Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences. Transposable elements comprise a substantial portion of many eukaryotic genomes. These mobile fragments of DNA can directly mutate genes through insertions into coding regions but may also affect the gene regulation through nearby insertions. There is evidence that the majority of transposable elements are epigenetically silenced, and in some cases this silencing may spread to neighboring sequences. This spreading of heterochromatin could create a significant fitness tradeoff between transposon silencing and gene expression. The maize genome has a complex organization with many genes flanked by retrotransposons, providing an opportunity to study the interaction of retrotransposons and genes. To survey the prevalence of heterochromatin spreading associated with different retrotransposon families, we profiled the spread of heterochromatin into nearby low copy sequences for 150 high copy retrotransposon families. While many retrotransposons exhibit little to no spreading of heterochromatin, there are some retrotransposon families that do exhibit spreading. Genes located near retrotransposons that spread heterochromatin have lower expression levels. The families of retrotransposons that spread heterochromatin marks to nearby low-copy sequences may have increased fitness costs for the host genome due to their suppression of genes located near insertions. A substantial fraction of most eukaryotic genomes is composed of transposable elements (TEs) agouti and Axin locus in mouse FLCFWATEs could influence neighboring genes by providing regulatory elements or promoters that would alter expression levels or patterns The complex organization of the maize genome, with interspersed genes and TEs The existence of heavily silenced retrotransposons interspersed with genes throughout the maize genome provides ample opportunities for TEs to exert epigenetic regulation on surrounding sequences. We were interested in further documenting the extent of heterochromatin spreading from maize retrotransposons to neighboring sequencings. Genomic profiling of DNA methylation and H3K9me2 found that heterochromatic spreading is only observed for a small number of specific retrotransposon families. These families tend to be enriched in pericentromeric regions of chromosomes. The analysis of haplotypes lacking specific retrotransposon insertions provides evidence that the adjacent heterochromatin is the result of spreading rather than insertion site bias.DNA methylation and chromatin modifications were profiled for low-copy sequences in the maize genome using methylated DNA immunoprecipitation (meDIP) and chromatin-immunoprecipitation (ChIP) with antibodies specific for H3K9me2 or H3K27me3, respectively. The fractions of the genome enriched for DNA or histone modifications were hybridized to a high-density microarray containing ∼2.1 million long oligonucleotide probes derived from the unmasked, non-repetitive fraction of the maize genome. The probes are spaced every 200 bp in the low-copy portions of the maize genome and can provide a profile for the chromatin state in these regions A large number of class I TEs (retrotransposons) have been identified within the maize genome A subset of the retrotransposon families also exhibit elevated levels of DNA methylation and H3K9me2 in regions more than 200 bp away from their insertion sites. In general, levels of H3K9me2 and DNA methylation were well correlated, but there were some families with different enrichment for these two marks. As expected, there was no evidence for enrichment (or depletion) of the facultative heterochromatin mark, H3K27me3, in regions flanking the retrotransposons . To idenThe analysis of the whole-genome bisulphite sequencing data supports the classifications of different retrotransposon families . Both CGmop1 mutant of the maize Rdr2 gene mop1 mutation on the expression levels of spreading or non-spreading retrotransposon families. Indeed, spreading retrotransposon families include examples of both up- and down-regulation in mop1 mutant individuals relative to wild-type  gene, which contributes substantially to CHG methylation Spreading retrotransposons exhibit higher levels of CHG methylation within the retrotransposon themselves . Spreadiftp://ftp.jgipsf.org/pub/JGI_data/Zea_mays_Mo17/) were aligned to the B73 reference genome sequence. Empty sites were defined as being those as which at least three Mo17 sequence reads cover a low-copy sequence flanking an insertion but do not align to the retrotransposon itself and for which no Mo17 reads cover the junction between the low-copy sequence and the retrotransposon. In total, 668 empty sites were identified for the spreading (both) retrotransposon families and 29 empty sites for the spreading (H3K9) retrotransposon families for which we had DNA methylation data in the unique regions flanking the insertion. The lack of the specific insertion in Mo17 was confirmed at 13 of the 14 empty sites that were tested using site-specific PCR primers to confirm the presence/absence of specific insertions. This suggests that there is a low false-positive rate in the identification of empty sites in Mo17. However, given the low coverage of the WGS data and challenges associated with aligning polymorphic sequences it is likely that many of the true empty sites were not identified in this analysis.The observation that certain families of retrotransposons have high levels of heterochromatic modifications in adjacent regions could reflect insertion site biases for these families or indicate that these families cause local spreading of heterochromatin. Examples of “empty” sites in the Mo17 haplotypes were identified and used to assess whether the high levels of DNA methylation would be observed in these regions when the retrotransposon was absent. Mo17 whole-genome shotgun WGS) sequences (generated by the DOE's Joint Genome Institute (JGI) and downloaded from The level of DNA methylation at the probe nearest to the empty site was used to assess relative DNA methylation levels with (B73) and without (Mo17) each insertion . The lowThe finding that only a subset of maize class I retrotransposon families are associated with local spreading of heterochromatin suggested that there might be intrinsic differences among different retrotransposon families that would explain this variation. We proceeded to characterize these families to ascertain whether there were specific common attributes of spreading families. None of the LINE families exhibit evidence for spreading of heterochromatic marks. RLG (gypsy) families are over-represented among spreading (both) retrotransposon families, while the spreading (H3K9) retrotransposons have more RLC (copia) families than expected . SpreadiThe relative abundance of spreading (both) retrotransposons is higher in the middle of the chromosome than the other families suggesting that these retrotransposons may be enriched in pericentromeric regions . HoweverThe finding that some retrotransposon families exhibit spreading of heterochromatic marks to surrounding sequences while others do not led us to hypothesize that these families may influence expression of nearby genes. RNAseq was used to estimate transcript abundance in three tissues of B73 and Mo17 including the identical leaf tissue samples used for profiling DNA methylation levels. All maize genes were annotated to identify the first retrotransposon 5′ of the transcription start site and to determine the distance between the retrotransposon and the transcription start site. Genes that are located near retrotransposons that exhibit spreading (both or H3K9) have significantly (p<0.001) lower expression levels in all genotypes and tissue examined . This reEpigenetic variation in low-copy sequences can be the result of pure epigenetic changes (no correlation with DNA sequence polymorphisms) or occur in a facilitated or obligatory fashion such that DNA sequence differences contribute to the epigenetic changes There is also evidence that differences in interspecific variation in transposon insertions contributes to gene expression diversity between related species Drosophila retrotransposons contain insulator elements that reduce the spreading of chromatin states bns locus in Arabidopsis have suggested the presence of an active mechanism to prevent the spreading of heterochromatin from retrotransposons Barbara McClintock proposed the concept that transposons could serve as “controlling” elements that would influence nearby genes rd leaf tissue of B73 and Mo17 was performed as described [39 – GSE29099]. Briefly, methylated DNA was immunoprecipitated with an anti-5-methylcytosine monoclonal antibody from 400 ng sonicated DNA using the Methylated DNA IP Kit . For each replication and genotype, whole genome amplification was conducted on 50–100 ng IP DNA and also 50–100 ng of sonicated DNA (input control) using the Whole Genome Amplification kit . For each amplified IP input sample, 3 ug amplified DNA were labeled using the Dual-Color Labeling Kit  according to the array manufacturer's protocol (Roche NimbleGen Methylation UserGuide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3. H3K9me2 and H3K27me3 profiling were performed on three replicates of B73 and Mo17 seedlings using antibodies specific for H3K27me3 (#07-449) and H3K9me2 (#07-441) purchased from Millipore . For each replicate, 1 g of plant material was harvested on ice, rinsed with water, and crosslinked with 1% formaldehyde for 10 minutes under vacuum. Cross-linking was quenched by adding glycine solution to a final concentration of 0.125 M under vacuum infiltration for 5 minutes. Treated tissue was frozen in liquid nitrogen and stored at −800 C until chromatin extraction. Chromatin extractions were performed using EpiQuik Plant ChIP Kit  according to manufacturer's recommendations. Extracted chromatin was sheared in 600 µl of the EpiQuik buffer CP3F with 5 10-second pulses on a sonicator. To test and optimize sonication conditions, cross-linking was reversed in a sample of sheared chromatin and the resulting products were analyzed on agarose gel. Sonication conditions were optimized to yield predominantly 200–500 bp DNA samples. Chromatin immunoprecipitations, reverse cross-linking, and DNA cleanup was performed using EpiQuik Plant ChIP Kit (Epigentek) according to manufacturer's recommendations. For each genotype, antibody, and replicate, 50–100 ng of input and immunoprecipitated (IP) DNA was amplified with a whole genome amplification kit . The amplification of no antibody control (negative control) was always 5–10 fold less efficient confirming specificity of immunoprecipitation. For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit  according to the array manufacturer's protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3. Samples were hybridized to the custom 2.1 M probe array (GEO Platform GPL13499) for 16–20 hrs at 42 C. Slides were washed and scanned according to NimbleGen's protocol. Images were aligned and quantified using NimbleScan software (Roche NimbleGen) producing raw data reports for each probe on the array. The histone modification and methylation mutants array data can be obtained from GEO accession (GSE39460). The resulting microarray data were imported into the Bioconductor statistical environment (http://bioconductor.org/). Microarray data channels were assigned the following factors: B73 immunoprecipitation, Mo17 immunoprecipitation, B73 input, or Mo17 input depending on sample derivation. Non-maize probes and vendor-supplied process control probes were configured to have analytical weights of zero. Variance-stabilizing normalization was used to account for array-specific effects. Factor-specific hybridization coefficients were estimated by fitting fixed linear model accounting for dye and sample effects to the data using the