on sequence with TaqIa, BstUI or HpyCH4IV , or cloned by using pGEMTeasy vector  and TAM1 competent cells . After cloning, individual clones were sequenced.5'-AGCAACTGTGCTATCCGAGGGAT and 5'-ATCCCTCGGA) overnight at 16°C with T4 ligase (New England Biolabs). Genome amplification was performed in the exponential phase with Taq polymerase (Qiagen). For MIRA quality control, real-time PCR with 10 ng amplicon and FastTaq polymerase (Qiagen) was done by using specific primers complementary to unmethylated gene promoters  or methylated targets . Primer sequences are available on request. For DNA methylation and chromatin profiling, NimbleGen tiling microarrays of human chromosome 19 were used. Labeling, hybridization, and scanning of NimbleGen tiling arrays were performed at the City of Hope microarray core facility. The log2 ratios of MIRA signal vs. input were generated using NimbleScan v 5.0 (NimbleGen) with default settings. All microarray data is MIAME compliant. The raw microarray data have been deposited in the Gene Expression Omnibus repository (accession number GSE26020), which is a MIAME compliant database, as detailed on the MGED Society website http://www.mged.org/Workgroups/MIAME/miame.html.MBD2B and MBD3L1 protein purification and MIRA were done as previously described ChIP experiments were done as previously described http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/) was used for annotation. The promoter region was defined as +/−500 bp relative to the transcription start site, and ‘gene body’ was defined as from the transcription start site to the end of the transcript. For DNA methylation and histone modifications, the average log2 ratios of probes falling into promoters or gene bodies were calculated. For CpG count in the gene body, the number of CpG dinucleotides within gene bodies was counted and divided by the length of the gene body and then scaled to counts per 1 kb.The hg18 RefSeq database , and those located on chr19 were kept for subsequent analysis. The top 15% exons with the highest expression level and bottom 15% exons with the lowest expression level were selected based on their log2 intensity distribution, and denoted as exons with high and low expression, respectively. For the genes covered with either H3K36m3 and MIRA signal or with H3K36m3 and H3K9m3 signal, the average log2 ratio signal of the probes falling into the two expression categories were calculated. For genes with at least two exons, the average log2 ratio signal of the probes falling into the first exon, last exon, and each intron were calculated. For the genes on chr19 with at least two coding exons, the average log2 ratio signal of the probes falling into the first coding exon and last coding exon were calculated. The distribution of these average probe signals was presented as boxplots .http://david.abcc.ncifcrf.gov/) and genes on chr19 were used as background.The gene lists were uploaded to DAVID functional annotation tools  are indicated. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with TaqIa, recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were separated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. B. COBRA analysis of an intragenic region of the POLD1 gene showing DNA methylation and H3K36me3 occupancy. The analyzed region is indicated by a blue box. C. COBRA analysis of an intragenic region of the NFKBIB gene showing DNA methylation and H3K36me3 occupancy. The analyzed region is indicated by a blue box. D. Negative correlation between unmethylated DNA (UMC) and H3K36me3 in HBEC. Averages of UMC signal in the gene body for each gene were spotted versus average of H3K36me3 signal in the gene body. E. Positive correlation between methylated DNA (MIRA) and H3K36me3 in HBEC. Averages of MIRA signal in the gene body of each gene were spotted versus average of H3K36me3 signal in the gene body. F. Negative correlation between histone H3 acetylation (H3Ac) and DNA methylation (MIRA) in promoters (-1000 to +500 bp of the transcription start sites). G. Negative correlation between unmethylated DNA signal (UMC) and methylated DNA signal (MIRA) in promoters.(PDF)Click here for additional data file.Figure S2Crosstalk between DNA methylation and H3K27me3 in HBEC.A. Gene bodies marked by DNA methylation and H3K27me3 on human chr19 in HBEC. The numbers indicate genes with gene bodies marked by H3K27me3 or DNA methylation or by both marks with at least 20% of gene body length coverage . B. Composite profile of genes with gene bodies marked by H3K27me3 and DNA methylation with at least 20% of gene body length coverage. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3′ gene end were divided into 10 bins. The average signal for each single bin is plotted. Both MIRA and UMC signals are enriched within H3K27me3-covered gene bodies indicating partial methylation. C. Representative epigenetic profile of a chr19 region containing H3K27me3-marked genes (boxes) in HBEC. D. DNA methylation level in gene bodies of different epigenetic gene groups, genes marked by H3K27me3 together with DNA methylation, genes marked by H3K9me3 and H3K36me3, and genes marked by DNA methylation together with H3K36me3. The average of signal in each gene body was plotted for each epigenetic group. A gene was considered marked by a specific epigenetic modification if this mark was present along at least 20% of gene body length. Multiple comparison tests show that all three pairs show statistical difference at p<0.001. E. CpG density in gene bodies for genes marked by H3K27me3 and DNA methylation, by H3K9me3 and H3K36me3, and by DNA methylation together with H3K36me3. Average of CpG density for each gene body was plotted for each epigenetic group. Comparison between H3K27me3-MIRA and H3K36me3-H3K9me3 shows statistical difference (p<0.001). Comparison between H3K36me3-MIRA and H3K36me3-H3K9me3 also shows statistical difference (p<0.001). There is no statistical difference between H3K27me3-MIRA and H3K36me3-MIRA (p>0.05).(PDF)Click here for additional data file.Figure S3Epigenetic characteristics of H3K9me3-associated genes on chr19 in HBEC.A. Partial DNA methylation in gene bodies of genes occupied with H3K9me3. A representative epigenetic profile of an H3K9me3-enriched gene is shown. Direction of transcription, gene coordinates and the region analyzed by COBRA in the NAPL1 gene are indicated. DNA methylation analysis was performed by COBRA for three CpG-poor and H3K9me3-occupied genes  with partial DNA methylation as indicated by UMC signal. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. B. Gene ontology terms associated with H3K9me3- and H3K36me3 dual-occupied genes according to analysis via DAVID.(PDF)Click here for additional data file.Figure S4Gene body DNA modifications and chromatin marks in relation to exon and intron structure. Genes on chromsome 19 were subdivided into two categories. Panels A and B show genes co-occupied with H3K36me3 and DNA methylation. Panels C and D represent genes co-occupied with H3K36me3 and H3K9me3. Using data from Affymetrix exon arrays, we determined which exons are expressed at low levels (bottom 15% of expressed genes) and which ones are expressed at high levels (top 15% of expressed genes). The MIRA, H3K36m3 or H3K9m3 average signal (log2 ratio) of probes in the selected exons or introns is plotted. We also plotted the signals for the first and last exons and the first and last coding exons, respectively. A. H3K36me3 levels in H3K36me3 and DNA methylation co-occupied genes. B DNA methylation levels in H3K36me3 and DNA methylation co-occupied genes. C. H3K36me3 levels in H3K36me3 and H3K9me3 co-occupied genes. A. H3K9me3 levels in H3K36me3 and H3K9me3 co-occupied genes.(PDF)Click here for additional data file.Figure S5DNA methylation and epigenetic profiles in HCT116-DKO and HCT116-WT cells.A. DNA methylation in LINE1 elements in HCT116-DKO and HCT116-WT cells. Using LINE1-promoter-specific primers, bisulfite-converted DNA was amplified. After cutting with HinfI, recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated by size on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation, which is almost completely lost in DKO cells B. MIRA and H3K36me3 distribution profile along the DNMT1 gene in HCT116-DKO and HCT116-WT cells. Note the loss of signal between exons 3 and 5 (yellow box) in DKO cells due to gene disruption but persistent transcription as indicated by the H3K36me3 profile. C. Verification of retained DNA methylation in some gene bodies in HCT116-DKO cells. Representative profile of persistent DNA methylation in gene bodies in HCT116-DKO cells and comparison to the profile in HCT116-WT cells. Red, DNA methylation; green, H3K36me3. The gene coordinates and location of the analyzed region in the NFKBIB gene are indicated. DNA methylation analysis was performed by COBRA for two different genes retaining DNA methylation in the gene body according to the MIRA data. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with TaqIa, recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated by size on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation.(PDF)Click here for additional data file.Figure S6Epigenetic profiles of H3K9me3-associated genes that are either upregulated or downregulated in HCT116-DKO cells.A. Composite profile of upregulated H3K9me3-covered genes in HCT116-WT and DKO cells. The profile was created for genes with gene bodies covered by H3K9me3 with at least 20% of gene body length coverage in HCT116-WT cells and upregulated transcription by at least log2 of 0.5 in comparison to HCT116-WT cells. B. Composite profile of downregulated H3K9me3 genes in HCT116-DKO. The profile was created for genes with gene bodies covered by H3K9me3 with at least 20% of gene body length coverage in HCT116-WT cells and downregulated transcription by at least log2 of 0.5 in comparison to HCT116-WT cells.(PDF)Click here for additional data file.


Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.In April 2009, a novel triple-reassortant swine influenza A H1N1 virus  was detected and spread globally as the first influenza pandemic of the 21−3 amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493–757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.By Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades  were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47×10These findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics. Orthomyxoviridae) possess a segmented, negative polarity, single-stranded RNA genome and lipid-enveloped virions st century. During the 2009–2010 pandemic, A/H1N1pdm became the dominant circulating virus. Efficient human-to-human transmission was observed, with clinical attack rates of 20–50% Influenza viruses ; nucleoprotein (NP); nonstructural (NS1 and NEP (formerly known as NS2)) and matrix (M1 and M2) proteins, and two glycoproteins, hemagglutinin (HA) and neuraminidase (NA) The Canadian provinces of Quebec (PQ) and Ontario (ON) first identified cases of influenza-like illness (ILI) with travel histories or exposure to travelers returning from Mexico within 2 weeks of outbreak detection . Thereafter, A/H1N1pdm was detected in British Columbia (BC), Alberta (AB) and Nova Scotia (NS) n = 178 and 57 unique virus genomes, respectively. We then conducted analyses to gain insight into 1) how A/H1N1pdm influenza spread spatially and temporally during the nationwide epidemic, and 2) whether mutated A/H1N1pdm protein residues influenced infection severity. Understanding the spatial and temporal spread of A/H1N1pdm lineages in Canada may provide insights into disease trajectory for future influenza pandemic planning. Determining any correlation between mutated protein residues and infection severity is important for vaccine development, and could be important for resource allocation should identified mutations arise in at risk populations.The A/H1N1pdm virus continues to circulate worldwide, giving rise to concerns about genotypic diversity that might evolve to enhance virus transmissibility and/or pathogenicity and affect vaccine efficacy. An earlier study noted early diversification of the A/H1N1pdm virus into seven distinct lineages, designated as clades 1–7, with spatial patterning within 19 weeks of the pandemic's initiation n = 235) were subjected to phylogenetic analysis. Our phylogenetic inference supports that multiple A/H1N1pdm lineages were introduced into Canada generating complex spatial patterns  based on 433 HA and NA global outbreak sequences, RV1595/09 exhibited inferred ancestry dating to March 30 2009 The first Canadian specimen (RV1595/09 (PQ) representing a mild case acquired April 16 2009 or week 03 of the global outbreak) belonged to clade 2  and S2. Clade 3 (represented by A/Arizona/01/2009) was detected globally several weeks after clades 1 and 2; although no fixed amino acid changes are common to all clade 3 isolates Clade 5, characterized by amino acid changes in the NP (V100I) and NA (V106I and N248D) was only observed twice in Canada (RV1758/09 and RV1954/09) in PQ during outbreak weeks 06 and 08, respectively  with infn = 40) up to outbreak week 11. Sequenced clade 6 viruses also were detected in 8 other regions , Newfoundland and Labrador (NFL), NS, Nunavut (NU), Northwest Territories (NWT), ON, and SK), but were fewer (range: 1–6) , appeared later in Canada starting in week 06 (RV1765/09 - MB). Clade 6 viruses accounted for 24.3% (57/235) of the sampled Canadian viruses. Amongst this study's sequenced data set, clade 6 viruses dominated in MB (ge: 1–6) .Clade 7 originated late in March 2009 and appeared in New York a few weeks after clades 1 and 2 were isolated in Mexico and California n = 168) and additional sampling of circulating A/H1N1pdm viruses during weeks 23 through 31 (n = 42)  , which j(n = 42) . The dis2 , which(n = 42) . Overall(n = 42)  and 2, i , which (n = 42) .−4 nucleotide changes per gene site and 9.00×10−4 coding substitutions per protein site. Coding variants were non-uniformly distributed across the segments: HA (352); NA (178); PB1 (172); NP (127); PB2 (32); PA (28); NS1 (18); M1 (4); M2 (4), and NS2/NEP (2)  . HoweverAside from expected diversity for the antigenic viral glycoproteins (HA and NA), high ns/s (coding/non-coding) ratios also were observed in the genes encoding nucleoprotein (NP) and polymerase basic protein 1 (PB1) . PB1 divIn order to track the antigenic evolution of A/H1N1pdm, we investigated potential antigenic drift in HA. Amino acid substitutions were observed in a limited number (9) of the Canadian viruses at characterized hemagglutinin antigenic sites: Ca (7) - R205K in Ca1 (6) and P137S Ca2 (1); S74N in Cb (1); and A186T in Sb (1)  [11], . Highest variability was observed in clade 2 and clade 7 viruses within PB1 spanning residues 566 through 652; this PB1 region corresponds to the viral genomic RNA (vRNA)-binding domain (residues 493–757) It is worth noting that under new selective pressures, such as oseltamivir antiviral treatment, recombinant viruses with enhanced viral polymerase activity have been found capable of rapidly generating adaptive mutations that may enhance viral fitness Globally, 25% to 50% of severely ill A/H1N1pdm patients had no coexisting medical conditions n = 77) for which infection status remained unknown. Thus, benefits of a proactive and systematic process to link laboratory-generated molecular data and epidemiological data for enhanced surveillance purposes during such a pandemic were highlighted during the course of this study.Notwithstanding the analysis of 133 unique wave 1 viruses and 158 wave 1 and 2 viruses (combined) for which infection status was known, our findings did not reveal any linkages between virus genotype and increased influenza infection severity. Although best attempts were made to achieve rigorous virus selection; lack of randomness in specimen sampling may have introduced bias  that precluded us from achieving findings with significance. For example, we are aware that the sequenced Canadian viruses associated with severe A/H1N1pdm infections were not optimally distributed. Moreover, our entropy analysis was restricted given large numbers of specimens (n = 5) in 3 distinct clades/regions: namely clade 7 - AB (3); clade 3 - PQ (1) and clade 6 - ON (1). All resistant cases were associated with oseltamivir treatment. Such low frequency resistance is reassuring given the pervasive application in Canada of antivirals in clinically-diagnosed A/H1N1pdm cases. This H275Y mutation does not cause resistance to zanamivir (Relenza®). Although recent data suggests that oseltamivir-resistance does not alter viral fitness and may induce severe disease at least in ferrets All sampled Canadian viruses encoded the A/H1N1pdm signature S31N (M2) allele that conveys antiviral resistance to Amantadine. The neuraminidase H275Y (H274Y in N2 numbering) mutation known to confer resistance to oseltamivir (Tamiflu®) and to peramivir, a drug used intravenously under an emergency use authorization, also was detected but only sporadically  underscore the complexity of influenza A virus genetics and advocate for intensified global surveillance applying whole-genome sequence data. Although evaluating effects of vaccination on viral evolutionary trends and vaccine efficacy over time are important, this study was not designed to address such issues. Continued molecular surveillance studies are indeed warranted to monitor A/H1N1pdm virus genotypic and phenotypic diversity in the post-vaccination era, providing information to enable timely epidemic or pandemic detection, inform health policy decisions and influenza management strategies, and to guide influenza vaccine development and prioritization. With the advent of worldwide sentinel surveillance in humans and animals as well as advances made in rapid whole-genome sequencing, mathematical modeling, global alerting, immunopathogenicity, and drug and vaccine therapeutics, public health officials now have assembled necessary tools to intervene effectively to prevent major morbidity and mortality from influenza disease. Fortunately, this marks a major turning point in the approach to management of influenza.2 incubator and observed daily for cytopathic effect. Influenza A/H1N1pdm virus was confirmed by molecular evaluation  and antiviral sensitivities inferred from DNA sequences were confirmed by phenotypic analyses.Virus specimens were collected from subjects with influenza-like illnesses as part of the ongoing Canadian influenza surveillance program. This program continuously monitors genetic and antigenic changes in influenza viruses circulating in Canada. As a national public health surveillance program, informed consent from subjects is not required. However, as some additional specimens were collected nationally from subjects that required hospitalization and participated in specific H1N1-related studies, ethics approval was acquired from the University of Manitoba Health Ethics Board to investigate these viruses. For these patients, written informed consent was obtained from each patient or their surrogate. Specimens originated from eleven of the twelve provinces and territories of Canada, namely Alberta (AB), British Columbia (BC), Manitoba (MB), New Brunswick (NB), Newfoundland and Labrador (NFL), Nova Scotia (NS), Northwest Territories (NWT), Nunavut (NU), Ontario (ON), Quebec (PQ), and Saskatchewan (SK); missing: Yukon. For virus propagation, 50 µL of clinical specimens was inoculated into the Madin-Darby canine kidney cell line (MDCK)  and examined for cytopathic effect. The MDCK cells were maintained in Eagles's MEM containing 100 U/mL Penicillin, 100 µg/mL Streptomycin, 0.292 mg/mL Glutamine, 25 mM Hepes buffer, 0.1 mM MEM non essential amino acid, 1 mM MEM sodium pyruvate and 2 µg/mL TCPK-trypsin. Cells were incubated at 37°C in a COn = 158), and were distributed geographically and temporally , representing week 03 of the global outbreak . AlthougThe influenza A (H1N1) 2009 vaccine produced for Canada by GlaxoSmithKline, called Arepanrix H1N1, was approved on October 21 2009; and vaccinations began for high-risk groups on October 26 2009 , with the general public following thereafter  and S2. n = 240) are available for download  extracts were directly processed. Otherwise, viruses were first propagated in MDCK cells, as described. Viral RNA was extracted, in the presence of supplied carrier RNA, with the MagMAX™ Viral RNA Isolation Kit  using a MagMAX™ Express-96 Magnetic Particle Processor according to the manufacturer's instructions. Extractions were performed on 140 µL for the cultures and 265 µL for the primary specimens. Complementary DNA was synthesized by reverse transcription reaction, and amplification by PCR was performed using specific primers for each gene segment for a sum of 14 amplicons. RT-PCR reactions were generated using the OneStep RT-PCR kit  with either the universal influenza A generic primer, Uni12, as reported elsewhere n = 1198) sequences were downloaded from the influenza sequence database . Accession numbers for the downloaded global genome references are provided in http://www.nescent.org/informatics/download.php?software_id=4, accessed June 2, 2010) also was applied to infer or assess the reliability of the phylogenetic trees In addition to the Canadian human-isolated A/H1N1pdm virus genomes sequenced in this study, other full-length sequences (10 coding sequences) available as of April 1 2009 to June 2 2010 (inclusive) (P<0.05) amino acid differences with randomization test Mutations within A/H1N1pdm genes were determined by Lasergene® v8.1 SeqMan Pro software  and displayed by Jalview v2.4.0 A/H1N1pdm sites exhibiting positive selection were identified using the codon-based maximum likelihood method, Fixed Effects Likelihood (FEL) within the Hypothesis Testing Using Phylogenies (HyPhy) package Viral sequences from this study have been deposited into GenBank , British Columbia (BC), Manitoba (MB), New Brunswick (NB), Newfoundland and Labrador (NFL), Nova Scotia (NS), Nunavut (NU), Northwest Territories (NWT), Ontario (ON), Quebec (PQ), and Saskatchewan (SK). Distribution of infection case status: When known, the infection status associated with each virus is depicted with coloured spheres: teal (mild); severe (red). Otherwise, unknown case status is depicted with triangles (black).(TIF)Click here for additional data file.Figure S2Regional and temporal distribution of A/H1N1pdm infections in Canada. A. National case incidence according to the global outbreak initiated April 1 2009, corresponding to the date of collection for the first global A/H1N1pdm virus. B. Regional incidence for Canada during Wave 1. C. Regional incidence for Canada during Wave 2.(TIF)Click here for additional data file.Table S1Descriptions for A/H1N1pdm viruses sampled in Canada.(XLS)Click here for additional data file.Table S2Amino acid variations and clades detected for A/H1N1pdm viruses sampled in Canada.(XLS)Click here for additional data file.Table S3Cumulative hospitalized A/H1N1pdm cases in Canada during Wave 1, by region.(DOC)Click here for additional data file.Table S4Entropy difference, with randomization test, between severe and mild sequence populations for A/H1N1pdm viruses sampled in Canada .(DOC)Click here for additional data file.Table S5Summary of A/H1N1pdm infection incidence in Canada.(DOC)Click here for additional data file.Table S6Oligonucleotides used to amplify and sequence A/H1N1pdm viruses in this study.(DOC)Click here for additional data file.Table S7Accession numbers for Influenza A/H1N1pdm viruses sequenced in this study.(DOC)Click here for additional data file.Table S8Accession numbers for the global outbreak A/H1N1pdm virus reference sequences used for generating (DOC)Click here for additional data file.Table S9Accession numbers for the global outbreak A/H1N1pdm virus reference sequences used for clade assignment in (DOC)Click here for additional data file.


