This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10−10). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10−5). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions.Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique  Supplementary Material, Fig. S1) /v-akt murine thymoma viral oncogene homolog 1 (AKT) growth regulatory cascade  regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B’, beta (PPP2R5B), protein phosphatase 2, regulatory Subunit B’, gamma (PPP2R5C) and protein phosphatase 2, regulatory Subunit B’, delta (PPP2R5D).yndromes ,3. To fuSupplementary Material, Table S1) using Illumina exome enrichment kits and sequencers, performing alignment and variant calling with our in-house pipeline, identifying a total of 94 putative de novo coding variants. We first considered genes with more than one de novo mutation, amongst which was PPP2R5D (RefSeq accession number NM_006245), in which closely located de novo non-synonymous mutations, c.592G>A (p.Glu198Lys) and c.598G>A (p.Glu200Lys), were identified in two individuals ; PPP2R5B, PPP2R5C and protein phosphatase 2, regulatory Subunit B’, epsilon(PPP2R5E), which encode B56α, B56β, B56γ and B56ε, respectively. Given our initial findings, we reviewed the exome data at the other PP2A-B56 subunit genes, which revealed de novo mutations in PPP2R5B  and PPP2R5C  in a further two individuals. Notably, these mutations were at similarly positioned residues to those identified in PPP2R5D . Additionally, none of the mutations were present in 1000 population controls (the ICR1000 series) in which exome sequencing had been performed using the same laboratory and analytical pipelines . Furthermore, the identification of 4 de novo mutations in 3 closely related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates .C>T p.Ser61Leu; RePPP2R5B, PPP2R5C and PPP2R5D in a separate series of 152 individuals with overgrowth phenotypes for whom parental DNA is not available . We identified one individual with PPP2R5D p.Glu200Lys, which is likely causative of their phenotype as the identical mutation had occurred de novo in one of the trio series . Three other rare non-synonymous variants were detected, but in the absence of additional evidence, a causal relationship with overgrowth cannot be inferred for these variants .We next analysed exome data at P = 1.6 × 10−5; Fig. We undertook structural analysis to gain insights into the possible impact of the mutations we identified in overgrowth cases on PP2A-B56 function. The mutations were all located within a highly conserved region of the PP2A-B56 subunits; indeed, they clustered within a nine amino acid region in intra-repeat loop 2 . The facial features were not distinctive . To our knowledge, de novo mutations in PPP2R5B and PPP2R5C have not been reported.We next evaluated the phenotypic data for individuals with pathogenic PP2A-B56 subunit mutations . Foremost amongst these is phosphatase and tensin holog (PTEN), inactivating mutations of which cause increased head circumference, a core feature of all the PTEN-associated syndromes, such as Bannayan Riley Ruvalcaba syndrome (MIM 153480) and Cowden syndrome (MIM 158350)  mutations and germline activating phosphoinositide-3-kinase, regulatory subunit 2 (beta) (PIK3R2) mutations (both encoding PI3K regulatory subunits) cause conditions characterized by increased head circumference; megalencephaly-capillary malformation (MCAP [MIM 602501]) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH [MIM 603387]) syndromes, respectively  (AKT1 cause Proteus syndrome (MIM 176920), a highly variable disorder of asymmetric overgrowth, tissue hyperplasia affecting multiple organs and increased tumour risk   . In addiectively . Activat615937]) . FinallyFig. S1) . These fSupplementary Material, Table S1. A full list of collaborators is given in the Supplementary Material.We recruited individuals with overgrowth through the COG study. The research was approved by the London Multicentre Ethics Committee (reference MREC/01/2/44), and informed consent was obtained from all participants and/or families. We extracted DNA from peripheral blood. We obtained phenotypic information through