limma package www.maizesequence.org. Each probe was only associated with the closest repeat and all probes located within 5 kb of a repeat were retained for further analyses. The probes were assigned based on distance to the retrotransposon and include both upstream (5′) and downstream (3′) sequences together. The distribution of retrotransposons along the length of the chromosome was performed as described in http://genomics.tacc.utexas.edu/data/rte_methylation_spreading/.DNA methylation profiling on three replicates of 3DNA was extracted from the outer tissues of B73 ears whose silks had emerged but had not been fertilized. Sodium bisulfite-treated Illumina sequencing libraries were prepared using a method similar to that of Lister et al Approximately 63M Mo17 454 whole-genome shotgun sequencing reads generated by the DOE's Joint Genome Institute (JGI) were trimmed and aligned to Maize B73 reference genome (AGPv2) and reads aligned uniquely (single loci) were filtered for subsequent analysis. A retrotransposon insertion site was classified as empty if we identified at least 3 WGS reads supporting the site that aligned to the insertion site that included>50 bp of aligned sequence outside of the repeat region in B73 with similarity of ≥94%, relatively short unaligned tails (≤20 bp), and contained a long overhang of >20 bp that begins ±3 bp from the annotated retrotransposon insertion site. PCR primers were designed to amplify the sequence at the “empty” sites using the B73 sequence (which contained the insertion) and the Mo17 sequence (which lacks the insertion) . These srd leaf, embryo, endosperm, and immature ear) for both B73 and Mo17. Samples were prepared at the University of Minnesota BioMedical Genomics Center in accordance with the TruSeq library creation protocol (Illumina). Samples were sequenced on the HiSeq 2000 developing 6–17 million reads per replicate. Raw reads were filtered to eliminate poor quality reads using CASAVA (Illumina). Transcript abundance was calculated by mapping reads to the maize reference genome (AGPv2) using TopHat under standard parameters www.iplantcollaborative.org). RPKM values were calculated per gene. All genes within 500, 1000, 2500, and 5000 bases of the closest upstream annotated transposable element (ZmB73_5a) using BEDtools RNA–seq was performed on three biological replicates of four tissues  The efficient enrichment of DNA associated with H3K9me2 was assessed using qPCR. The copia sequence is known to be enriched for H3K9me2 while the GAPC sequence is not associated with H3K9me2 . Primers for these regions were used to perform qPCR using three technical replicates. The percent of input DNA recovered after IP with the H3K9me2 antibody or a noIG control was determined for both sequences. (B) Several regions were selected for validation based on ChIP-chip profiling. Two regions enriched for H3K9me2 (H1 and H2) and four regions with no evidence for H3K9me2  were used to design primers for qPCR. The percent of input DNA recovered by ChIP using an H3K9me2 antibody or a noIG control was determined for three replicates of B73 using these primers. The H1 and H2 sequences were enriched by ChIP while the L1, L2, L3 and L5 sequences showed much lower levels of recovery.(TIF)Click here for additional data file.Figure S2Levels of DNA methylation within retrotransposons. Whole-genome bisulphite sequencing data was used to assess the average level of methylation within retrotransposons. DNA methylation levels within each sequence context  were determined for each family of retrotransposon. The average levels of methylation for elements classified as having spreading of both 5 mC and H3K9me2, spreading of H3K9me2 only and non-spreading were determined and plotted. The error bars indicate standard deviation among retrotransposon families and “*” indicate significant (p<0.001) differences for a group relative to the non-spreading families. The level of internal methylation at CG sites is similar for retrotransposons with and without spreading of heterochromatin although there is a significant difference in spreading (both) relative to non-spreading families. CHG methylation is slightly lower in non-spreading families. The non-spreading families have slightly elevated levels of CHH methylation relative to the other families.(TIF)Click here for additional data file.Figure S3Chromatin modifications in regions flanking maize retrotransposon superfamilies including RIX – LINE (black), RLC – copia (red), RLX – unknown LTR (blue) and RLG – gypsy (green). We identified probes located in low-copy DNA flanking retrotransposons in maize. The number of probes for each class is indicated within the Figure legend. The average level of DNA methylation (A–B), H3K9me2 (C) or H3K27me3 (D) is shown for the 5,000 bp adjacent to each superfamily. The level of chromatin modifications are based on ChIP-chip experiments and the y-axis represents the average log ratio for the immunoprecipitated samples relative to genomic input DNA.(TIF)Click here for additional data file.Figure S4Profiles of chromatin surrounding spreading (both) retrotransposons. Black lines indicate DNA methylation. Blue lines indicate H3K9. Green lines indicate H3K27 levels. Chromatin values calculated include probes flanking both ends of retrotransposable elements. Copy number of repeat fragments in the B73 reference genome as well as the total number of probes flanking each repeat are displayed. The y-axis provides the distance (in bp) from the retrotransposon insertion.(TIF)Click here for additional data file.Figure S5Profiles of chromatin surrounding spreading (H3K9) retrotransposons. Black lines indicate DNA methylation. Blue lines indicate H3K9. Green lines indicate H3K27 levels. Chromatin values calculated include probes flanking both ends of retrotransposable elements. Copy number of repeat fragments in the B73 reference genome as well as the total number of probes flanking each repeat are displayed. The y-axis provides the distance (in bp) from the retrotransposon insertion.(TIF)Click here for additional data file.Figure S6Similar profiles of DNA methylation enrichment adjacent to retrotransposon families in different tissues and genotypes of maize. The relative level of DNA methylation in each 200 bp bin is shown for each of the 144 retrotransposon families. The red color indicates enrichment for the modification while blue indicates depletion of the mark. Black indicates levels of the modification similar to genome-wide average values. The color intensity is based on the average log ratio of immunoprecipitated DNA relative to input DNA. The retrotransposons are grouped according to whether they show spreading for DNA methylation and H3K9me2, just H3K9me2 or neither of the marks. The profiles are shown for B73 leaf (A), Mo17 leaf (B), B73 endosperm (C) and B73 embryo (D).(TIF)Click here for additional data file.Figure S7Histone modification patterns in different genotypes. The profile of several histone modifications is shown for both B73 and Mo17. The relative level of H3K9(di)- or H3K27(tri)-methylation in each 200 bp bin is shown for each of the 144 retrotransposon families. Modification levels calculated include probes flanking both ends of retrotransposable elements. The red color indicates enrichment for the modification while blue indicates depletion of the mark. Black indicates levels of the modification similar to genome-wide average values. The color intensity is based on the average log ratio of immunoprecipitated DNA relative to input DNA. The retrotransposons are grouped according to whether they show spreading for DNA methylation and H3K9me2, just H3K9me2 or neither of the marks. (A) and (B) display the profiles for H3K9me2 in B73 and Mo17 seedlings, respectively. (C) and (D) display the profiles for H3K27me3 in B73 and Mo17 seedlings, respectively. (E) shows the H3K27me3 profile for immature ear tissue from B73.(TIF)Click here for additional data file.Figure S8Similar levels of H3K9me2 within spreading and non-spreading retrotransposons. The ChIP-chip assay does not provide information on the abundance of H3K9me2 within repetitive regions. In order to assess whether the level of H3K9me2 was similar within retrotransposons with, and without, spreading we designed primers for internal sequences of 10 retrotransposon families including six with spreading (both) and four that were classified as non-spreading. The qPCR protocol described by Haring et al. (2007) was used to assess the percent input DNA recovered by immunoprecipitation. The percent of input DNA recovered after IP with the H3K9me2 antibody or a noIG control was determined for both sequences and the standard deviation is indicated with error bars. There were high levels of H3K9me2 in each of these retrotransposons but there was no significant difference between the spreading and non-spreading retrotransposons.(TIF)Click here for additional data file.Figure S9Small RNA coverage of retrotransposon families. B73 shoot small RNAs (TIF)Click here for additional data file.Figure S10Characteristics of retrotransposon families with spreading of heterochromatic marks. In each of the plots the retrotransposon families are grouped into both (5 mC and H3K9), H3K9 only and non-spreading columns and the superfamily is indicated by the color. The data points are jittered to allow visualization of all families. (A) The genomic copy number of each family is shown using a log-scale. The families with spreading of both marks tend to have higher copy numbers. However, there is an overlap in the range of copy number for families with and without spreading. (B) The total Mb of the B73 genome comprised by each family is shown. (C) The average insertion date for each family is plotted. While the non-spreading families include both young and old retrotransposons the families with spreading of both marks tend to be younger families.(TIF)Click here for additional data file.Figure S11DNA methylation levels in flanking sequences are similar throughout the chromosome. The level of DNA methylation in sequences flanking retrotransposons was determined from bisulphite sequencing data. Only flanking sequences that did not contain any repetitive sequences within 1 kb of the retrotransposon were used. The proportion of methylated cytosines in CG (A) or CHG (B) contexts was determined for 1 kb of low copy sequences flanking spreading (both), spreading (H3K9) and non-spreading retrotransposon families.(TIF)Click here for additional data file.Figure S12Genes near spreading retrotransposons show lower expression than genes near non-spreading retrotransposons. (A) Average RPKM values  for all genes falling within 0.5 kb, 1 kb, and 2.5 kb from the respective class of retrotransposons were determined. (B) Proportion of genes expressed (RPKM>0) in B73 leaf tissue for genes near 5 mc and H3K9 spreading elements, H3k9 spreading only, non-spreading, and no TE nearby. Red asterisks indicate highly significant (p<0.001) difference from the non-spreading elements within the distance classification. Black asterisks indicate significant (p<0.001) difference from genes not near any TE. (C) Average RPKM values in B73 leaf tissue for expressed genes  falling within 0.5 kb, 1 kb, and 2.5 kb from the respective class of retrotransposons were developed. Red asterisks indicate significant (p<0.05) difference from the non-spreading elements (green) within the distance classification. Black asterisks indicate significant (p<0.05) difference from genes not near any retrotransposons (purple).(TIF)Click here for additional data file.Table S1Attributes and classification of retrotransposon families. A list of 145 retrotransposon families with information and statistics about each family in the B73 genotype.(XLSX)Click here for additional data file.Table S2Primers used for empty site validation.(XLSX)Click here for additional data file.