The mutations of LHBs in pre-S, especially in pre-S2, are definitive in hepatocellular carcinoma (HCC) associated with HBV. However, the mechanisms of the N-glycosylation modification in LHBs are unclear. The N-glycosylation modification of LHBs affects Endoplasmic Reticulum stress, cell proliferation and its secretion which was further studied.The objectives of our studies was to indentified that modification of LHBs by N glycosylation modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation.The LHBs was mutated; then expression of proteins related to endoplasmic reticulum stress and EAED path of L02 cells affected by LHBs and its mutations was evaluated. LHBs proteins bound to multiubiquitin chains and its glycosylation motif were studied. The subcellular localization and secretion of LHBs and its mutations were identified. The effect on cell cycle and proliferation by LHBs and its mutations were detected.These data demonstrated that the N-glycosylation motifs of LHBs were associated with ER stress. The N15S, N123S, and N177S mutated LHBs proteins could induce overexpression of EDEM in L02 cells. LHBs and its mutated proteins contained p62-derived UBA domain, which could affect expression of cyclins. The subcellular localization of LHBs in endoplasmic reticulum was similar to its mutations. The secretion of LHBs was blocked by N320K mutation, which could induce an increase in G1 phase and inhibition of S phase, and inhibited mitotic entry.In conclusion, our studies powerfully demonstrated that modification of LHBs by N glycosylation could modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation. The N320K may be the key sites N-linked glycosylation modification of LHBs. It may be a mechanism of HBV-induced HCC. HBV (Hepatitis B virus) is the most common infectious agent affecting the liver world-wide , 2. PatiHCC was significantly related to two pre-S deletion mutants of LHBs -12, whicN-glycosylation is the key modification type of proteins, and modulates their crucial functions in cells , 17. ProWe previously demonstrated that pre-S2 LHBs could interact with C53; it is an unprecedented regulator of checkpoint response . Then prThe objectives of our studies was to indentified that modification of LHBs by N glycosylation modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation.L02 was purchased from the American Type Culture Collection (ATCC) (USA). Cell culture medium Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin medium supplement, glutamine and fetal bovine serum (FBS) were obtained from Gibco life Technologies (UK). Western blotting materials were purchased from Roche Applied Sciences (USA). cDNA Synthesis Kit was purchased from Fermentas, EU. SYBR green DNAPCR Master Mix was purchased from the Applied Biosystem (ABI) Company . Site-directed mutagenesis was used the TransformerTM Site-Directed Mutagenesis kit (Clontech). All antibodies were obtained from the Santa Cruz (The USA). Protein G Plus/Protein AAgarose Suspension was obtained from Calbiochem (the USA). LHBs ELISA Kit was purchased from Auszyme . Lipofectamine 2000 was purchased from Invitrogen (USA). The flow cytometer was purchased from BD (The USA). Dimethyl sulfoxide (DMSO) and all other chemicals were obtained from the Sigma-Aldrich (The USA).SPSS 11.0 software (USA) was used for the statistical analyses.LHBs were amplified from serum of a patient with chronic hepatitis B (subtype B), using upstream Primer (5'-cggaattccgatgggaggttg-3') and downstream primer (5'-ccaagcttggtcaaatgtataccc-3'). The restriction sites, EcoRI and Hind III, were incorporated into the primer sequences for cloning purposes. The PCR reaction conditions were initial denaturing at 95 °C for 5 min, denaturing at 94 °C for 40 sec, annealing at 58 °C for 45 sec, and extension at 72 °C for 1 min. A total of 40 reaction cycles was performed. The PCR product was purified and cloned into pCDNA-3.1(-) vector, then obtained the plasmid pCDNA-3.1(-) - LHBs.Informed consent was obtained from each patient included in the study, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by our institution's human research committee.LHBs was cloned into pEGFP-C1 expression vector using upstream Primer (5'-aagcttatgggaggttgg-3') and downstream primer (5'-ggatccaatgtatacccaaagac -3'). The restriction sites, BamHI and Hind III, were incorporated into the primer sequences for cloning purposes. The PCR reaction conditions were initial denaturing at 95 °C for 5min, denaturing at 94 °C for 40 sec, annealing at 58 °C for 45 sec, and extension at 72 °C for 1 min. A total of 40 reaction cycles was performed. The PCR product was purified and cloned into pEGFP-C1vector, then obtained the plasmid pEGFP-C1- LHBs.CALR was cloned using forward primer (5’- ctcgagatggcgggatcc -3’) and reverse primer (5’- ggtaccggaaagaattttttggc -3’). The restriction sites, Xho I and Kpn I, were incorporated into the primer sequences for cloning purposes. The PCR reaction conditions were initial denaturing at 95 °C for 5 min, denaturing at 94 °C for 40 sec, annealing at 54 °C for 40 sec, and extension at 72 °C for 1 min. A total of 40 reaction cycles was performed. The PCR product was purified and cloned into the pDS-RED1-N1 expression vector, then obtained the plasmid pDS-RED1-N1- CALR.Method for site-directed mutagenesis of LHBs was based on the manual of the Transformer TM Site-Directed Mutagenesis kit. The N-glycosylation sites were predicted, and the mutated base and amino acid sequence of the LHBs protein are showed in The resulting product was cut with SacI. The EcoR I-Hind Ш-fragment containing the N15S, N123S, N177S and N320K mutations were subcloned into the pCDNA-3.1(-) vector and pEGFP-C1.For determining the role of LHBs or mutation of LHBs in endoplasmic reticulum stress, we cultured L02 cell for 24 hrs, then transfected with pCDNA3.1 (-), pCDNA3.1 (-)-LHBs, pCDNA3.1 (-)-N15S, pCDNA3.1 (-)-N123S, pCDNA3.1 (-)-N177S, and pCDNA3.1 (-)-N320K respectively. Plates were sealed and incubated for 48 hrs at 37℃. Total proteins were extracted from the separated L02 cells respectively. The expression of GADD34 and GRP78 were detected by western blot respectively.L02 cells were grown on culture in 6-well plates for 48 hrs; cells were lysed in lysis buffer with Phenylmethanesulfonyl fluoride (PMSF). The extract was centrifuged, then each supernatant was added 50 μL of Protein G Plus/Protein AAgarose Suspension for 4 hrs at 4°C. The supernatant was added respectively, 10 μL antibody rotated mildly overnight at 4°C, then 60 μL Protein G Plus/Protein A Agarose Suspension was added, and rotated mildly for 9 h at 4°C, centrifuged for 5 min at 4°C. Immunoprecipitates were washed three times in lysis buffer, and then further examined by western blot.L02 cells were harvested after culturing for 24 hrs, then transfected with pCDNA3.1 (-), pCDNA3.1 (-)-LHBs, pCDNA3.1 (-)-N15S, pCDNA3.1 (-)-N123S, pCDNA3.1 (-)-N177S, and pCDNA3.1 (-)-N320K respectively as described in the above methods. After 48 hrs, the total proteins of cells were extracted respectively.Mixed ubiquitin chains  were diluted in 50 mMTris, 0.1% BSA, pH 7.5 (50µL) and loaded on washed UBA-agarose beads (product code UW9010) (10µL). Following incubation UBA-agarose beads with proteins from cells respectively, bound proteins were occasional agitated at room temperature for 20 minutes, and then were eluted in SDS PAGE loading buffer (50µL). After run on an SDS-PAGE gel before being transferred for Western blotting, blots were probed with anti- LHBs antibodies. The bands were LHBs p39 (39 kDa).L02 cells were harvested after culturing for 24 hrs, then transfected with pCDNA3.1 (-), pCDNA3.1 (-)-LHBs, pCDNA3.1 (-)-N15S, pCDNA3.1 (-)-N123S, pCDNA3.1 (-)-N177S, and pCDNA3.1 (-)-N320K respectively as described in the above methods. After 48 hrs, the total proteins of cells were obtained and bound to Con A or LCA Sepharose CL-4B column respectively. Then the proteins bound to Con A or LCA column were eluted and run on an SDS-PAGE gel before being transferred for Western blot. Blots were probed with anti-LHBs antibodies.Lysates of cells were boiled for 10 min with denaturing buffer . Then the above lysates were incubated for 1 h at 37°C with 500 U N-Glycosidase F (PNGase F) (New England Biolabs). Then the proteins were run on an SDS-PAGE gel before being transferred for Western blot. Blots were probed with anti-LHBs antibodies.The plasmids pEGFP-C1-LHBs  and pDS-RED1-N1-CALR were cotransfected into L02 cells. After transfection for 48 hrs, cells were treated with 4% paraformaldehyde (PFA) for 10 min, washed 3 times with PBS, and stained with 0.1μg/mL 4',6-diamidino-2-phenylindole (DAPI) for 30min at 37℃. Those were observed with Zeiss LSM510 confocal laser scanning microscope.The supernatants of cultured cell were collected and detected using LHBs ELISA Kit . The method was according to the kit instruction manual.Cells were dissociated with trypsin, washed, and resuspended in PBS as a single-cell suspension. Cells were fixed in 70% ethanol overnight, stained with propidium iodide (25 ug/ml), and incubated for 30 min at 37°C with RNase A (20 ug/ml). The cells group treated with PBS was used as the controls. Cells were assessed by flow cytometer and the results were analyzed with Modifit software. The DNA content of the cells was then evaluated by fluorescence-activated cell sorting with a FACSCalibur (BD Immunocytometry Systems).To avoid potential carry-over effects of plasmids transfection-induced cell cycle defects in the previous cycle on the following mitotic entry during the next cycle, plasmids were transfected into cells during the interval between the two thymidine blocks, so that we were able to evaluate direct impact of LHBs and its mutations on mitotic entry. Cells were synchronized by double thymidine block. Briefly, cells were plated at 40% confluency and arrested with 2 mM thymidine. After 19 hrs incubation, cells were washed 4 times with