a standardized questionnaire. A summary of the overgrowth phenotypes used in this study is given in http://www2.le.ac.uk/projects/birthcohort), a continuing follow-up study of individuals born in the UK in 1 week in 1958. The ICR1000 series [Accession number: EGAS00001000971] is accessible from the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/datasets/EGAD00001001021).We used lymphocyte DNA from 1000 population-based controls obtained from the 1958 Birth Cohort Collection .We used Stampy (version 1.0.14) to map sequencing reads to the human reference genome (hg19). Duplicate reads were flagged using Picard version 1.60. We used Platypus version 0.1.5 to perform variant calling whereas annotation was performed by CAVA, a variant annotation tool written in Python that follows Human Genome Variation Society nomenclature and ensures consistent annotation of indels . Sequences were analysed using Mutation Surveyor software v3.20 (SoftGenetics) and verified by manual inspection.We used Sanger sequencing of M13 tagged-PCR products from genomic DNA to validate all variants identified in probands by exome sequencing and to confirm the absence in parental samples as appropriate. Primer hybridisation sequences used for PCR are given in PPP2R5B), NM_001161725 (PPP2R5C) and NM_006245 (PPP2R5D).Mutation positions were assigned according to the following human mRNA reference sequences: NM_006244  in complex with Shugoshin-like 1 (Sgo1) (Protein Data Bank accession code 3FGA) . Images HMG online.Supplementary Material is available at Wellcome Trust Award100210/Z/12/Z.I.W. and R.v.M. are supported by Cancer Research UK program grant C309/A11566 and The Institute of Cancer Research. Access to these resources was enabled via the 58READIE Project funded by Wellcome Trust and Medical Research Council (grant numbers WT095219MA and G1001799). N.R. acknowledges support from the NIHR RM/ICR Biomedical Research Centre. Funding to pay the Open Access publication charges for this article was provided by Wellcome Trust.This work was supported by


Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. The serine-threonine protein phosphatase, protein phosphatase 2A (PP2A), is constitutively expressed in all eukaryotes. This phosphatase is implicated in diverse functions such as metabolism, DNA replication, transcription, RNA splicing, translation, cell-cycle progression, morphogenesis, development, and transformation ,2. Protea, a substrate which was used to measure its catalytic activity [v-scr transfected fibroblasts, p56lck from transformed T cells, and the receptor kinases, for epidermal growth factor and insulin [The catalytic subunit of PP2A (PP2A-C) can be covalently modified at the C-terminus by carboxymethylation  and tyroactivity . Howeveractivity . Phosphoactivity ,14. Ther insulin . Reversi insulin ,16.in vitro. Sperm motility initiation and fertilizing ability develop during their passage through the epididymis [Testicular spermatozoa in mammals are immotile and lack the ability to fertilize eggs ididymis . Despiteididymis ,19. It iididymis –22. A roididymis ,24. Inclididymis .We have shown that the testis isoform of protein phosphatase 1, PP1γ2, is essential for spermatogenesis and is also involved in sperm motility regulation ,24. Immo4) [8 sperm/ml by resuspension in homogenization buffer with protease inhibitors  β-mercaptoethanol and 1 mM sodium orthovanadate). The sperm suspension was sonicated on ice with three 10-sec bursts of a Microson ultrasonic cell disruptor (Misonix Inc.) set at level three. The suspension was then centrifuged at 16,000 x g for 20 min. at 4°C. The supernatants were supplemented with 5% glycerol and stored at -20°C.Mature bull testis were obtained from Duma Meats, a local commercial slaughter house within couple of hours of sacrifice. Spermatozoa were isolated from proximal caput, distal caput and caudal epididymis and washed twice in Hepes buffer, pH 7.0  . Sperm pMicrocystin-agarose beads (Upstate) were washed two times with an equal volume of HB+ buffer supplemented with 5% BSA to prevent nonspecific binding. The Microcystin-agarose beads were mixed with 100 μl sperm extract at 4°C with