No clear information on the optimal dosage of Canakinumab in CAPS is available.To analyse the modification of dosage schedule of Canakinumab in CAPS in 12 months of routinely clinical practice.12 patients (9 children and 3 adults) with Muckle-Wells syndrome (3), MWS/CINCA overlap (3) and CINCA (6) were analyzed. Patients were previously enrolled in the CACZ885D2306 trial and studied for the following 12 months.At baseline, 7 patients were treated with the initial dosage of 2 mg/kg  every 8 weeks. In 5 patients  the dosage was 4 mg/kg (or 300 mg) every 8 weeks.During the following 12 months modification of dosage of frequency was performed in 7/12 patients. The 5 patients at higher dosage during the CACZ885D2306 study needed to increase the frequency of administration with a mean frequency of 6 weeks (range 4-8). The mean reason was the presence of mild clinical manifestation and/or persistent elevation of acute phase reactants. In one of these patients the therapy was subsequently discontinued due to persistent disease activity. An increased frequency (6 and 7 weeks) was also performed in 1 MWS and 1 CINCA patient, respectively.In 5 patients the treatment was not modified being effective in the control of the disease.This study confirms the efficacy of Canakinumab in CAPS. However, pediatric patients and those with a more severe phenotype require higher and more frequent dosage than previously described.


RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A, SRY and DYS14 was performed by real-time PCR.cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.Hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.Use of this assay for the detection of hypermethylated  Traditionally prenatal diagnosis of fetal genetic status has depended on the use of invasive diagnostic tests, either amniocentesis or chorionic villus sampling (CVS), which carry a small but significant risk of miscarriage de novo. NIPD for fetal sex determination can be carried out at an early gestational age for pregnancies known to be at risk of a sex-linked genetic condition Analysis of cffDNA in maternal plasma is now in routine clinical diagnostic use for non invasive prenatal diagnosis (NIPD) where the target fetal sequence is derived from the father or where the allele arises de novo) allele to make a definitive diagnosis. Absence of detection of the paternal target sequence is either indicative of a true negative result or could also be due the lack of amplification of the sequence due to low concentrations of circulating cffDNA or the complete absence of cffDNA in the sample. Amplification of a fetal specific marker that confirms the presence of cffDNA allows a negative result to be more accurately interpreted as either a true or false negative result. The use of several fetal identifiers has been reported including the hypermethylated RASSF1A promoter Currently, clinical applications of NIPD require the detection of the paternally inherited , at the early pregnancy unit (n = 4), at a routine ultrasound scan (n = 16) or for an invasive test procedure (n = 8) at Salisbury NHS Foundation Trust. The study was approved by the South West 1 Research Ethics Committee A (ref 09/H0104/59). Gestational age at blood collection was confirmed by routine ultrasound in all cases.g for 10 minutes and the plasma fraction transferred to a 2 ml centrifuge tube and re-centrifuged at 20,000 g for 10 minutes. The cell free plasma fraction was stored at −80°C. Cell free DNA was extracted from 3 ml plasma using the Circulating Nucleic Acid Kit (Qiagen) following the manufacturer’s instructions and resuspended in 70 µl AVE buffer.Maternal blood was collected into two 10 ml EDTA tubes and centrifuged at 1600 TseI and 4 U BsmI (methylation insensitive digest) and 2 U BstYI and 2 U BstUI (methylation sensitive digest). Samples were incubated at 60°C (undigested and methylation sensitive digest) and 65°C (methylation insensitive digest) for 2 hours. Additional restriction enzymes were then added to the reaction and the samples digested further at 37°C for 2 hours using no enzyme (undigested control), 8 U EcoRI, 8 U MspI and 4 U HaeIII (methylation insensitive digest) and 8 U EcoRI, 8 U HhaI and 4 U HpaII (methylation sensitive digest). All enzymes were supplied by New England Biolabs. cfDNA from male plasma was digested for each batch of samples analysed and performed as a digest control for the RASSF1A methylation insensitive and methylation sensitive digests. These digests should proceed to completion and therefore no RASSF1A amplification products should be observed following real time PCR. Further information regarding the selection of the restriction enzymes and details of the cleavage sites of the enzymes in the RASSF1A amplicon are given in Restriction digestion reaction and real time PCR conditions were optimised on a separate training set of 90 plasma samples obtained from the SAFE-RAPID sample bank at Great Ormond Street Children’s Hospital prior to commencing this study RASSF1A or SRY , DYS14  and RASSF1A . The cfDNA from male plasma also acts as a digest control for the RASSF1A methylation insensitive and methylation sensitive digests as digestion should proceed to completion and no RASSF1A amplification products should be observed.Samples that were negative for RASSF1A was determined by positive real time PCR amplification plots  and a correct melt profile showing a specific peak at 91.2°C  required 3 replicates to be positive for analysis to be undertaken. For the methylation insensitive digest (control for complete enzyme digestion of RASSF1A target sequence) complete digestion was assumed to have occurred if no replicates were positive. For the methylation sensitive digest, ≥2 out of 3 replicates were required to be positive to confirm the presence of hypermethylated fetal DNA. Samples with fewer than 2 positive replicates were not considered to have sufficient cffDNA present for reliable interpretation of the SRY result. Nine SRY (or DYS14) replicates were analysed for each patient sample (three from each digest) and the sample was considered to be male if 6 or more replicates showed amplification  and a correct melt profile showing a specific peak at 80.5°C  and gestational ages ranged from 5–23.2 weeks (mean = 9.1 weeks). All blood samples were received in the laboratory within 72 hours of collection.RASSF1A in the undigested sample indicating that total cell free DNA had been extracted successfully in all cases. Hypermethylated RASSF1A, was amplified in ≥2 out of 3 replicates for the methylation sensitive restriction enzyme digest for 79 samples (88%) indicating the presence of cffDNA. Of these, 45 showed amplification of SRY (≥6 out of 9 replicates positive) and were male at delivery and 33 had no detectable SRY (0 out of 9 replicates positive) and were female at delivery (including the XX twins). One sample was male at delivery but only showed amplification of SRY in only 3 out of 9 replicates and therefore this result would have been classified as inconclusive despite the detection of sufficient cffDNA. Hypermethylated RASSF1A was amplified in <2 replicates in 11 cases and these would have failed the data analysis parameters for reporting a clinical result. Of these, 10 samples had gestational ages less than 7 weeks 2 days which could explain the low level or absence of cffDNA in the samples SRY (i.e. an inconclusive result) and two samples had 6 replicates positive for SRY. One male sample showed 0 replicates positive for both hypermethylated RASSF1A and SRY and therefore could have been mis-reported as female if the RASSF1A assay had not been used. All samples showed complete enzyme digestion of RASSF1A in the methylation insensitive digest reactions . Eleven female samples showed 0/9 replicates positive for DYS14 but the remainder (n = 26) showed ≥1 replicate positive  with an average Ct value of 39.2 (SD 1.7). The male samples (n = 24) had 9/9 DYS14 replicates positive with an average Ct value of 32.5 (SD 1.8). The male sample that had no amplification of hypermethylated RASSF1A or SRY showed 9/9 replicates positive for DYS14 however the mean Ct value was 38.2. RASSF1A, SRY and DYS14 for male and female pregnancies.Samples that had fewer than 9/9 replicates positive for RASSF1A promoter sequences can be used to confirm the presence of cffDNA in diagnostic samples. The RASSF1A promoter sequence is very GC rich and is therefore problematic to fully digest and amplify by real time PCR. We have made several modifications to previously published protocols RASSF1A sequences, monitor that the digestion of the target has gone to completion and also improve the efficiency and interpretation of RASSF1A real time PCR. In preliminary experiments using published protocols we observed that incomplete digestion of RASSF1A was common in plasma samples derived from men and non-pregnant women where RASSF1A should not be observed following digestion with methylation sensitive enzymes  indicating the presence of cffDNA. For these samples the SRY real time PCR results and fetal sex at delivery were 100% concordant indicating that the test is 96–100% accurate with a 95% confidence interval RASSF1A) were observed in most (n = 10) samples that had a gestational age of 7 weeks or less. This is concordant with a recently published study of a clinical audit of fetal sex determination that showed that discordant results could be obtained if testing is performed before 7 weeks RASSF1A assay should be considered for all samples to ensure that sufficient cffDNA is present to enable confident interpretation of a test result. For example, in this study two samples of gestational age <7 weeks had reportable levels of hypermethylated DNA and 7 replicates positive for SRY analysis and were phenotypically male at birth. Conversely a sample of gestational age 10 weeks 4 days showed unreportable levels of hypermethylated RASSF1A and an inconclusive number of positive SRY replicates. Using digital droplet PCR to quantify fetal load (RASSF1A) and fetal sex (SRY) it has been shown that the Pearson’s correlation coefficient between SRY and RASSF1A fetal loads is 97.3 with the RASSF1A fetal loads measured for some samples being lower than those determined using SRYSRY result but negative RASSF1A result. In all cases where unreportable levels of hypermethylated RASSF1A are obtained we recommend that repeat blood samples should be requested and testing repeated.Using the modified protocol we assessed the utility of the assay for confirmation of cffDNA when undertaking NIPD for fetal sex determination. Hypermethylated DYS14 has been reported as having a higher sensitivity of detection DYS14 amplification although the Ct values for these samples (mean 39.2) were significantly higher than those obtained for male samples (mean Ct 32.5)  . This haRASSF1A is applicable to all pregnancies since is it polymorphism independent and can be performed at the same time as the fetal sex determination on the same aliquot of cffDNA. The SRY and RASSF1A assays have identical reaction conditions and can be amplified in the same plate and therefore results can be reported within 48 hours of receipt of the maternal blood sample. The assay could also be used for NIPD of single gene disorders and fetal RhD status provided that the restriction enzymes do not cut within the target amplicon. Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.This modified protocol for detection of fetal specific hypermethylated Figure S1SRY and RASSF1A real time SYBR green PCR assays.Examples of PCR amplification plots and melting profiles for the (PDF)Click here for additional data file.Figure S2(a) Amplification plots showing the difference in efficiency of three methylation sensitive RASSF1A restriction enzyme digest protocols using cell free DNA extracted from the plasma of a non-pregnant female. (b) Amplification plots showing an example of the difference in efficiency of two methylation sensitive RASSF1A restriction enzyme digest protocols using cell free DNA extracted from the same plasma sample from a pregnant female.(PDF)Click here for additional data file.Figure S3Details of restriction enzyme digests.(PDF)Click here for additional data file.