fresh medium and transfected with plasmids using Lipofectamine 2000 (Invitrogen). After incubation with DNA-lipid mixture for 3 hrs, cells were washed twice and incubated in fresh medium for additional 5 hrs. Subsequently, cells were cultured in medium containing 2 mM thymidine and 2 ug/ml puromycin for the second arrest and drug selection. After 16 hrs incubation, cells were released into the cell cycle by incubation in fresh medium. Cells were collected or fixed at indicated time points and subjected to specific analyses.BrdU labeling was used to evaluate DNA synthesis. After released from the second thymidine arrest at indicated time points, cells grown in 12-well plate were pulse labeled with BrdU (50 uM) for 30 min. After three washes of PBS, cells were fixed with 1 ml of Carnoy’s fixative  at -20°C for 20 min, and followed by three washes of PBS. Subsequently, DNA was denatured by incubation of 2M HCl at 37°C for 60 min, followed by three washes in borate buffer . After incubation with the blocking buffer, cells were stained with anti-BrdU antibody  overnight at 4°C. After washes in PBS, cells were incubated with Texas Red-conjugated anti-mouse goat IgG for 30 min at RT. After washes, cells were mounted and BrdU positive cells were manually scored under immunofluorescence microscope.Cells were lysated with Lysate buffer. Samples were loaded on a 12% SDS-PAGE gel, and transferred to PVDF membranes. Membranes were incubated overnight at 4 ℃with rabbit polyclonal anti-GRp78 (1:500), GADD34 antibody , β-actin , Cyclin A , Cyclin B1 , Cyclin D1 , Cyclin E , LHBs , EDEM .Using SPSS 11.0 software, a 2-tailed Student’s t test was used to compare the statistical difference between the two groups, and a 1-way ANOVA Newman-Keuls Multiple Comparisons Test was used to compare the differences between the three groups or more. Probabilities of 0.05 or less were considered to be statistically significant.In previous works we have focused on the interaction between pre-S2 LHBs and CDK5RAP3 that is the important mechanism about HBV-induced HCC . To analLHBs present unique doublet types, non-glycosylated LHBs  and single-glycosylated . It is due to posttranslational N-glycosylation in S domain of LHBs A. Because LHBs protein is localized in ER, we further determined whether the mutation protein of LHBs at N15S, N123S, N177S and N320K was associated with ER stress. The expressions of GADD34 and GRp78 were detected by western blot analysis. In contrast to the markers of ER stress, the expression of GADD34 and GRp78 was significantly upregulated in L02 cells transfected with LHBs and its mutations compared to control, especially N320K mutation A. But, tTwo reports in Science  , 21  havAfter run on an SDS-PAGE gel before being transferred for western blot, blots were probed with anti-LHBs antibodies. The bands were LHBs p39 (39 kDa). The study indicated that LHBs and its four mutated proteins were able to bind to ubiquitin-binding protein p62 multiubiquitin chains D. But, nIt confirmed that LHBs and it four mutated proteins contained p62-derived UBA domain, especially in N15S, N123S and N320K mutations, but not hHR23B-derived UBA2 domain, NBR1-derived UBA domain and NUB1/NUB1L UBA domain.To determine the effect on cyclins related to N-linked glycosylation modification of LHBs, the expressions of cyclins were detected by western blot analysis.Our results showed that the N15S, N123S and N177S mutated LHBs proteins could induce overexpression of cyclinA in L02 cells. The LHBs and N123S, N177S, N320K LHBs mutation proteins could induce overexpression of cyclin B1 in L02 cells, especially in LHBs. The N15S, N123S and N177S LHBs mutation proteins could induce overexpression of cyclin D1 in L02 cells, especially in N15S and N123S LHBs mutations. The N320K LHBs mutation proteins could induce overexpression of cyclin E . The effect of overexpression of LHBs and its mutations on cell cycle distribution was determined in L02 cells by flow cytometry. LHBs and its mutations induced an increase in G1 phase and inhibition of S phase, especially in N320K (Supplement 2A).Cells were synchronized at the G1/S boundary by double thymidine block, and then released into mitosis. After 24 hrs, BrdU was added into the medium at indicated time points to evaluate DNA synthesis. As shown in Supplement 2, incorporation of BrdU into the control, accumulation of mitotic L02 cells was delayed by LHBs and its mutations, especially in N320K B. To test for Nglycosylation, lysates of LHBs-expressing L02 cells were treated with Con A, LCA and Peptide: N-Glycosidase F (PNGase F) respectively. As shown in To demonstrate the localization of LHBs and its mutations in L02 cells, they were cultured then cotransfected with plasmids. The confocal images were acquired using Zeiss 510 META confocal microscope. The images showed that CALR protein (red) was localized in the endoplasmic reticulum . LHBs or its mutation  was localized on endoplasmic reticulum too. The nuclear of cells  were stained by DAPI. The overlaid images indicated that CALR overlapped with LHBs or its mutation on endoplasmic reticulum. The subcellular localization of LHBs was similar to its mutations .To demonstrate the secretion of LHBs and its mutations in L02 cells, they were cultured then transfected with plasmids. The LHBs levels in cell culture supernatants were detected by ELISA kit. The results showed that the secretion of LHBs was significantly lower than others in cell culture supernatants (P<0.01) . HBV has been considered the crucial cause of HCC. Wang et al. have found that deletions at pre-S1 or pre-S2 of LHBs domains could be observed in ground glass hepatocytes . The preOur study reported the consensus motifs of LHBs mutants . Then, we focused on those motifs and occurrence of those putative asparagines residues by serine or lysine. Our results demonstrated that the modification of N-glycosylation of LHBs was related to ER stress, especially in N-glycosylation sites N177S and N320K. The markers of ER stress, GADD34 and GRp78 expression were significantly upregulated by LHBs and its mutations, especially N320K A. It mayWe found that the N15S, N123S and N177S mutated LHBs proteins could induce overexpression of EDEM in L02 cells B. The N1Our results confirmed that LHBs and its four mutated proteins contained p62-derived UBA domain, especially in N15S, N123S and N320K mutations. Most studies on p62 have indicated that it could induce inhibition of apoptosis and proteasomal . The ubiThe research showed that the N15S, N123S and N177S mutated LHBs proteins could induce overexpression of cyclinA in L02 cells. The LHBs and N123S, N177S, N320K LHBs mutation proteins could induce overexpression of cyclin B1 in L02 cells, especially in LHBs. The N15S, N123S and N177S LHBs mutation proteins could induce overexpression of cyclin D1 in L02 cells, especially in N15S and N123S LHBs mutations. The N320K LHBs mutation proteins could induce overexpression of cyclin E . The N-lConcanavalin A (ConA) has been used to study characterization of glycoproteins . LHBs anBut the subcellular localization of LHBs was similar to its mutations; it indicated that modification of N-glycosylation had no effect on the localization of LHBs. The secretion of LHBs or its mutations were blocked, especially in N320K. LHBs and its mutations induced an increase in G1 phase and inhibition of S phase, especially in N320K A. As shoSupplement 1.The base (A) and amino acid (B) sequence of N 15 S-LHBs mutated on the point Asn15 of N- glycosylation. The base (C) and amino acid (D) sequence of N 123 S- LHBs mutated on the point Asn123 of N- glycosylation. The base (E) and amino acid (F) sequence of N 177 S -LHBs mutated on the point Asn177 of N- glycosylation.The base (G) and amino acid (H) sequence of N 320 K-LHBs mutated on the point Asn320 of N- glycosylationSupplement 2.To demonstrate the localization of LHBs and its mutations in L02 cells, cells were cultured then co-transfected with plasmids. After 48 hrs, cells were deal with PFA and DAPI, confocal images were acquired using Zeiss 510 META confocal microscope. CALR protein (red) localized to the endoplasmic reticulum . LHBs or its mutation  localized on endoplasmic reticulum too. The nuclear of cells  were stained by DAPI. The overlaied images indicated that CALR overlapped with LHBs or its mutation in endoplasmic reticulum (down right). LHBs (A), N15S (B), N123S (C), N177S (D) and N320K (E)


Narcolepsy cataplexy syndrome, characterised by excessive daytime sleepiness and cataplexy, is strongly associated with a genetic marker, human leukocyte antigen (HLA) DQB1*06:02. A sudden increase in the incidence of childhood narcolepsy was observed after vaccination with AS03-adjuvanted Pandemrix influenza vaccine in Finland at the beginning of 2010. Here, we analysed whether the coinciding influenza A H1N1pdm pandemic contributed, together with the Pandemrix vaccination, to the increased incidence of childhood narcolepsy in 2010. The analysis was based on the presence or absence of antibody response against non-structural protein 1 (NS1) from H1N1pdm09 virus, which was not a component of Pandemrix vaccine.Non-structural (NS) 1 proteins from recombinant influenza A/Udorn/72 (H3N2) and influenza A/Finland/554/09 (H1N1pdm09) viruses were purified and used in Western blot analysis to determine specific antibody responses in human sera. The sera were obtained from 45 patients who fell ill with narcolepsy after vaccination with AS03-adjuvanted Pandemrix at the end of 2009, and from controls.Based on quantitative Western blot analysis, only two of the 45 (4.4%) Pandemrix-vaccinated narcoleptic patients showed specific antibody response against the NS1 protein from the H1N1pdm09 virus, indicating past infection with the H1N1pdm09 virus. Instead, paired serum samples from patients, who suffered from a laboratory confirmed H1N1pdm09 infection, showed high levels or diagnostic rises (96%) in