rotation overnight. Following this incubation the flow-through (unbound) fraction was collected by centrifugation. The bead pellet (bound PP2A) was washed by centrifugation twice with TTBS  and two times with HB+ buffer. The flow-through and pellet fractions were boiled with Laemmli sample buffer and stored at -20°C for Western blot analysis.For alkali mediated PP2A demethylation, PP2A in sperm extracts was first bound to microcystin. A 10 μl volume of microcystin-agarose bead slurry (prepared as described above) was incubated with sperm extracts prepared from the proximal caput, distal caput and caudal regions of the epididymis. The extracts were incubated for 150 min, washed in TTBS wash buffer  twice and in HB+ once. The washed microcystin-agarose beads were then resuspended in 30μl of HB+. Microcystin bound PP2A from each of these three regions was divided into two equal aliquots; one part was treated with 0.2 M NaOH while the other was left untreated as control. After 8 minutes at 4°C the alkali was neutralized with 0.2 M Tris-HCl (pH 6.8). An equal volume of neutralized alkali was added to the control suspension . MicrocySperm extracts, denatured by boiling for 3 min in Laemmli sample buffer, were separated by SDS-gel electrophoresis using 12% polyacrylamide slab gels in a Mini-Protean II system (Bio-Rad Laboratories). After electrophoresis, proteins were electrophoretically transferred to Immobilon-P, PVDF membrane (Millipore Corp). Nonspecific protein binding sites on the membrane were blocked with 5% nonfat dry milk in TTBS . The blots were washed with TTBS and then incubated overnight with primary antibody at 4°C. The following primary antibodies were used during this study: PP2A-C demethyl-sensitive antibody 4b7  (1:2000), anti-PP2A-C  (1:2000), anti-PP2A-C  (1:1000), anti-phospho-PP2A-C Y307  (1:500), anti-PP1γ2  (1:5000) , anti-PMa was used as a substrate to measure catalytic activity of PP2A [32P released from phosphorylase a. Enzyme activity is expressed in mmol of PO4 released/minute/2x105 sperm.Radio-labeled phosphorylase  of PP2A . The sub7 sperm/ml. Motility was analyzed on 20 μm capillary microscope slides by the computer assisted analyzer (CASA) of Hamilton Thorne Biosciences installed with the CEROS version 12.2g software. Three random fields were chosen for each sample and motility was recorded for 90 frames at 60 frames per second. The data was analyzed with the following settings: minimum contrast 30, minimum cell size 4 pixels, static cell size 8 pixels, static cell intensity 60, low size gate 0.17, high size gate 2.26, low intensity gate 0.35, high intensity gate 1.84, minimum static elongation gate 0, maximum static elongation gate 90, minimum VAP 50μ/s, minimum STR 50%, VAP cut off 10μ/s and VSL cut off 10μ/s.Sperm from the caudal epididymis were isolated and washed twice by centrifugation with HEPES buffer. Sperm were then incubated at 37°C in HEPES containing 20mM glucose and 20% BSA for control measurements and along with1 mM L-homocysteine in addition to 1 mM adenosine to elevate intracellular S-adenosylhomocysteine (SAH). For motility determination, samples were taken from sperm preparations at a concentration of 2X10Western blot analysis of soluble extracts of sperm isolated from proximal caput, distal caput, and caudal regions of the epididymis when probed with anti-PP2A antibody each showed one immunoreactive band at ~36 kDa, presumably due to PP2A. There was equal PP2A immunoreactivity in the three extracts obtained from each region . Little Since PP2A, in somatic cells is known to undergo tyrosine phosphorylation , we nextData in Though demethyl PP2A antibodies are well characterized, we sought to determine by an independent method that sperm PP2A methylation is different in the three regions of the epididymis. Carboxy-methyl groups in proteins can be removed by sodium hydroxide treatment ,31. PP2AThe enzyme PP2A methyl transferase, LCMT1 responsible for transferring the methyl group from S-adenosyl methionine specifically to PP2A is detected by Western blot analysis of mouse testis and sperm extracts probed with Anti-LCMT1 antibody at ~38Kd . HoweverS-Adenosyl-L-methionine (SAM) is a universal methyl donor in methylation reactions. Following transfer of its methyl group SAM is converted to S-adenosylhomocysteine (SAH). Cellular SAH is broken down to homocysteine and adenosine by the enzyme SAH hydrolase. Homocysteine and adenosine are further metabolized or transported out of the cell. Elevated intracellular SAH is a feedback inhibitor of methyl transferases preventing methylation reactions. Incubation of cells including spermatozoa with adenosine and homocysteine results in increased intracellular SAH levels. L-homocysteine by itself also increases the intracellular SAH levels but to a lesser extent than when adenosine is also present .8 /ml) were incubated at 37°C with 1 mM L-homocysteine with 1mM adenosine, 1 mM L-homocysteine alone, or 1 mM adenosine alone for 10 min in a suspension buffer containing glucose and BSA. Western blot analysis of extracts of sperm following this incubation is shown in Caudal spermatozoa  inhibits PP1 (IC50 of 1 nM) but not PP2A [4 released/minute/2x105 sperm, respectively. As seen in We measured catalytic activity of PP2A in sperm extracts using phosphorylase and PP2A . The linand PP2A ,24. Simiand PP2A . Focusinnot PP2A . PhosphaNext we examined if demethylation of PP2A or PP2A inhibition could initiate sperm motility in distal caput sperm. Distal caput sperm were treated with 1 mM L-homocysteine and 1 mM adenosine to demethylate PP2A or 5 nM okadaic acid to inhibit PP2A. These treatments could not initiate motility in immotile caput epididymal sperm even when L-homocysteine and adenosine concentrations were increased to 10 mM and up to four hours of incubation. However, as previously reported, higher concentration of the okadaic acid (1 uM), a concentration that inhibits both PP1 and PP2A was able to initiate motility in immotile distal caput sperm .°. Motility was quantified with a Computer Assisted Sperm motility Analyzer (CASA) system. For both the L-homocysteine and adenosine- treated and okadaic acid-treated sperm, all motility parameters were increased compared to control  phosphorylation is associated with sperm maturation and motility . It appeWe previously identified that PP2A is one of the two serine/threonine phosphatases in sperm. The other is PP1γ2, which is present both in the soluble and insoluble fractions of sperm sonicates . Here wein vivo and in vitro  and PP2A specific methyl esterase 1 (PME1). The fact that PP2A methylation changes tro Figs  also impin vivo, it is puzzling that the activity of PP2A is lowered, rather than increased, in caudal epididymal sperm following incubation of sperm with adenosine and homocysteine in vitro which results in demethylation of PP2A. It is possible that SAH is both an inhibitor of methyl transferase and of PP2A activity. It is interesting in this regard that pharmacological inhibition with okadaic acid also causes demethylation accompanied by increased tyrosine phosphorylation of PP2A  Sperm extracts from caudal regions of epididymi were prepared by sonication. The soluble fraction of the extracts was serially diluted and analyzed for phosphatase activity with phosphorylase a as the substrate. A 4ul sperm extract aliquot initially containing 4X106 sperm were serially diluted to 0.25X106 sperm. The combined activity of PP1 and PP2A denoted in mmol/min decreases in a linear fashion with decreasing sperm numbers in the extract.  A caudal sperm concentration of 2x106/ml was further used to measure phosphatase activity at various time points with intervals of approximately 3mins. The total phosphatase activity shown in counts per minute (CPM) increases linearly with time. The 10 minute time point was used in all our experiments.(TIF)Click here for additional data file.S2 File without any treatment (Control),  treated with 1mM L-homocysteine and adenosine (L-Hcy + Ado) and  5 nM okadaic acid (OA) after 10 minutes of incubation at 37°C. Sperm treated with 1mM L-homocysteine and adenosine or 5nM okadaic acid show increased percentage motility, velocity and lateral head amplitude.CASA recordings of Bovine sperm in HEPES buffer supplemented with glucose and BSA (WMV)Click here for additional data file.