The finding that APECED syndrome is due to a failure in negative thymic selection has suggested that central tolerance could play a role also in polygenic autoimmunity and that autoimmune patients should have an increase in RTEs. The aim of this work was to assess the frequency of RTEs, transitional B cells and Th17 lymphocytes in MS.PBLs from 55 MS patients  and 32 healthy controls were analysed by FACS for RTEs, naïve and memory T cells as well as Th17 cells and transitional B cells.+CD24hiCD38hiCD27-)  were found between controls and MS. A significant decrease in the percentage of CD4 effector memory (CD45RA-CCR7-CD27+) , as well as an increase in Th17 cells  was observed in MS vs controls.No differences in the percentage of RTEs (CD45RA+CD31+PTK7+)  or B transitional cells .The results of this preliminary study do not favour a failure in central tolerance in MS but confirm involvement of Th17 cells which are associated with a higher degree of disability.


AbstractCimex lectularius Linnaeus, 1758 from the Czech Republic and other European countries, hosted on Myotis Kaup, 1829 (4) and Homo sapiens Linnaeus, 1758 (57). The karyotype of all the specimens of Cimex lectularius analysed contained 26 autosomes and a varying number of the sex chromosomes. The number of sex chromosomes showed extensive variation, and up to 20 fragments were recorded. Altogether, 12 distinct karyotypes were distinguished. The male karyotypes consisted of 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 42 and 47 chromosomes. The females usually exhibited the number of chromosomes which was complementary to the number established in the males from the same sample. However, 11 polymorphic samples were revealed in which the karyotypes of females and males were not complementary each other. The complement with 2n = 26+X1X2Y was found in 44% of the specimens and 57,4% samples of bed bugs studied. The karyotypes with higher chromosome numbers as well as individuals with chromosomal mosaics were usually found within the samples exhibiting particularly extensive variation between individuals, and such complements were not found within samples contaning a few or single specimen. The occurrence of chromosomal mosaics with the karyotype constitution varying between cells of single individual was observed in five specimens (4.3%) from five samples. We assume that polymorphism caused by fragmentation of the X chromosome may result in meiotic problems and non-disjunction can produce unbalanced gametes and result in lowered fitness of individuals carrying higher numbers of the X chromosome fragments. This effect should be apparently enhanced with the increasing number of the fragments and this may be PageBreakthe reason for the observed distribution pattern of individual karyotypes in the studied samples and the rarity of individuals with extremely high chromosome numbers. The assumed lowering of the fitness of individuals carrying higher numbers of the X chromosome fragments could affect population dynamics of variable populations.Variation in the number of chromosomes was revealed in 61 samples of  Cimex Linnaeus, 1758 is the best known taxon of the family Cimicidae (Heteroptera) which contains up to 110 described species of haematophagous ectoparasites exploiting mostly bats and birds as hosts .The bed bug, ome e.g. . Variatiome e.g. , 1967 stome e.g. , 2011 exCimex pipistrelli Jenyns, 1839 is known as an obligate parasite of bats which may share its hosts with Cimex lectularius. The karyotype of Cimex pipistrelli is similar to the standard complement of Cimex lectularius but contains a higher number of autosomes  depending on information available from the collectors. Individual sites within a single city are differentiated by numerals . Bugs identified as Cimex lectularius were also collected at four sites PageBreakPageBreakof bat roosts in the Czech Republic and Slovakia. The complete list of the collecting sites is shown in The studied specimens of 010–2012 . The kar1-nY where n stands for the additional X chromosomes. The corresponding karyotypes of females were characterized by the formula 2n=26+2X1-n.The chromosome preparations were made from gonads or midgut using the spreading technique described by After withdrawing of tissues for cytogenetic methods, the material was preserved in 96% ethanol and used in parallel molecular studies. Their results have approved the original specimens determination according to morphological characters , 2013. TCimex lectularius analysed contained 26 autosomes and a varying number of the sex chromosomes. The relative length of chromosomes in the complement was successively diminishing from 5.3 to 1.7%. No distinct size groups of chromosomes could be differentiated; however, the largest and the smallest autosomal pair could be usually recognized according to their size. The original sex chromosomes X1X2Y were medium-sized whereas their supposed fragments occurring in the karyotypes with higher chromosome numbers were the smallest elements of the set.The karyotype of all the specimens of Cimex lectularius studied, 12 distinct karyotypes were differentiated  consisted of a single specimen only. The results recorded in individual collecting sites are summarized in 1X2Y) and 2n=30 in females PageBreakPageBreak(2n=26+X1X1X2X2)  . This cot roosts .1-3Y) and 2n=32 in females (2n=26+2X1-3)  . This chPageBreakKaryotype 3 included complements with four X chromosomes; 2n=31 in males (2n=26+X1-4Y) and 2n=34 in females (2n=26+2X1-4) (6+2X1-4) . This co1-5Y) and 2n=36 in females (2n=26+2X1-5)  . It was 1-6Y) and 2n=38 in females (2n=26+2X1-6)   and it w1-7Y) and 2n=40 in females (2n=26+2X1-7)   and it w1-8Y)   and it wPageBreakKaryotype 8 included a complement with nine X chromosomes and 2n=36 in males (2n=26+X1-9Y) (6+X1-9Y)  and it w1-10Y)   and it w1-13Y)  . This kaPageBreakKaryotype 11 included a complement with 15 X chromosomes and 2n=42 in males (2n=26+X1-15Y) (+X1-15Y) . This ka1-20Y)  . This coThe females exhibited the number of chromosomes which was usually complementary to the number established in the males from the same sample. However, 11 polymorphic samples were revealed in which the karyotypes of females and males were not complementary one another. Two females showing karyotypes with odd numbers of X chromosomes  were recorded .PageBreakrespectively). Mosaicism with two and five X chromosome fragments was also revealed in a male from Italy (Venezia 3) .The occurrence of chromosomal mosaics with the karyotype constitution varying between cells of single individual was observed in five specimens  from five samples. A female from Slovakia (Trnava) had two karyotypically different cell types. The complement with 14 X chromosome fragments (2n=40) was found in mesenteron cells, whereas 17 X chromosome fragments (2n=43) were observed in germinal cells from ovarium. In other individuals showing mosaic, variation was recorded between germinal cells derived from gonads only. In a single female from Austria (Melk), the karyotypes with 12 and 14 X chromosome fragments were recorded . A male from Janov in the Czech Republic showed cells with six or seven X fragments . A similar mosaic constitution was recorded in a female from České Budějovice (4) . The Trnava sample contained five males with different karyotypes  and a female showing a mosaic karyotype with different chromosomal numbers observed in both examined tissues. The Melk sample included a male with 2n=26+X1-10Y, and two females, one with 2n=26+2X1-7 and another with a mosaic karyotype constitution 2n=26+2X1-12/14.The karyotypes with higher chromosome numbers as well as individuals with chromosomal mosaic were usually found within the samples exhibiting particularly extensive variation between individuals. A sample from Liberec contained two males with karyotypes 2n=26+XCimex pipistrelli included 28 autosomes and the sex chromosome trivalent X1X2Y . This represents the highest X chromosome number recorded within Cimicidae, Heteroptera and probably also Insecta.We have obtained certain findings that are at variance with the previously published results. The distribution pattern of incidence of the X chromosome fragments reported by PageBreakThe results obtained in Cimex pipistrelli confirm the previously published data  has already become widely fixed in the extant populations. However, it is not sure that the assumed original fission resulted always in the formation of the same fragments. Similarly, the nature of subsequent fissions producing successively other fragments is not clear and may vary. The karyotypes of females with higher numbers of the X chromosome fragments could be heterozygous with varying constitution of fragments derived from parents. This possible variation cannot be evidenced with the use of classical cytogenetic techniques and molecular approach should be employed in clarifying this question.Therefore, the most plausible explanation of the origin of the supernumerary elements in the bed bug complements remains fragmentation of the X chromosome. This mechanism produces a complicated system of multiple sex chromosomes and it was proposed already in previously published papers  not related to humans (The causes of the origin and maintenance of extensive fragmentation of the X chromosome of bed bugs remain unclear. Populations of bedbugs have been exposed to various insecticides all over the world for decades . Potentio humans .PageBreakThe irregular meiotic division or meiotic drive may enhance segregational variation in the chromosome number in progeny as well as mitotic segregation disturbances may contribute to the origin of mosaics in somatic cells. Mating of individuals of different origin and possibly different genetic constitution may initiate and increase the occurrence of segregation problems. We can assume that non-disjunction can produce unbalanced aneuploid gametes and result in lowered fitness of individuals carrying higher numbers of the X chromosome fragments. This effect should be apparently enhanced with the increasing number of the fragments and this may be the reason for the observed distribution pattern of individual karyotypes in the studied samples and the rarity of individuals with extremely high chromosome numbers. On the other hand, meiotic drive could cause preferential transmission of certain karyotype variants to the offspring. We found an extraordinarily wide extent of karyotype variation between specimens in a few population samples only. We assume that this extreme variation might result from random mixing of individuals of different origin at a single site. Mating between geographically unrelated individuals can easily be imagined in a parasite such as the bed bug transmitted by migrating people. However, it is difficult to explain why these highly variable samples usually included specimens with an extreme karyotype constitution and the highest numbers of the X chromosome fragments. It is obvious that mating of parents with different karyotypes can produce great variety of recombinant complements in offspring, particularly in females. Variation in the number of chromosome fragments may be associated with abnormalities occurring in chromosome segregation during the cell division. The regular course of meiosis in the bed bug may be influenced by the holokinetic nature of chromosomes, completely achiasmatic male meiosis and inverted meiosis of the sex chromosomes. We can only speculate about relationships between the system of transmission of the fragmented sex chromosomes and the unusual features of reproductive biology of bed bugs. The assumed lowering of the fitness of individuals carrying higher numbers of the X chromosome fragments could potentially affect population dynamics of variable populations. It is apparent that more intensive cytogenetic screening combined with data on molecular variation in DNA sequences might shed light to this question.