H1N1pdm virus NS1-specific antibodies and very high cross-reactivity to H3N2 subtype influenza A virus NS1 protein.Based on our findings, it is unlikely that H1N1pdm09 virus infection contributed to a sudden increase in the incidence of childhood narcolepsy observed in Finland in 2010 after AS03-adjuvanted Pandemrix vaccination. Narcolepsy with cataplexy is characterised by an excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep. In addition, patients may have other rapid eye movement (REM) sleep-related symptoms such as hypnagogic hallucinations and sleep paralysis Previously, a 17-fold increase in the annual incidence of narcolepsy, as compared to previous years in children aged under 17, was observed in Finland in 2010 As the onset of narcolepsy has been shown to be seasonal, and the cases of narcolepsy increased following the 2009 H1N1 pandemic in China The study protocol was reviewed and approved by the Institutional Review Board of the National Institute for Health and Welfare (THL), Helsinki. Children with narcolepsy diagnosed after Pandemrix vaccination were recruited to the immunological narcolepsy study at the neurological outpatient clinics in Finnish children's hospitals during the years 2011 and 2012. The study protocol was approved by the Ethics Committee of participating hospitals and informed written consent was given both by children and their guardians.The collection of serum samples from individuals suffering from an upper respiratory tract infection was approved by the Ethical Committee of the Pirkanmaa Hospital District (ETL-code numbers R09152M and R10075M). All patients gave their written informed consent for serum samples.Control serum specimens for the study included serum samples collected in 2004/2005 prior to the 2009 influenza pandemic. The samples were made anonymous by destroying all person identification data before they were shipped to the National Institute for Health and Welfare (THL); only the age of the subjects and the sample collection dates were retained. Permission to use these anonymous serum specimens was based on ethical permissions given by the Helsinki-Uusimaa Health District Ethical committee: HUS 315/E0/05  and 199/13/03/00/09 . The studies fully comply with the rules concerning national and international regulations, privacy and data protection relevant to medical- and patient-oriented studies.The present study comprised 45 narcoleptic patients aged 5 to 18 years at onset who fell ill in 2009 or 2010 after receiving Pandemrix vaccination in November or December 2009. The description of the patients, their date of Pandemrix vaccination, time of onset of narcolepsy and the time of serum sample collection is shown in Serum specimens from all 45 narcoleptic patients were collected during 2011. Paired serum specimens from 28 adults suffering from a clinical, laboratory-confirmed H1N1pdm09 virus infection were collected during the epidemic season 2009–2010 and 2010–2011 in the Tampere city area. H1N1pdm09 infection was confirmed by identifying viral RNA in nasopharyngeal stick samples collected during the acute phase of the disease using a diagnostic polymerase chain reaction (PCR) test with the NS gene as the PCR target Paired serum specimens were collected at the acute (at the time of the diagnosis) and convalescent phases (14–21 days later) of the disease. All patients showed a diagnostic antibody rise in the H1N1pdm09 virus-specific hemagglutination inhibition assay  that was initially collected from diagnostic left-over serum specimens from the HUSLAB  diagnostic laboratory. Serum specimens were previously analysed and they were found to be negative for anti-H1N1pdm09 antibodies as analysed by means of the hemagglutination inhibition (HI) test (NS) genes were modified by PCR to create N- and C-terminal BamHI sites for further cloning into an E. coli GST fusion vector pGEX-3X . All DNA manipulations were performed according to standard protocols, and the newly created gene constructs were fully sequenced.Regions coding for influenza A virus non-structural protein 1 (NS1) in the cDNAs for wild-type (wt) A/Udorn/72 (H3N2)  and wt A/Finland/544/09 (H1N1pdm09) (GQ283489.1) E. coli BL21 cells. For GST and GST-NS1 protein purification, 5 mg of sonicated, cellular protein samples in Laemmli sample buffer GST and chimeric GST-NS1 fusion proteins were expressed under isopropyl-beta-thiogalactopyranoside (IPTG) induction in To analyse humoral immune responses against NS1 proteins, serum specimens were obtained from 45 narcoleptic patients. Twenty eight paired serum samples were obtained from patients who suffered from a laboratory confirmed H1N1pdm09 virus infection, and 50 control sera were obtained from age-matched children. For Western blot analysis, 3 µg of purified GST (26 kDa) and GST-NS1 A/Udorn/72  or A/Finland/544/09  proteins were loaded on 12% SDS-PAGE gels. Proteins, separated on gels, were stained with Coomassie Brilliant Blue or transferred onto Immobilon-P membranes  with an Isophor electrotransfer device . The membranes were sliced and stained with serial dilutions of tested human sera  in PBS, containing 5% nonfat milk at room temperature for 2 h. After washing with PBS, secondary peroxidase-conjugated anti-human IgG immunoglobulins  were allowed to bind at room temperature for 1 h. After washing with PBS, the bands were visualised by 3-amino-9-ethylcarbazole (AEC) Serum specimens, obtained from 45 narcoleptic patients, from 28 patients (56 paired serum specimens) suffering from influenza infection and 50 age-matched control individuals, were analysed by the HI test against influenza A/California/07/2009 vaccine strain and epidemic A/Finland/814/01 (H1N1) and A/Finland/715/00 (H3N2) virus strains isolated in Finland. The HI tests were performed in accordance with World Health Organization (WHO) guidelines with established procedures Anti-NS1 titers in human sera were obtained by Western blotting. These values were used for calculating the geometric mean titers with 95% confidence intervals for each patient group. The significances of the differences between the mean H1/H3 titers of narcoleptic patients and other patient groups were calculated with Student’s t-test .To compare the genetic relationship of various human influenza A virus NS1 proteins the sequences for N1N1pdm09, H1N1, H2N2 and H3N2 virus NS1 genes were obtained from Genebank. The software programme Mega version 4 In order to study whether the patients who developed narcolepsy after Pandemrix vaccination in 2009–2010 were also suffering from an H1N1pdm09 virus infection simultaneously or after the vaccination, we carried out a computer search for potential virus proteins that could form the basis for an immunological distinction of vaccine or influenza infection-induced immunity. Since the vaccine contained the H1N1pdm09 virus  surface glycoproteins and internal viral proteins from a vaccine backbone virus PR8 (A/Puerto Rico/34 H1N1), it is not possible to distinguish vaccine or infection-induced immunity by conventional immunological methods like the HI test, the virus neutralization test or Western blotting against viral structural proteins. However, since the Pandemrix vaccine did not contain viral NS1 protein, which is abundantly produced during virus infection, we analysed whether measuring an immune response against the NS1 protein could serve as a method to distinguish H1N1pdm09 virus infected and vaccinated individuals from those that were only vaccinated.NS genes of the 20th century human H1N1 and H3N2 influenza A viruses have evolved from the A/Brevig mission/18 virus NS genes of the pandemic H1N1pdm viruses, however, originate from classical swine influenza viruses NS genes  and influenza A/Finland/554/09 (H1N1pdm09) NS1 proteins by Western blot technique. Western blot analysis was used since the purification of NS1 protein in a native form is very difficult due to its insolubility and stickiness. In addition, we had previously good experience in analyzing specific antibody responses against recombinant microbial proteins by Western blotting Serum specimens showed antibody reactivity against influenza A virus NS1 proteins (GST-NS1 fusion protein) but hardly any reactivity against the GST protein . All tesAntibody titers against H1N1pdm09 subtype and H3N2 subtype influenza A virus NS1 proteins in serum specimens were analysed from 45 narcoleptic patients , S2. Thest and 2nd pandemic season in 2009/2010 and 2010/2011, when the epidemic influenza A virus was almost solely of the H1N1pdm09 subtype. Almost all (89%) patients who had suffered from an H1N1pdm09 virus infection showed high or very high levels (>600) or a significant rise (>2 x) in antibody titers against the H1N1pdm09 subtype influenza A virus NS1 protein , and/or different excipients included in the suspensions could contribute to the induction or enhancement of autoimmunity, leading to the onset of symptomatic narcolepsy. Theoretically, it is possible that any pro-inflammatory signal, whether it originates from a microbial infection or vaccination, such as inflammation inducing Pandemrix vaccine, could trigger further development of autoimmunity by a non-specific “bystander” mechanism, leading to clinical onset of narcolepsy with cataplexy. We did not, however, find evidence for influenza infection as a significant contributing factor in the etiology of narcolepsy in our patients.Table S1Clinical and laboratory data of 45 narcoleptic patients.(DOC)Click here for additional data file.Table S2Anti-NS1 and virus-specific hemagglutination inhibition (HI) titers from 45 narcoleptic patients.(DOC)Click here for additional data file.Table S3Anti-NS1 and virus-specific hemagglutination inhibition (HI) titers from 28 paired serum samples.(DOC)Click here for additional data file.Table S4Anti-NS1 and virus-specific hemagglutination inhibition (HI) titers from 50 age-matched controls.(DOC)Click here for additional data file.