Hepatitis C virus roots a chronic liver disease. Currently approved treatment strategy includes administration of alpha interferon and ribavirin combined therapy for 24-48 weeks. One of the predictor of sustained virological response is an early virological response to treatment characterized as rapid response. Hyper variable region 1 (HVR1) of E2 protein is responsible for viral entry and acts as a target for neutralizing antibodies. Any mutation in this region would effect virus interaction with target cell and viral persistence.Thirty one clones of six pre-treatment samples subjected to combination therapy were investigated. Three of the patients were rapid responders  and two were breakthrough responders (BT1 and BT2). Envelope 2 gene was amplified, cloned and sequenced. Amino acid substitution, frequency, composition and antigenic properties of HVR 1 of E2 protein were studied.In both rapid responders (R.R) (14 amino acid sites) and breakthrough responders (BT.R) (13 amino acid sites) half of the amino acid sites were either conserved or resistant to any physiochemical change due to amino acid substitution. It also indicated that average composition of hydrophilic and basic amino acids were comparatively lower in rapid responders than other samples affecting probable interaction of virus with target cells. A central non antigenic region was constant among the breakthrough responders but differed in length significantly among rapid responders reflecting the adaptive nature of HVR1 to the immune response.We observed that although HVR1is quite variable region in HCV 3a patients responding differently to treatment it still maintains its physiochemical properties for its proper functioning and viability. HCV roots a persistent infection which advances towards chronic hepatitis, liver steatosis, cirrhosis and hepatocellular carcinoma -3. It isThe standard therapy for HCV treatment adds up to taking Alpha-interferon 3 MU three times a week  and 1000effect the outcome of treatment. Rapid responders have recently been identified as positive predictors of sustained virological response in Pakistan [In HCV E1 and E2 protein are heterogeneous, especially hyper variable regions which include HVR1, a 27 amino acid sequence (81 nucleotides) ,12. ManyPakistan . TherefoIn HVR1, four amino acids position 2, 6, 19 and 23 were conserved in all the 31 variants. Conserved and variable sites among variants of rapid responders and among variants of breakthrough responders were also analyzed  at amino acid position 21.In breakthrough responders variable sites that maintained their physiochemical properties were amino acid 5 and 8 that were exclusively polar neutral sites. Amino acid 16 was exclusively non polar and hydrophobic, this amino acid is a conserved site in rapid responders as well. Amino acid 27 was also exclusively polar and hydrophilic in breakthrough responders, whereas in rapid responders this site was almost conserved with polar hydrophilic properties and very rare non polar amino acid substitution (1 out of 15 in our case).Amino acid 16 and 27 maintained their physiochemical properties despite of amino acid substitution in breakthrough responders. Amino acid 16 was at conserved site while amino acid 27 maintained its physiochemical properties in rapid responders thus amino acid 16 and 27 can be considered as sites which are resistant to a functional change.Similarly amino acids 9, 20 and 21 maintained their physiochemical properties in rapid responders were either at conserved sites in breakthrough responders or were resistant to physiochemical changes.Five amino acids sites that were conserved sites in both rapid responders and breakthrough responders are 2, 6, 10, 19 and 23. Five sites that were either conserved sites in one of the two groups or that maintained their physiochemical properties in both the groups were 16, 27, 9, 20 and 21. In total ten amino acids sites were exclusively conserved between samples responding differently to the treatment. Remaining seventeen sites were displaying different physiochemical properties.In rapid responders ten amino acid sites were conserved. Four amino acid sites were not conserved but maintained their physiochemical properties. Therefore among rapid responders more than half part of the HVR1 (fourteen amino acid sites) were stable functionally. In breakthrough responders nine amino acids were conserved. Amino acid substitution in four amino acid sites resisted any physiochemical change. Therefore like rapid responders almost half of the region of HVR1 remained stable functionally.Frequency of individual amino acid in HVR1 was compared between samples  than R.R group (17.6%). Acidic amino acids composition was slightly greater in breakthrough responders (2.22%) than in rapid responders (0.23%). . In R2 antigenic region extended from position 6 to 11 and remained constant in all variants of R2. In sample R3 antigenic region started a bit earlier and ended a bit earlier from position 4 to 8  and two breakthrough responders (BT1 and BT2) were compared at amino acid level. Total number of conserved and variable sites remained almost same in both the groups opposing the common assumption that responders harbors less variability than the non responders. Fourteen out of twenty seven amino acid positions were resistant to any change in physiochemical properties in rapid responders. On the other hand half of the amino acids were conserved or resistant to physiochemical shifts in breakthrough responders. Although number of conserved and variable sites was same in both groups their positions varied between rapid responders and breakthrough responders. Ten amino acids sites were exclusively conserved between samples responding differently to the treatment. Half of these sites were either conserved in one of the two groups or retained their physiochemical properties. Other half of the amino acids did not harbor any substitution at all. Another study confirmed that despite of high variability in HVR1, at most of the position hydropathic characters remained conserved . AnotherIt has been already noted that neutral amino acids are more frequent in HVR1 than in common proteins . It has Hydrophilic residues are exposed on the surface and are involved in interactions with other molecules whereas hydrophobic amino acids anchor E2 to core and maintains conformation of HVR1 . In the Most frequent amino acids in HVR1 were glycine (G). threonine (T), serine (S) and alanine (A)  and ribavirin (10 mg/kg per day) for 6 months. Serum samples taken before the onset of treatment were examined. Three patients  showed a rapid response to treatment as defined by negative HCV RNA (<500 IU/ml) after 1 month of treatment. Two patients (BT1 and BT2) showed a breakthrough virological response during treatment followed by reappearance of HCV RNA at the end of treatment. One of the patient showed negative HCV RNA at the end of treatment and was defined as end of treatment response (ETR). The subjects gave informed consent to participate and the study was conducted in accordance with the 1964 Declaration of Helsinki and Guidelines for Good Clinical Research Practice in Pakistan. The study was approved by Ethics Committee of Molecular Virology Division.Hepatitis C viral RNA was extracted from 100 μl of stored serum of the patients using Gentra RNA isolation Kit  according to the kit protocol. cDNA synthesis of extracted RNA (10 μl) was done at 37°C for 50 min and 42°C for 10 min using 200 units of Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RTase) (Invitrogen) and 8 Units of RNase inhibitor in a 20 μl of reaction with E2 outer anti sense primer (GTATTACGAGGTTCTCCAAAGC). For nested PCR amplification of entire E2 gene two sets of primers were optimized. The first round of PCR was done with 10 pM of E2 outer antisense primer and with 10 pM of outer sense primer (TCCTGGTAGTGCTGCTGCTA). Four microliter of the first round PCR products was used as a template for second round of nested PCR using inner sense (GAAACCCACGTCACCGGGGGAA) and inner antisense primers (CGCCTCCGCTTGGGATATGAGTAACA). The amplification conditions were same for both PCR rounds: initial denaturation at 94°C for 2 min followed by 35 cycles consisting of denaturation at 94°C for 45 sec, primer annealing at 50°C for 45 sec and primer extension at 72°C for 3 min followed by a final extension of 10 min. The amplified products were visualized on 1.2% agarose gel stained with ethidium bromide under UV transilluminator after electrophresis. The required bands were excised and DNA was purified from the agarose gel with Silica Bead DNA Gel Extraction Kit (Fermentas Inc. Germany). Purified DNA was suspended in depc treated water and used for cloning and sequencing.Purified E2 amplified products were directly ligated to 50 ng of PCR 2.1 vector . The product was transformed into chemically competent TOP10F' cells by incubating the entire ligation reaction along with competent cells on ice for 30 min then a 1 minute heat shock was given to cells at 42°C after which the cells were immediately transferred to ice. Then the cells were incubated with 500 ul of Luria-Bertani (LB) medium at 37°C for 1 hour. Successfully transformed cells were selected on LB agar plate supplemented with 100 μg/ml ampicillin and 12.4 ug/ml tetracycline. Plates were subjected to overnight incubation at 37°C. Clones carrying E2 gene were confirmed by colony PCR. 50 ul of culture was centrifuged at 13000 rpm for 2 min. supernatant was discarded and pellet was resuspended in 50 μl of 1X T.E. resuspended pellet was subjected to heat shock at 100°C for 10 min followed by centrifugation at 13000 rpm for 2 min. Supernatant that was supposed to contain the plasmid was used as a template for the PCR reactions. These PCR reactions were prepared both with 10 pmol vector-specific primers M13 forward (TAATACGACTCACTATAGGG) and M13 reverse (TAGAAGGCACAGTCGAGG) and with gene specific primers (E2 inner sense and inner antisense). Bands of size approximately 1056 bp with gene specific primers and 1183 with vector specific primers were visualized under UV confirmed successful cloning. Plasmid was isolated from cultures containing E2 clones using a Plasmid Miniprep Kit, (Fermentas Life Science technologies USA) according to manufacturer's protocol. From each patient 5-10 positive clones were randomly selected for sequencing.For each clone, both strands of E2 gene were sequenced with M13 forward and reverse primers by Applied biosystems prism dye termination method according to the manufacturer's instructions . Sequencing was performed on automated sequencer . Sequencing PCR reaction was performed with profile; 96°C for 15 sec, 50°C for 10 sec, 60°C for 4 min repeated 35 times. Labeled DNA was then purified with 2 μl of 3M Sodium Acetate and 2 μl of 125 mM EDTA followed by precipitation with 26 μl 100% ethanol. After 15 min incubation at room temperature DNA was centrifuged for 30 minutes at 2800 rpm at 4°C. Washing was done with 36 μl of 70% ethanol. Pellet was air dried and rehydrated in 12 μl formamide. Pellet was chilled on ice after it was heat shocked at 95°C for 5 minutes. Air dried pellet was then analyzed using an ABI prism sequencer. A set of 31 variants was analyzed containing 2-10 clones selected from each patient. . Sequences were analyzed with the Chromas LITE programme .http://www.clcbio.com.Amino acid heterogeneity analysis was done by aligning amino acids using Bioedit clustal W programme. These aligned sequences were then analyzed using MEGA version 4.1 . Number Antigenic regions were identified within the HVR1 of Envelope protein by analyzing antigenic profiles of all the variants by the method of Parker et al. .HM590012 to HM590017, HM584119 to HM584121, HM462252, HQ184929 to HQ184935, HQ202715 to HQ202721 and HQ189124 to HQ189130Accession numbers of nucleotide sequences submitted to GenBank are The authors declare that they have no competing interests.MI conceived the study, participated in its design and coordination and gave a critical view of manuscript writing. MA performed and analyzed the results. SA, AH, MI, MA, SB and BK participated in results analysis. All the authors read and approved the final manuscript.


Mild hypoxia is common after stroke and associated with poor long-term outcome.Oxygen supplementation could prevent hypoxia and improve recovery. A previousstudy of routine oxygen supplementation showed no significant benefit at 7 and12 months. This pilot study reports the effects of routine oxygensupplementation for 72 hours on oxygen saturation and neurological outcomes at 1week after a stroke.Patients with a clinical diagnosis of acute stroke were recruited within 24 hof hospital admission between October 2004 and April 2008. Participants wererandomized to oxygen via nasal cannulae (72 h) or control . Clinical outcomes were assessed byresearch team members at 1 week. Baseline data for oxygen(n = 148) and control (n = 141)did not differ between groups.The median (interquartile range) National Institutes of Health Stroke Scale(NIHSS) score for the groups at baseline was 6 (7) and 5 (7) respectively.The median Nocturnal Oxygen Saturation during treatment was 1.4%(0.3) higher in the oxygen than in the control group (p<0.001) during theintervention. At 1 week, the median NIHSS score had reduced by 2 (3) in theoxygen and by 1 (2) in the control group. 31% of participants in theoxygen group and 14% in the control group had an improvement of ≥4NIHSS points at 1 week doubling the odds of improvement in the oxygen group(OR: 2.9).Our data show that routine oxygen supplementation started within 24 hours ofhospital admission with acute stroke led to a small, but statisticallysignificant, improvement in neurological recovery at 1 week. However, thedifference in NIHSS improvement may be due to baseline imbalance in strokeseverity between the two groups and needs to be confirmed in a larger studyand linked to longer-term clinical outcome.ISRCTN12362720; European Clinical Trials Database 2004-001866-41Controlled-Trials.com  Hypoxia is common after acute stroke, affecting up to 63% of patients at sometime after admission invitro studies that oxygen encourages the formation of toxic freeradicals leading to further damage to the ischaemic brain Oxygen treatment is not without side effects There is only one large (n = 550) quasi-randomized study ofoxygen supplementation for acute stroke, and this suggests that routine oxygentreatment to unselected stroke patients does not reduce morbidity and mortalityThe evidence for oxygen treatment after acute stroke from experimental and clinicalstudies is conflicting, and, unsurprisingly, stroke management guidelines differwhile based on the same sparse evidence In this paper we report on a pilot study examining the effects of routine fixed doseoxygen supplementation at a rate of 2 or 3 L/min dependent on baseline oxygensaturation for 72 hours on oxygen saturation and neurological recovery at 1week.The protocol for this trial and supporting CONSORT checklist are available assupporting information; see This is a randomized controlled single blind pilot study of routine oxygensupplementation after acute stroke. A double blind study could not be used sincemedical staff would know the group allocated due to the presence/absence ofnasal cannulae. Patients were recruited from the University Hospital of NorthStaffordshire (UHNS), UK, a large teaching hospital admitting about 800 patientswith stroke per year. Depending on bed availability, most stroke patients areadmitted to the acute stroke unit within 24 hours of presentation. Patients withan admission diagnosis of stroke or possible stroke were identified by a memberof the stroke research group, who checked the medical admissions unit log bookevery morning and was contactable by the medical admissions team for new strokesin the day. Adult patients with a clinical diagnosis of acute stroke Recognised indications for oxygen treatment such as oxygen saturation onair <90%, acute left ventricular failure, severe pneumonia,pulmonary emboli, and chronic respiratory failure treated with long termoxygen at home.Recognised contraindications to fixed dose oxygen treatment , e.g. type II respiratory failure.Stroke was not the patient's primary clinical diagnosis. Patientswith other serious life-threatening illnesses likely to lead to deathwithin the next few months were also excluded.2) was greater than93% or at a rate of 3 L/min if baseline SpO2 was 93% orless. This dose was based on the results of earlier studies 2 were assessed regularly (at least three times a day) as partof routine clinical care. Those who developed indications for oxygen, or neededa higher concentration of oxygen than the protocol prescribed, were given theconcentration of oxygen they needed by the treating clinician, irrespective ofthe treatment group they were in.Participants were randomized to one of two treatment groups, the oxygen group andthe control group. Participants in the oxygen group were given oxygen at a flowrate of 2 L/min if baseline oxygen saturation , partial anteriorcirculation syndrome (PAC), lacunar syndrome (LAC) and posterior circulationsyndrome (POC) using the Oxfordshire Community Stroke Project (OCSP)Classification 2 every second producinga moving average every 5 seconds using an internal algorithm. Hands wereinspected to ensure that the fingers were warm and well-perfused. Nail varnishwas removed and long fingernails were clipped, where necessary. The pulseoximeter was attached to the wrist and the sensory probe was fitted to the indexfinger. To reduce movement artifacts the hemiparetic side was used for oximetry2<90%) was corrected to a notional 8 h period usingthe formula: T<90 (in minutes)  =   x 480. The same corrections were usedfor T<80 and T>98.Oxygen saturation and heart rate were assessed using a Pulsox-3I pulse oximeter(Konica Minolta). The instrument records SpOThe protocol was approved by the North Staffordshire Research Ethics Committee on06.10.2004 (ref: 04/Q2604/73). It is registered in the International StandardRandomized Controlled Trial Number Register (ISRCTN12362720) and the EuropeanClinical Trials Database (EudraCT number 2004-001866-41). Written informedconsent was sought from all study participants. Assent from the next of kin wasaccepted if the patient agreed to take part but was unable to give fullyinformed consent. Participants who were incompetent at the time of recruitment,but competent when followed-up at 1 week were asked to confirm consent. Patientswho were recruited to the study by assent and refused consent at 1 week werewithdrawn from the study.Descriptive statistics and counts were performed using Microsoft Excel, MicrosoftOffice XP, Microsoft Corporation, US, and tests of significance (as detailed inthe text) were carried out using SPSS statistical software version 15.0 . Statistical significance was accepted if thep-value was <0.05.Three hundred and one patients were recruited to the study from October 2004 toApril 2008, with a mean recruitment rate of 7 (range 1 to 23) per month; theflow of patients through each stage of the study is shown in Baseline demographic and clinical characteristics, stroke type and severity, andoxygen saturation at randomization were similar in the control and oxygen group.The mean age of patients included in the study was 72.3 (SD 11.6) years, 137(47%) were male, 243 (84%) were independent for activities ofdaily living before the stroke, and 113 (39%) were living alone.Concomitant medical problems included ischaemic heart disease in 71(25%), congestive cardiac failure in 34 (12%), atrial fibrillationin 53 (18%), and chronic obstructive pulmonary disease in 26 (9%)of participants. The majority of patients were normoxic with a mean oxygensaturation of 96.1%  atrandomization. The mean time from stroke onset to randomization was 17∶12(SD 8∶42) hh∶min. Bamford classification data is included in Inclusion was via fully informed consent in 115 (78%) participants in theoxygen group and 106 (75%) in the control group and by assent in 33(22%) participants in the oxygen group and 35 (25%) in the controlgroup. At the 1 week reassessment, patients who were included via assent wereasked to confirm consent if they had regained competence. One patient in thecontrol group and 1 patient in the oxygen group refused consent at that pointand were withdrawn.2 of less than 90% . Twelve patients (8%) in the oxygen group and 16 patientsin the control group (11%) were prescribed oxygen for clinicalindications during the treatment period (p = 0.4Fisher's Exact test).Oxygen saturation was assessed 3 times a day during the first 72 hours as part ofroutine clinical practice. In 48 (32%) patients in the oxygen group and52 (47%) patients in the control group one or more readings showed anSpO2of less than 80% and 90% and the number of participants who spentmore than 1 hour with a saturation of less than 90%. The median MeanNocturnal Oxygen Saturation was 1.3% higher in the treatment group thanin controls . A lower proportion of patients inthe oxygen group spent more than 5 min and more than 30 min with anSpO2 of <90% during the recording night . There was no significant difference in the proportion of patientswho spent more than 60 min with an SpO2 of less then 90%, orin the time spent with an SpO2 of less than 80%, since suchvery severe hypoxia was uncommon, and would have been treated as soon asidentified by the clinical team.Pulse oximetry was conducted successfully in 98 patients in the oxygen group andin 100 controls. All parameters of oxygenation were significantly better in theoxygen than in the control group except the time spent with an SpOEighty-nine participants (29%) had no or insufficient oximetry data foranalysis. This equated to missing values of 41 and 48 for the controls andoxygen group respectively. There was no significant difference in theproportion of patients returning no or insufficient oximety data between the twogroups. Participants who did not complete oximetry were older than those whocompleted (74.1 vs. 71.4 years), less likely to have been independent before thestroke (73% vs. 89%), less likely to have full scores on the GCSverbal response item (57% vs. 71%), less likely to be able to walk(8% vs. 17%), and were recruited sooner after stroke onset(15∶20 vs. 17∶10 hh∶mm). Otherwise there were no significantdifferences in baseline. Further information on oxygen saturation is provided inAnalysis was by intention to treat. Neurological scores improved from baseline to1 week in both groups. At baseline the median (IQR) NIHSS was 6 (7) in theoxygen and 5 (7) in the control group. By 1 week the median NIHSS scores(including deaths) had improved more in the oxygen than in the control group−2 (3) for oxygen vs. −1 (2) for control, p<0.001 Mann WhitneyU-test). A significant improvement in neurological scores was defined asreduction of 4 or more points in the NIHSS from baseline to week 1. The oddsratio (95% CI) for significant improvement with oxygen supplementationwas 2.9 (1.59–5.4). Forty five (31%) participants showed asignificant improvement in neurological score in the oxygen group compared to 18(14%) participants in the control group . There wOxygen treatment might be more effective in older patients with a history ofheart and lung problems, those with lower baseline oxygen saturation, patientswith more severe strokes, or those with an impaired level of consciousness. Itmight also be affected by treatment with oxygen before randomization and theaetiology of the stroke (infarct or