We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired  Salmonella serovars. Consequently, S. enterica serovar Typhimurium, which infects a wide range of animals, expresses PmrA-dependent genes and displays antibiotic resistance in conditions that activate the PmrA and/or PmrD proteins. By contrast, the human-adapted S. enterica serovar Paratyphi B only does so in the presence of both PmrA- and PmrD-activating conditions. Bacteria harboring the Paratyphi B pmrA gene also exhibited enhanced biofilm formation, which may contribute to serovar Paratyphi B's persistent infection of the gallbladder. Our findings demonstrate that the ability of horizontally acquired genes to confer new traits can be affected by ancestral proteins, even within one bacterial species. Therefore, a protein's function in a given organism must be appreciated in the context of other proteins operating within the same genetic network.Horizontally acquired genes are typically viewed as independent units that confer new traits when introduced into different bacterial species. However, preexisting proteins in a bacterium can impact the ability of horizontally acquired gene products to bring about new functions when they target ancestral pathways. Here, we establish that a single amino acid difference in the ancestral transcription factor PmrA alters its dependence on the horizontally acquired gene product PmrD to promote gene expression within closely related  Vibrio choleraelocus of enterocyte effacement – by enteropathogenic Escherichia coli (EPEC) E. coli by experimental introduction of the relevant DNA Salmonella serovars to display resistance to the antibiotic polymyxin B under different environmental conditions.The phenotypic properties that distinguish closely related bacterial species are often ascribed to differences in gene content S. enterica serovar Typhimurium is controlled by the ancestral PmrA/PmrB two-component system, the major regulator of lipopolysaccharide (LPS) modification genes 3+ or Al3+2+ indirectly activates the PmrA/PmrB system in a process that requires the horizontally acquired pmrD gene 2+ is an inducing signal for the PhoP/PhoQ two-component system pmrD transcription S. typhimurium displays polymxyin B resistance when experiencing low Mg2+ and/or the presence of Fe3+.Inducible resistance to polymxyin B in S. enterica vary in the degree to which the horizontally acquired pmrD gene activates the PmrA/PmrB system S. enterica serovars accounting for the observed phenotypic diversity in polymyxin B resistance S. enterica serovar Paratyphi B does not activate the PmrA/PmrB system in response to low Mg2+ and that activation of PmrA/PmrB in response to Fe3+ requires the horizontally acquired pmrD gene product. We establish that this disparity from S. typhimurium is due to a single amino acid difference between the PmrA proteins, which dramatically alters PmrA's affinity for its target promoters and the levels of PmrA-P in vivo. The Paratyphi B PmrA allele confers enhanced biofilm formation, which may aid survival of this human-adapted serovar in its particular habitat. Our work provides a singular example whereby quantitative differences in the biochemical properties of an ancestral transcription factor dictate the ability of a horizontally acquired gene product to confer new traits.We previously reported that natural isolates of S. enterica isolate belonging to the Paratyphi B serovar and is classified as a member of the systemic pathovar (SPV) that causes paratyphoid fever in humans 2+  isolates (S. paratyphi B strains belonging to the enteric pathovar (EPV), which cause local enteric infections pbgP in low Mg2+ regardless of the presence/absence of Fe3+  isolates harboring mutations in pmrD, like other natural Salmonella isolates S. paratyphi B and S. typhimurium specify identical PmrD proteins. Moreover, pmrD transcription in S. paratyphi B was stimulated in low Mg2+  other than pmrD. Because PmrA-P constitutes the only known target of the PmrD protein S. paratyphi B PmrA protein differs from the S. typhimurium homolog. Thus, we sequenced the pmrA gene from 32 natural isolates originating from the Salmonella reference collections A S. paratyphi B (SPV) strains has a glutamate residue at position 211 (PmrA E211) whereas most other analyzed S. enterica isolates, including S. typhimurium and S. paratyphi B (EPV) strains, bear a glycine residue at that position (PmrA G211)  . S. typhtion 211 , constitnditions .S. paratyphi B's inability to transcribe pbgP in low Mg2+, then replacing its pmrA (E211) by the pmrA (G211) allele should restore expression. To test this notion, we engineered isogenic S. paratyphi B SARA46 strains bearing a pbgP-lac transcriptional fusion and either the pmrA (G211) or pmrA (E211) alleles under the control of the S. paratyphi B pmrCAB promoter at its normal chromosomal location. When grown in low Mg2+, the S. paratyphi B strain (pmrA G211) produced 10 times more β-galactosidase activity than the isogenic pmrA (E211) strain (pmrD eliminated pbgP expression in both S. paratyphi B strains when grown in media containing low Mg2+ (S. typhimuriumpmrA mutant under any growth conditions (If a difference in PmrA is solely responsible for ) strain . As expelow Mg2+ , as descnditions .pmrD gene prevented S. paratyphi B (pmrA E211) from expressing pbgP during growth in low Mg2+ + high Fe3+ (3+ is detected directly by the PmrB sensor S. typhimuriumS. paratyphi B (pmrA G211) supported pbgP transcription in a pmrD mutant incubated in low Mg2+ + high Fe3+  or pmrA (E211) alleles to survive killing by polymyxin B when grown on N-minimal media agarose plates containing low Mg2+ + high Fe3+. All four strains survived killing by polymyxin B (pbgP under this condition (pmrD if the strains harbor the pmrA (E211) allele but not if they carry the pmrA (G211) allele  allele ; this is) allele . Similarigh Fe3+ . CollectS. paratyphi B and S. typhimurium abolish low Mg2+-dependent expression of PmrA-activated genes in the former but not the latter serovar? To address this question, we first carried out homology modeling of the PmrA DNA-binding domain in complex with DNA using the structure of the DNA-binding domain of the Escherichia coli PhoB response regulator in complex with its DNA target pbgP promoter, which is fully conserved in S. paratyphi B and S. typhimurium. Using an electrophoretic mobility shift assay, the purified phosphorylated PmrA (G211) protein bound more effectively to the pbgP promoter fragment than the purified phosphorylated PmrA (E211) protein  or PmrA (E211) from the normal chromosomal location grown under low Mg2+ conditions. Western blotting with anti-HA antibodies demonstrated that the S. typhimurium (pmrA E211) strain produced much less PmrA protein than the isogenic pmrA (G211) strain (Because PmrA-P autogenously controls its own expression and that of its cognate sensor PmrB from a PmrA-activated promoter located upstream the ) strain ; by cont) strain , which ipmrA (E211) allele do not accumulate high enough levels of PmrA (E211) protein and are unable to transcribe PmrA-dependent genes in low Mg2+, unlike those expressing the pmrA (G211) gene.Taken together, these results indicate that the reduced affinity of PmrA (E211) for target promoters impedes positive autoregulation of the PmrA/PmrB system and production of PmrA (E211). Consequently, bacteria harboring the S. paratyphi B and S. typhimurium render transcription of PmrA-activated genes dependent on PmrD in the former but not in the latter serovar when Fe3+ is present? And how does S. paratyphi B overcome the lower affinity of its PmrA protein for target promoters in order to stimulate PmrA-dependent expression under such conditions? When bacteria experience inducing conditions for the PmrA/PmrB system, the sensor PmrB phosphorylates the DNA binding protein PmrA, increasing PmrA's affinity for target promoters and resulting in transcription of PmrA-activated genes 2+, promotes the phosphorylated state of PmrA by protecting it from dephosphorylation by PmrB, an activity primarily present under PmrA non-inducing conditions in vivo.Why does the single amino acid difference between the PmrA proteins from c) because it retains all the known enzymatic activities of the full-length PmrB protein c-mediated dephosphorylation in the absence of PmrD  or PmrA (E211) from the normal chromosomal location were separated on a Phos-tag gel, which retards phosphorylated proteins more than their unmodified forms and has been used to examine phosphorylated response regulators in vivoS. typhimurium (pmrA E211) strain had a higher proportion of PmrA-P compared to the isogenic pmrA (G211) strain -P by PmrD led to higher levels of phosphorylated PmrA protein ) strain , despite) strain .c-mediated dephosphorylation, leading to increased amounts of phosphorylated PmrA (E211) in bacteria experiencing PmrD- and PmrA-inducing conditions. Because PmrA-P constitutes the active form of PmrA that binds target promoters in vivoCumulatively, these findings indicate that PmrD is more efficient in protecting PmrA (E211)-P than PmrA (G211)-P from PmrBS. typhimurium strain harboring the pmrA505 allele can transcribe PmrA-activated genes in a pmrD mutant and under non-inducing conditions for the PhoP/PhoQ system in vitroin vivo. We hypothesized that this increase might be sufficient to overcome the DNA-binding defect of the PmrA (E211) protein, enabling it to activate expression of PmrA-dependent genes in response to the low Mg2+ signal. As predicted, the R81H substitution rescued the ability of PmrA E211 to promote pbgP transcription. When bacteria experienced low Mg2+, the S. typhimurium strain with the pmrA (R81H E211) gene transcribed pbgP to levels similar to those produced in response to Fe3+ (pmrA (R81H E211) strain expressed pbgP when grown in high Mg2+, though not to the levels displayed by the isogenic strain harboring the pmrA (R81H G211) allele (pbgP transcription was ∼5-fold higher in a pmrA (R81H E211) derivative deleted in pmrD than in the isogenic pmrA (E211) ΔpmrD strain when encountering low Mg2+ + high Fe3+ (pmrA (R81H G211) ΔpmrD strain (An  to Fe3+ . The pmr) allele . Furtherigh Fe3+ . Yet, thD strain . These rS. typhimurium (pmrA G211) strain experiences Fe3+, there is a surge in the mRNA levels of PmrA-activated genes, which increase, peak and then decrease to reach new steady-state levels in a manner reflecting the amount of PmrA-P protein pbgP promoter in vitro than the PmrA (G211) protein (pmrA (E211) allele might differ in the kinetics with which PmrA-dependent transcripts are produced in vivo.We previously reported that when an  protein , we reas2+ + high Fe3+ and incubated for different extents of time. In the pmrA (G211) strain, the pbgP and pmrC mRNAs peaked at 5 min and 10 min, respectively, before decreasing to steady-state levels (pmrA (E211) strain, the transcripts increased steadily over 60–90 min (pmrD gene abolished pbgP and pmrC expression in the pmrA (E211) strain (pmrA (G211) strain  strain , but it ) strain . Thus, tS. paratyphi B can cause chronic infections by persisting in the gallbladder for many years S. typhi to form biofilms on cholesterol-coated gallstones is believed to facilitate