haemorrhage). We therefore carried out anexploratory subgroup analysis to identify subgroups which might benefit more orless from oxygen treatment. The odds ratios for significant improvement withoxygen treatment (NIHSS reduction from baseline of 4 or greater) There was no significant difference in the group means for oxygen and controlrespectively for highest systolic blood pressure (167 SD 29 mmHg vs. 167 SD 28mmHg), highest diastolic blood pressure (93 SD 19 mmHg vs. 91 SD 15 mmHg), andheart rate (92 SD 16 BPM vs. 92 SD 19 BPM) during the 72 hours of the treatmentperiod. The highest mean temperature during the first week was similar in bothgroups (37.2% SD 0.6 vs. 37.1% SD 0.7 C). There was no differencein the proportion of patients requiring antibiotics in the first week(n = 27 (18%) vs. n = 22(15%) for oxygen and control respectively) or sedative medications(n = 9 (6%) vs. n = 9(6%) for oxygen and control respectively).The main finding of this study is that routine oxygen supplementation, given for 72hours at a rate of 2 or 3 L/min, dependent on baseline oxygen saturation leads, to asmall but statistically significant improvement of neurological recovery at 1 week.The odds of improving by 4 or more NIHSS points at 1 week were doubled in the oxygengroup. While statically significant the difference in the improvement in NIHSS issmall and may be due to the non-significant baseline imbalance in stroke severitybetween the two groups (baseline NIHSS was 1 point higher in the oxygen than in thecontrol group). The NIHSS is not designed as an outcome tool, and the relevance of asmall change in NIHSS during the first 7 days on longer-term functional outcome isunclear.However, other studies have shown that baseline NIHSS is a strong predictor ofrecovery and long-term outcome Our data contrasts with an earlier study by Ronning and Guldvog et alA more recent, but small, study by Singhal While we aimed to include a representative sample of all stroke patients admitted tothe hospital, the majority of subjects included had relatively mild strokes. Themedian NIHSS at baseline in this study was 6 and 5 in the oxygen and control groupsrespectively, while the median NIHSS of patients admitted to the local stroke unitis about 8 . The main reason forthis is the requirement of informed consent or assent and the short time betweenadmission and recruitment. While patients with milder strokes can give informedconsent, those with more severe strokes can only be included if assent from the nextof kin can be gained. By the time subjects are identified for the study, relativeshave usually left hospital and are then contacted during visiting times or have totravel back in to discuss the study. Patients with more severe strokes were morelikely to benefit from oxygen in the Ronning and Guldvog study The results of the overnight pulse oximetry showed that oxygen supplementationeffectively improved all parameters of oxygenation. Mean Nocturnal Oxygen Saturationwas 1.3% higher in the oxygen group than in the control group. In two smallerpreparatory studies we have shown that oxygen supplementation at a rate of 2 L/minduring the day with compliance ensured by continuous observation raises oxygensaturation by 2% Oxygen supplementation is simple, cheap, and applicable even in the most basicclinical environments. The results of this study suggest that oxygen given at 2L/min for 72 h may improve neurological recovery at 1 week. The results of thispilot study should be confirmed in a larger fully powered trial, which is nowongoing (The Stroke Oxygen Study) Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.During November-December 2009, we collected leftover serum from a blood bank, a pediatric children's hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults. The 2009 pandemic influenza A (H1N1) virus (pH1N1) was first identified in April 2009 and caused widespread illness in the United States and around the world During the 2009 pandemic, Florida employed a surveillance system that tracked the percentage of Emergency Department (ED) visits for influenza-like illness (ILI) throughout the state. According to surveillance data, Tampa Bay experienced a gradual increase in influenza activity in the spring and summer of 2009, followed by a large fall wave of influenza activity that peaked in late October and decreased steadily thereafter . EstimatSerosurveys, which estimate the prevalence of antibodies to a specific pathogen, can be a valuable tool in determining the proportion of the population infected with a novel virus. Unlike most influenza surveillance, which relies on presentation of clinical illness, serosurveys capture persons that experienced symptomatic or asymptomatic illness, and can provide information on total infections which may be underestimated with traditional surveillance methodologies. However, serosurveys are limited by the sensitivity and specificity of the assay employed to detect antibody titers To date, one published study has reported on the prevalence of pH1N1 antibodies among residents in one region of the United States In November and December 2009, after pH1N1 virus activity in Tampa Bay had peaked , we collWe sought to collect 160 specimens from each of six age groups: <5 years, 5–17 years, 18–24 years, 25–49 years, 50–64 years and ≥65 years. Infants less than 6 months were excluded due to the potential for maternal antibody transmission. The required sample size was calculated using relative standard error measurements. We estimated that the lowest seroprevalence for all groups would be among adults aged ≥65 years. At the time of the serosurvey, we estimated that 15% of this age group would be seropositive, requiring a sample size of approximately 160 to maintain a relative standard error less than 20%. We were able to collect at least 160 samples from all ages groups except for children aged <5 years for which we were only able to collect 60 samples. We estimated that the seroprevalence among this age group would be high (30%), and therefore despite the small sample size, would meet the relative standard error criteria of less than 20%.This study was proposed to the Florida Department of Health Institutional Review Board (IRB) and Centers for Disease Control and Prevention (CDC) IRB who considered the investigation as public health response, and therefore not subject to IRB review and approval.Leftover serum specimens for Tampa Bay residents aged ≥16 years were collected from a large blood bank testing facility during a 4-day period from November 30 -December 3, 2009 . For resAntibodies against pH1N1 virus were detected by the hemagglutination-inhibition (HI) assay as previously described using A/California/07/2009 virus Using sera from patients with pH1N1 laboratory-confirmed infections and non-exposed United States residents, a previous study determined that a threshold HI titer ≥40 yielded a sensitivity of 75% and specificity of 97% in determining previous infection with the pH1N1 virus among persons <60 years of age Because serology cannot differentiate between antibodies produced by virus infection and response to vaccination, we developed a simple statistical model to estimate the proportion of seropositive results due to pH1N1 vaccination coverage  . MonthlyOverall, 27% of the study sample had pH1N1 antibody titers ≥40 with an age-standardized seroprevalence of 25% . A cumulThe overall BRFSS/NHFS vaccination coverage estimate for Florida two weeks prior to the time of specimen collection was 9.0%, with the highest vaccination coverage among children aged <5 years (17%) and school-aged children aged 5–17 years (15%) . Based oThe proportion of Tampa Bay residents that were estimated to be seropositive due to infection with pH1N1 virus after the peak of the fall wave was 25% . The higBy December 2009, an estimated 30% of Tampa Bay's population had elevated levels of antibodies against the pH1N1 virus (25% from infection and 5.9% from vaccination). Our estimates indicate that half of young adults aged 18–24 years and more than half of school-aged children had antibodies to the pH1N1 virus at titers of ≥40 at that time. Thus, the proportion of the Tampa Bay population among these age groups that remained susceptible to pH1N1 virus infection by December 2009 was markedly decreased.Our seroprevalence estimates are similar to previously published studies from Pittsburgh, PA Though the availability of pH1N1 vaccine was still limited by the time of the serosurvey , school-aged children comprised one of the target groups for initial vaccination campaigns, and thus were more likely to receive the vaccine earlier Our survey has several potential limitations. First, we did not have baseline serum specimens prior to the 2009 pandemic for comparison and therefore were not able to test for a four-fold rise in antibody titers or adjust for pre-existing, cross-reactive antibody titers. Previous studies have suggested that cross-reactive antibodies were most common among persons aged ≥60 years In summary, we performed a serosurvey in Tampa Bay to determine the prevalence of antibodies to the pH1N1 virus after the fall wave of the pandemic, and during the early phases of the pH1N1 vaccination campaign in Florida. We adjusted seroprevalence results to account for HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination. Our results provide evidence for substantial immunity against the pH1N1 virus among the Tampa Bay population. Though disease activity decreased after December 2009, vaccination levels continued to increase in Florida; thus the proportion of the population with immunity to the pH1N1 virus by the end of the pandemic was probably higher than the estimates presented herein.Appendix S1Method of seroprevalence adjustment to account for hemagglutination-inhibition (HI) assay sensitivity and specificity.(DOCX)Click here for additional data file.Appendix S2Simple statistical model used to estimate the proportion of seropositive results due to vaccination.(DOCX)Click here for additional data file.Table S1Statistical model to estimate the proportion of Tampa Bay residents with vaccine-induced pH1N1 virus seropositivity in November- December 2009, including all components and equations.1 Estimated from Behavioral Risk Factors Surveillance System (BRFSS) and National pH1N1 Flu Survey (NHFS)2 Vaccine immunogenicity estimates based on published immunogenicity studies 3 Estimated proportion of population with vaccine-induced seropositivity (≥1∶40 GMT)  =  (vaccine coverage) x (proportion with ≥1∶40 antibody response)4 Seroprevalence adjusted for assay sensitivity and specificity. For children and adults aged <65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 97%. For adults aged ≥ 65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 94% 5 Estimated proportion with pH1N1 virus infection prior to vaccination  =   x (estimated proportion of population with vaccine-induced seropositivity)6 Proportion with vaccine-induced seropositivity not infected prior to vaccination  =  (estimated proportion of population with vaccine-induced seropositivity) minus (estimated proportion of population with pH1N1 virus infection prior to vaccination)7 Proportion infected with pH1N1 virus  =   minus (proportion with vaccine-induced seropositivity not infected prior to vaccination).(DOCX)Click here for additional data file.


There was an error in the last paragraph of the Discussion section. The third and fourth sentences should read:"Interestingly methylation of Trypanosoma brucei H3K76, which is orthologous to histone H3K79 in mammals, is required for replication initiation and overexpression of the Trypanosoma Dot1A methylase results in over-replication[44]. Although this observation suggests that methylation of H3K76 in Trypanosoma achieves the opposite effect than the methylation of H3K79 in mammals, and it is yet unclear whether Trypanosoma H3K76 methylation associates with replication origins, both enzymes seem to be involved in regulating replication re-initiation events and thus might both be involved in processes that mark genomic regions for initiation. "


Institutional affiliations 2 and 3 for authors Khalid Belkhir and Nicolas Bierne are incorrect. Affiliations 2 and 3 should be: UMR 5554, ISEM Université Montpellier II.ST.In addition, the term "FST" was incorrectly typeset multiple times in the "Genetic Diversity and Population Differentiation" section of the Results. The correct typesetting of this term is F


To determine clinical characteristics of patients hospitalized in the United Kingdom with pandemic (H1N1) 2009, we studied 1,520 patients in 75 National Health Service hospitals. We characterized patients who acquired influenza nosocomially during the pandemic (H1N1) 2009 outbreak. Of 30 patients, 12 (80%) of 15 adults and 14 (93%) of 15 children had serious underlying illnesses. Only 12 (57%) of 21 patients who received antiviral therapy did so within 48 hours after symptom onset, but 53% needed escalated care or mechanical ventilation; 8 (27%) of 30 died. Despite national guidelines and standardized infection control procedures, nosocomial transmission remains a problem when influenza is prevalent. Health care workers should be routinely offered influenza vaccine, and vaccination should be prioritized for all patients at high risk. Staff should remain alert to the possibility of influenza in patients with complex clinical problems and be ready to institute antiviral therapy while awaiting diagnosis during influenza outbreaks. In addition, we included infants who had not left the hospital since birth in whom pandemic (H1N1) 2009 had developed. We included transfers from other hospitals when a transfer was for a reason other than influenza and when the history of influenza clearly indicated that it had been acquired at another hospital. FLU-CIN procedures were reviewed and approved by the Ethics and Confidentiality Committee of the National Information Governance Board for Health and Social Care in England for collection, storage, and use of personal data for surveillance purposes.From this source cohort, we defined patients with nosocomial pandemic (H1N1) 2009 as those admitted to a hospital for a reason other than acute respiratory infection in whom respiratory symptoms developed Of 1,520 patients in the FLU-CIN cohort, illnesses in 30 (2.0%) (15 children) met the criteria for nosocomial influenza (Twelve (80%) adults and 14 (93%) children had serious underlying illnesses. The most common illnesses were hematologic malignancy for adults (5), and congenital abnormality or prematurity (7) or malignancy (4) for children.Of the 15 adults, 2 had been admitted for elective surgical procedures; 1 for emergency surgery; and 8 for deterioration of chronic conditions, including complications caused by chemotherapy, malignancy, or transplantation. Two patients were admitted for pancreatitis (1 of whom had underlying myeloma); 1 patient was admitted for obstetric complications, and another patient was admitted for psoriasis. Of 15 children, 3 were admitted for elective procedures, 6 had been in the hospital since birth , 1 was transferred from another hospital, and 5 had acute conditions .None of the patients had received pandemic influenza vaccine. Although 14 adults were eligible because of concurrent conditions, influenza symptoms developed in 11 either before vaccine became available or before they would have seroconverted if vaccinated at the earliest opportunity (vaccine became available in the United Kingdom at the end of October 2009). Four children were eligible because of age and concurrent conditions, and symptoms developed in 3 before vaccine became available or before they would have seroconverted. Only 2 patients (both adults) had received seasonal influenza vaccine.Pseudomonas aeruginosa in an unspecified intravenous line in 1 patient. Twelve patients received antibacterial drugs during their respiratory illness, 2 of whom had Haemophilus influenzae in sputum samples and 1 (co-infected with rhinovirus) who had had a blood culture positive for Klebsiella sp.Twenty-one (72%) of 29 patients (10 children) received antiviral medication as inpatients (data were unknown for 1 patient); all initially received oseltamivir as monotherapy. Therapy for 2 patients (nos. 13 and 18) was switched from oseltamivir to zanamivir after 4 days and 10 days, respectively, because drug-resistant virus carrying the H275Y mutation was identified. Administration of antiviral drugs ranged from 0 to 8 days after symptom onset; 12 (57%) of 21 patients who received therapy did so within 48 hours. Sixteen patients were already receiving antibacterial drugs when influenza symptoms began. Two of these patients had a bacterial co-infection: coagulase-negative staphylococci in a blood culture for 1 patient and The most common signs were fever (8 [53%] adults and 12 [80%] children), cough , coryza , and dyspnea (7). Fewer patients had malaise (4); myalgia (3); anorexia, nausea, diarrhea (2 each); and arthralgia, sore throat, headache, vomiting, altered consciousness, sneezing, and rash (a child) (1 each).Median length of hospitalization before onset of influenza symptoms was 11 days for adults (range 4–78 days) and 13 days (range 6–54 days) for children, excluding infants in a hospital since birth. For infants in a hospital since birth, the interval from birth to onset of influenza signs ranged from 41 to 123 days (median 78 days).Results of chest radiography ≤3 days after onset of influenza symptoms were documented for 8 adults and 5 children (43%). Of these patients, 4 adults and 1 child (38%) had radiologically confirmed pneumonia.>2 organ systems. This level includes all patients with complex conditions requiring support for multiorgan failure (ICU).Level 0 is care given to patients whose care needs can be met through normal ward care. Level 1 care is given to patients at risk for a deteriorating condition or recently relocated from higher levels of care whose needs can be met in an acute-care ward with additional advice and support from the critical-care team. Level 2 care is given to patients requiring more detailed observation or intervention, including support for a single failing organ system and those changing from higher levels of care (high dependency unit). Level 3 care is given to patients requiring advanced respiratory support alone or basic respiratory support and support for Seven adults and 8 children (50%) required level 3 care . One (3%) adult required level 2 care. Six adults required mechanical ventilation and 1 required noninvasive ventilation (data for ventilatory support were unknown for 1 adult). Three children required mechanical ventilation and 1 required noninvasive ventilation. The remaining 4 children who received level 3 care were 3 infants and 1 child, each of whom required a period of close monitoring, but did not ultimately require ventilation. The remaining 7 adults and 7 children required level 0 or 1 care.<7 months, all remaining adults recovered from influenza and were discharged from the hospital.Five (33%) of 15 adults died in the acute-care hospital that provided treatment, 2 within 30 days after symptom onset. Of adults who died, 3 had underlying malignancy  or were immunocompromised and 2 had diabetes (type I and type II respectively). Pandemic (H1N1) 2009 was included in the recorded causes of death for all 5 adults. Although some patients had a prolonged hospital stay of Of 15 children, 3 (20%) were known to have died, although only 1  died at the hospital where surveillance was conducted; acute respiratory distress syndrome/lower respiratory tract infection was stated as a cause of death. Another child, with malignancy, whose death was expected, died at home shortly after discharge. The third child was transferred to another hospital, and cause of death is unknown. All other children recovered from pandemic (H1N1) 2009. Two children remained in the hospital for treatment of their underlying malignancy, and the other children were discharged.–,Although pandemic (H1N1) 2009 produced a generally mild illness, in the United Kingdom, as elsewhere, severe illness developed in a small proportion of relatively young patients who required hospitalization , many cases of nosocomial infection in other patient groups probably have been overlooked, particularly because influenza in these groups is likely to have been milder. Additionally, some patients are likely to have been discharged from a hospital during the incubation period of nosocomially acquired pandemic influenza. Thus, in this case series, detecting such patients would not have been possible.Nosocomial cases in this study occurred equally in adults and children. Consistent with previous findings  (Vaccine against pandemic (H1N1) 2009 became available at the end of October 2009. Assuming a 2-week period for vaccine administration, case-patients in groups at risk with influenza onset dates after November 30, 2009, could have been vaccinated and would have had time to seroconvert (14 days). Using these criteria, we determined that 4 cases (in 3 adults and 1 child) (13%) were potentially preventable by vaccination; 2 of these patients required escalated care, and 1 patient died. Although 72% of patients received antiviral therapy, similar to 75% in the source cohort  2009 in this case series were associated with high rates of illness and death. This finding highlights the need for adherence to infection control guidelines for staff and visitors , staff vaccination, maintenance of clinical suspicion for influenza in areas of high risk, prompt  antiviral treatment for vulnerable patients in whom influenza is possible or likely, and consideration of postponing nonurgent procedures for hematology patients during periods of known high influenza activity. This report demonstrates that nosocomial transmission is a recurrent problem when the prevalence of influenza is high and the total effect of nosocomial influenza is underestimated by outbreak reports alone.