colonization of the gallbladder pmrA allele altered S. enterica's ability to form biofilms on cholesterol-coated surfaces using an assay developed by the Gunn laboratory S. paratyphi B (pmrA E211) compared to the isogenic pmrA (G211) strain (pmrD gene further increased biofilm formation in the S. paratyphi B (pmrA E211) strain, which reached levels similar to those displayed by a pmrA null mutant (S. paratyphi B (pmrA E211) to express PmrA-dependent genes requires the PmrD protein (S. paratyphi B (pmrA G211) strain deleted for pmrD displayed low levels of attachment to cholesterol-coated surfaces, like the isogenic +pmrD strain (pmrA gene and do not appear to involve genes that are specific to S. paratyphi B because they can be recapitulated in an S. typhimurium strain background (Biofilm formation was higher in ) strain . Deletiol mutant . This wa protein . By cont+ strain . The gro+ strain  and therckground .S. enterica  isolates that cause paratyphoid fever, which is in contrast to S. typhimurium and other natural isolates of S. enterica that contain a glycine residue at that position (pmrA (E211) allele might be sufficient for S. paratyphi B to proliferate in its particular ecological niche. Furthermore, bacteria harboring the pmrA (E211) allele exhibited enhanced biofilm formation on cholesterol-coated surfaces  allele in S. paratyphi B natural populations  for target DNA  versus the pmrA (E211) allele lead to distinct cellular behaviors, as previously demonstrated in other signal transduction systems We suggest that the dissimilar affinities displayed by the PmrA orthologs for target promoters distinguish the ability of bacteria to survive in their particular niches upon experiencing the presence of Ferget DNA  might reB operon , a propee levels . Our finE. coli strain B and the E. coli K12 arginine repressor results in transcription of arginine biosynthesis genes in E. coli strain B even when arginine is present, whereas repression of these very genes is selected for in E. coli K12 Our work provides a singular example of how different alleles of a conserved transcription factor can display disparate signal prerequisites for activating gene expression. Importantly, these differences are independent of both the signal-sensing domain of the upstream sensor protein that controls the activity of the transcription factor c-mediated dephosphorylation than PmrA (G211)-P when PmrD was present  gene from the normal chromosomal location, was constructed by combination of the one-step inactivation method + selection. A PCR fragment encompassing the coding region of the pmrA (E211) gene was amplified using primers 2426 and 2428  with 0.1% casamino acids, 10 µM MgCl2, and 100 µM FeSO4 and supplemented with 1.3% lactose as the sole carbon source.Strain EG14331, which has a MudJ transposon insertion in and 2428  and S. pS. typhimurium pmrA gene, was constructed using a modification of the one-step inactivation protocol R cassette was amplified from plasmid pKD3 using primers 7994 and 7995  gene from the normal chromosomal location, was constructed by combination of the one-step inactivation method + selection. A PCR fragment encompassing the coding region of the pmrA (G211) gene was amplified using primers 2426 and 2428  with 0.1% casamino acids, 10 µM MgCl2, and 100 µM FeSO4 and supplemented with 1.3% lactose as the sole carbon source.Strain EG16275, which has a MudJ transposon insertion in and 2428  and S. tS. paratyphi B derivative of strain SARA46 harboring the pmrA (G211) ORF from the normal chromosomal location (DC280), we used a modification of the one-step inactivation protocol R cassette was amplified using primers 11408 and 11409 (10 transposon in the phoP gene S. paratyphi B SARA46 containing plasmid pKD46 pmrA::tetR strain (DC167) containing plasmid pKD46 was kept at 30°C. A DNA fragment that encompassed the S. typhimurium pmrA ORF and a CmR cassette downstream of the pmrA gene was amplified with primers 12533 and 12534 (R cassette was removed using plasmid pCP20 as described To construct an nd 11409  and genond 12534  using DCS. paratyphi B derivative of strain SARA46 harboring the pmrA (G211) allele at the normal chromosomal location,we used a modification of the one-step inactivation protocol R cassette was amplified from plasmid pKD3 using primers 12235 and 12437  with a His6 tag at the C-terminus was constructed by cloning a PCR fragment generated with primers 2453 and 2454 (S. paratyphi B SARA46 DNA as a template between the NdeI and HindIII sites of plasmid pT7-7.Plasmid pT7-7-PmrA (E211) -His6 encoding the and 2454  and S. pS. paratyphi B and from S. typhimurium, and the N-terminally His-tagged cytoplasmic domains of the PmrB protein (PmrBc) or PmrBc T156R mutant from S. typhimurium were overproduced in E. coli strain EG13796 harboring plasmids pT7-7-PmrA (E211)-His6, pT7-7-PmrA (G211)-His6, pT7-7-His6-PmrBc and pT7-7-His6-PmrBc T156R as described C-terminally His-tagged PmrA proteins from 2 were washed two times with N-minimal medium containing no Mg2+, and added into the appropriate fresh media with 1∶50 dilution. The bacterial cultures were grown in a shaking water bath for 4 h at 37°C before the assay. Data correspond to the mean values of three independent experiments performed in duplicate.β-galactosidase assays were carried out in triplicate, and the activity was determined as described Biochemical assays were carried out as in pbgP, mgtA and ompX DNA fragments for gel mobility shift assays were generated by using PCR primer pairs 767 and 955, 7192 and 7195, and 9198 and 9202, respectively (S. typhimurium 14028s as template. The DNA fragments were gel-purified with QIAquick columns (Qiagen), and 150 ng of DNA was labeled using T4 polynucleotide kinase (NEB) and (γ-32P) ATP at 37°C. Unincorporated (γ-32P) ATP was removed using G-50 microcolumns (Amersham). 10 µM His-tagged PmrA-H6 was incubated for 60 min at room temperature with 5 µM H6-PmrBc T156R  (Amersham) and 0, 100, 200 or 300 pmol of phosphorylated or unphosphorylated His-tagged PmrA (E211) or PmrA (G211) proteins were mixed with binding buffer (20 mM Hepes (pH 8.0), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 50 µg/ml BSA, and 10% glycerol) in a final volume of 30 µl and incubated at room temperature for 20 min. Samples were run on 4–20% TBE gels (Invitrogen), dried and then autoradiographed using a BAS-5000 imaging system and phosphor imaging plate (Fuji Film).The ectively  and genoc T156R beads as described 2/1 mM DTT/10% glycerol. Proteins were then eluted in the same buffer at a flow rate of 0.5 ml min−1. Absorbance was monitored at 280 nm and fractions were analyzed by SDS-PAGE. The column was calibrated with a mixture of protein molecular mass standards , containing aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa) and conalbumin (75 kDa), which was applied to the column under similar conditions.180 µl PmrA (50 µM) was phosphorylated using pGEX-PmrB2 were washed twice with N-minimal medium containing no Mg2+ and added into fresh N-minimal medium at pH 7.7 with 10 µM MgCl2 and 100 µM FeSO4 with 1∶50 dilution. The bacterial cultures were grown to OD600 0.4 in a shaking water bath at 37°C before harvesting the cells at 4°C and resuspending the cell pellet with 1 ml ice-cold 20 mM Tris, pH 7.0 (Ambion). The samples were then added to a 2 ml Lysing Matrix Tube  and lysed three times for 40 s at 6 m/s using the FastPrep-24 instrument . The tubes were spun down to remove the Lysing Matrix beads and 100 µl cell lysate was added to 100 µl 2× Laemeli buffer (Biorad) and boiled for 3 min. Equivalent amounts of each sample  were run on a 4–12% Bis-Tris gel (Invitrogen) in 1× MES buffer (Invitrogen), transferred to a PVDF membrane, and analyzed by Western blotting with an anti-HA monoclonal antibody (Sigma) or an anti-RpoB antibody (Neoclone). Western blots were developed using anti-mouse IgG horseradish peroxidase-linked antibodies  and Supersignal west femto (Pierce).Bacteria from overnight cultures grown in N-minimal medium at pH 7.7 with 10 mM MgClin vivo, samples were prepared as described in the previous section. 100 µl of cell lysate was added to 100 µl chilled 2× Laemeli buffer (Biorad) (to detect phosphorylated PmrA) or to 100 µl 2× Laemeli buffer (Biorad) and boiled . Samples were analyzed on a Phos-Tag gel as described 2 were prepared. Stacking gels contained 4% (w/v) 19∶1 acrylamide/Bis solution and 130 mM Tris, pH 6.8. Equivalent amounts of each boiled or unboiled sample  were loaded onto the gel and run for 2 h at 4°C under constant voltage (150 V) using chilled running buffer containing 1% (w/v) SDS, 25 mM Trizma base and 192 mM glycine. Gels were then equilibrated for 10 min with chilled transfer buffer containing 20% (v/v) methanol, 192 mM glycine and 25 mM Trizma base, with 1 mM EDTA to remove Mn2+ from the gel. Gels were incubated for an additional 10 min in transfer buffer without EDTA. Transfer to nitrocellulose membranes was performed using the Bio-Rad wet transfer apparatus under constant voltage (100 V) for 60 min. Western blotting was carried out as described in the previous section.To determine the levels of PmrA-P 2 were washed twice with N-minimal medium containing no Mg2+ and added into fresh N-minimal medium at pH 7.7 with 10 mM MgCl2 with 1∶50 dilution. The bacterial cultures were grown to OD600 0.4 in a shaking water bath at 37°C, spun down and resuspended in 100 µl N-minimal medium at pH 7.7 with 1 mM MgCl2, and then added into N-minimal medium at pH 7.7 containing no Mg2+ and 100 µM FeSO4. 0.5 ml aliquots of cells were removed at the indicated time points, mixed with RNAprotect Bacteria Reagent (Qiagen) for stabilization of RNA, and total RNA was isolated using RNeasy Kit (Qiagen) with on-column DNase treatment. cDNA was synthesized using TaqMan (Applied Biosystems) and random hexamers. Quantification of transcripts was carried out by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) in an ABI 7500 Sequence Detection System (Applied Biosystems). The following primers , pbgP , pmrC  and pmrD . The relative amount of cDNA was determined using a standard curve obtained from PCR with serially diluted genomic DNA, and results were normalized to the levels of 16S ribosomal RNA. Data correspond to the mean values of at least three independent experiments.Bacteria from overnight cultures grown in N-minimal medium at pH 7.7 with 10 mM MgCl primers  were use2 and 2.5 µg/ml polymyxin B with or without 100 µM FeSO4 and incubated at 37°C overnight before examination of the plates for bacterial growth.The ability of bacteria to grow in the presence of polymyxin B was determined as follows. Bacteria were streaked onto N-minimal media plates, pH 5.8, containing 1% agarose, 38 mM glycerol, 10 µM MgClS. enterica strains to form biofilms on cholesterol-coated microcentrifuge tubes was determined as described 600 0.5. 100 µl of cells were added to cholesterol-coated Eppendorf tubes (Fisher Scientific) and incubated on a Nutator shaker at room temperature for 6 days. Each day, spent medium was removed, and the tubes were washed twice with LB medium before fresh LB medium was added. On day 6, after incubating tubes at 60°C for 1 hr to fix the attached bacteria, 200 µl of 0.1% crystal violet was added to stain cells for 5 min at room temperature. The tubes were then washed with 1 ml 1× PBS until the solution ran clear, and 200 µl of 33% acetic acid was added to extract the crystal violet dye, which was quantified at OD570 using a Victor 3 1420 Multilabel counter (Perkin Elmer). Data correspond to the mean values of three independent experiments performed in duplicate.The ability of pmrA gene was amplified with high fidelity AccuPrime Taq DNA polymerase (Invitrogen) by using primers 2876 and 2877 (pmrA ORF respectively. PCR products were purified with the QIAquick PCR purification kit (Qiagen). Sequencing reactions were initiated by using primers 2878, 2879 or 2880, performed using Big Dye 3.1 (Applied Biosystems) and analyzed on a 310 Genetic Analyzer (Perkin-Elmer). DNA sequences were translated by using Editseq 3.92 (DNASTAR). The sequences of these pmrA genes as well as those previously determined in pmrA gene sequences have been deposited at GenBank under the accession numbers listed in The and 2877 , which aFigure S1S. paratyphi B PmrA differs from that of other S. enterica strains at position 211. (A) Alignment of the deduced amino acid sequences of the pmrA gene from 33 S. enterica isolates. A red arrow indicates the amino acid residue at position 211. (B) Homology model of the PmrA DNA-binding domain in complex with DNA, which was predicted using a homology-modeling program (Phyre) E. coli PhoB (purple) complexed to DNA The (TIF)Click here for additional data file.Figure S2The PmrA (E211) protein has a lower propensity to form dimers in solution than the PmrA (G211) protein. Gel filtration chromatogram of phosphorylated PmrA (G211) or PmrA (E211) proteins that were individually applied to a Superdex 200 10/300 GL column. Absorbance was monitored at 280 nm.(TIF)Click here for additional data file.Figure S3c and their phosphorylated forms dephosphorylated by PmrBc. (A) Levels of PmrBc-P and PmrA-P following incubation of PmrBc-P (5 µM) with PmrA (G211) or PmrA (E211) (10 µM) proteins at the times indicated at the top of the figure according to the protocols described in c (5 µM) in the presence of 2.5 µM (C) or 1.25 µM (D) PmrD for the indicated times.The PmrA (G211) and PmrA (E211) proteins are similarly phosphorylated by PmrB(TIF)Click here for additional data file.Figure S4bcsA or csgA in S. typhimurium. (A–B) Growth of S. paratyphi B (A) or S. typhimurium (B) strains used for biofilm analyses in 600 was determined using a Victor3 1420 Multilabel counter (Perkin Elmer). (C) mRNA levels of the bcsA and csgA genes from wild-type (14028s) or pmrA (EG7139) S. typhimurium strains determined by reverse-transcription-qPCR analysis. Bacteria were grown in N-minimal medium containing 10 µM Mg2+ and 100 µM Fe3+ and harvested to prepare RNA. Expression levels were normalized to those of the 16S ribosomal RNA gene. Data correspond to at least two independent experiments and error bars show standard deviation.PmrA does not regulate the expression of (TIF)Click here for additional data file.Table S1S. typhimurium strains to polymyxin B.Susceptibility of (DOC)Click here for additional data file.Table S2Bacterial strains and plasmids used in this study.(DOC)Click here for additional data file.Table S3Primers used in this study.(DOC)Click here for additional data file.Table S4pmrA genes from S. enterica natural isolates.GenBank accession numbers for the (DOC)Click here for additional data file.


CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P). In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency.Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study.We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different approaches revealed nine haplotypes for deleted 21-hydroxylase deficiency alleles.An allele carrying a CYP21A1P/A2 chimeric genes in Brazil.This study demonstrated high allelic variability for 30-kb deletion in patients with 21-hydroxylase deficiency indicating that a founder effect might be improbable for most monomodular alleles carrying  CYP21A2 gene, which encodes the enzyme, are responsible for the disease :CYP21A1P[3.2] (1:4) fragments in the patient. The mother presented ratios of 2:1 for C4A[7.0]:C4B[5.4] and 1:3 for CYP21A2[3.7]:CYP21A1P[3.2] and was positive for p.P30L, IVS2-13A/C > G and Δ8 mutations in ASO-PCR experiments indicating a deleted allele with CL6N sequence. The daughter inherited the 30-kb deleted allele from the mother, therefore she should carry an allele with three copies of each C4B and CYP21A1P to produce the relative intensities observed in the Southern blot  indicated that the two pseudogenes in the paternal trimodular allele might carry the normal sequence in codon 318 . Therefore, p.P34L seems to be a novel mutation not derived from pseudogene, whereas p.H62L showed a frequency of 3.4% in pseudogenes.Sequencing 170 C4/CYP21 monomodular alleles evaluated by combining Southern blot, ASO-PCR, MLPA and sequencing techniques. The occurrence of different chimeric genes in Brazilian patients had not been reported before. Although Real-Time PCR and HLA-haplotypes would be alternative techniques to estimate, respectively, C4 and CYP21A2 copy number [This study reports two novel deletion alleles in Brazilian patients with 21-hydroxylase deficiency and describes the variability of y number -37 and fC4B fragment  in MLPA. This result indicate that exon 17 in the C4B[6.0] gene probably corresponded to C4A sequences and the patient had been described as having very low immune response toward C4B antiserum  gene ,40. Suchficiency  and woul alleles . It can d before , data obtivities . C4A genplotypes ,44. Diffplotypes , therefoulations ,20,46,47ntiserum . AlthougCYP21A1P pseudogene copy indicating that the recombination breakpoint should map within the 5 kb that separate 3'-end CYP21A2 and the exon 32 of TNXB gene. They differed because haplotype III showed the C allelic variants in both g.1645 and g.2110 (p.R356W) nucleotides whereas G and T, respectively, were observed in haplotype IV . Different mutations in exon 1 together with rare 5'-end SNP characterize a novel haplotype that seems to be frequent in Brazilian patients carrying monomodular alleles since it corresponded to 20% (4/20) of total 30-kb deleted alleles reported here as causing 21-hydroxylase deficiency. Probably, haplotype V have arisen in Brazil from an African ancestral since Brazilian population is highly miscigenated with populations from different origins including Africans of Benin Gulf region [A group of ten genes had recombination breakpoints identified between exon 3 and 4. Haplotype V diverged from the VI and VII because the alleles did not carry the pseudogene-derived p.P30L, but showed p.P34L and p.H62L mutations in exon 1. p.H62L has been recently described in association with p.P453S in patients with the NC form of 21-hydroxylase deficiency ,51. In of region . This haCYP21A1P promoter region and p.P30L mutation as pseudogene-derived sequences and they are not very frequent in other populations [Haplotypes VIII and IX were separated because they showed different recombination breakpoints within intron 2. Those two haplotypes presented the ulations ,53.CYP21A1P promoter region and p.P30L upon the enzyme activity as proposed before [CYP21A1P/A2 chimeric genes can be correlated with SW or SV forms of 21-hydroxylase deficiency depending on the mutations they carry ,20,46,47d before .CYP21A1P/A2 borders in almost all cases. However, to distinguish between deletions and large gene conversions in genetic studies searching for detailed allelic information, it would be more informative if more C4 and CYP21 probes were included in the analysis.Our study showed that the combination of Southern blot and ASO-PCR/direct sequencing with MLPA tests may constitute an option for mapping and better characterize chimeric genes on RCCX monomodular alleles, especially in populations with high allelic diversity such as that in Brazil. MLPA has been proposed as a candidate with good potential to be used in neo-natal screening and in pre-natal diagnosis because it can be performed with very low amount of DNA . EventuaCYP21A1P/A2 borders in almost all cases. However, to distinguish between deletions and large gene conversions in genetic studies searching for detailed allelic information, it would be more informative if more C4 and CYP21 probes were included in the analysis.Our study showed that the combination of Southern blot and ASO-PCR/direct sequencing with MLPA tests may constitute an option for mapping and better characterize chimeric genes on RCCX monomodular alleles, especially in populations with high allelic diversity such as that in Brazil. MLPA has been proposed as a candidate with good potential to be used in neo-natal screening and in pre-natal diagnosis because it can be performed with very low amount of DNA . EventuaThe authors declare that they have no competing interests.FBC carried out part of Southern blot, the MLPA and sequencing experiments and sequence alignment; FCS, RDB and RJP participated in ASO-PCR experiments for pseudogene-derived mutations and SNPs; MA, LCP and IFL participated in Southern blot and AS0-PCR studies of seven families; SHVLM, MTMB and GGJ were responsible for diagnosis and management of patients and participated in the design of the study; MPM conceived the study, and participated in its design and coordination and also 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/1471-2350/11/104/prepub


Drosophila, one of the earliest developmental decisions made in the embryo is that of determining its sex. This process involves regulating the expression of the X chromosome sensing promoter of Sex-lethal (Sxl) at its establishment promoter, peSxl/TM6C, Sb; E(z)[EY21318]; E(z)32A40/TM6C, Sb, Tb; Su(z)12[4]/TM6C, Sb, Tb; wild-type w[1118] and Ore R. In situ hybridization in 0-4 hour embryos was used to analyze peSxl expression levels using a Digoxygenin labeled RNA probe specific to peSxl transcripts; Chromatin Immunoprecipitations were performed with antibodies specific to H3K4me3, H3K27me3 and HP1a on chromatin prepared from 1-3 and 2-4 hour embryos, also from the above mentioned stocks. Quantitative real-time PCR quantified Sxl sequences in the DNA from each ChIP.sh1, Su(z)12 and E(z) mutant embryos show they affect both the timing and strength of transcription of the sex establishment promoter, pe.Sxl qRT-PCR analyses support the in situs, showing a change in SxlPe mRNA levels. We also observe that these proteins are necessary for proper histone 3 lysine 4 (H3K4) and histone 3 lysine 27 (H3K27) methylation at the promoter. Surprisingly, we find that embryos deficient in E(z), Su(z)12 or ASH1 protein also affect the binding of HP1a at PeSxl sequences.We find that the PcG/Trx-G proteins genetically interact with mutations in the sex determination pathway and influence the ability of females to determine their sex. In situ analysis of aOur data support the idea that PcG proteins function on a larger set of target genes with a broader role in gene regulation and development. Additionally, we find a novel heterochromatin role for PcG/Trx-G proteins which influences HP1a in the regulation of gene expression.