The direct effect of antigenic site mutations in influenza viruses on antigenic drift and vaccine effectiveness is poorly understood.To investigate the genetic and antigenic characteristics of human influenza A (H3N2) viruses circulating in Ontario during the early 2010–2011 winter season.We sequenced the hemagglutinin (HA) and neuraminidase (NA) genes from 41 A(H3N2) viruses detected in nasopharyngeal specimens. Strain typing was performed by hemagglutination inhibition (HI) assay. Molecular and phylogenetic tree analyses were conducted.HA and NA genes showed high similarity to the 2010–2011 vaccine strain, A/Perth/16/2009 (H3N2)-like virus (97·7–98·5% and 98·7–99·5% amino acid (AA) identity, respectively). Compared to A/Perth/16/2009 strain, HA gene mutations were documented at 28 different AA positions across all five H3 antigenic sites, with a range of 5–11 mutations in individual viruses. Thirty-six (88%) viruses had 8 AA substitutions in common; none of these had reduced HI titer. Among Ontario isolates, 11 antigenic site AAs were positively selected with an increase in glycosylation sites.The presence of antigenic site mutations with high frequency among 2010–2011 influenza H3N2 isolates confirms ongoing adaptive H3N2 evolution. These may represent early phylogenetic changes that could cause antigenic drift with further mutations. Clinical relevance of antigenic site mutations not causing drift in HI assays is unknown and requires further investigation. In addition, viral sequencing information will assist with vaccine strain planning and may facilitate early detection of vaccine escape. Influenza viruses are considered one of the most common causes of respiratory infection among humans.4Hemagglutinin is known to be a major target region of neutralizing antibodies which inhibit binding with sialic acid receptors effectively.Forty-one H3N2-positive samples were included in this study, which consisted of all early season samples and a random selection from later in the season. Real-time reverse transcription PCR (rRT-PCR) for influenza A and B was performed as an initial screen on samples from hospital (admitted patients only) and outbreak settings. Samples from ambulatory patients (office settings and emergency patients not admitted) underwent viral culture for respiratory viruses using rhesus monkey kidney cells [(RMK) ]. Following total nucleic acid extraction, overlapping primers were used to amplify four and three fragments encompassing the coding region of HA and neuraminidase (NA) gene, respectivelyA representative subset of Ontario's isolates was submitted to Canada's National Microbiology Laboratory for strain characterization by hemagglutination inhibition (HI) assay. This was performed using 4 hemagglutination units of virus, 0·5% v/v turkey red blood cells and post-infection ferret antisera against A/Perth/16/2009. HI titers were defined as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination of 0·5% v/v turkey red blood cells; an eightfold reduction in titer compared to the reference strain was considered significant.21In addition to 41 HA sequences obtained from Ontario's isolates, the analysis was performed with an enhanced dataset. A total of 1072 HA protein sequences of H3N2 (August 2010–January 2011) viruses of different circulating strains from Africa (73), Asia (372), Europe (133), Oceania (85), South America (60) North America , and Canada  were downloaded from the GISAID  database.HA and NA study sequences have been submitted to GenBank under accession numbers JQ658888 to JQ658927 and JQ658928 to JQ658967, respectively.HA1 sequence assembly was carried out using Vector NTI ContigExpress . BioEdit was used for raw sequence curation and for multiple alignments of protein and nucleotide sequences.mega 4.0) using the neighbor-joining method algorithm.dendroscope version 2.2.1.14A HA1 phylogenetic tree of Ontario's strains and a selection of global sequences were generated with Molecular Evolutionary Genetic Analysis (P > 95%) are inferred to be under positive selection.To assess whether Ontario HA1 of H3N2 virus underwent positive selection when compared to MDCK-grown A/Perth/16/2009 (GenBank accession # GQ293081.1), we assessed the site-specific dN/dS and likelihood-ratio tests (LRT) using phylogenetic analysis using maximum likelihood (PAML 4.4 version).Potential N-linked glycosylation sites were predicted using NetNGlyc 1.0 Server.pymol.17Three-dimensional structures of the HA proteins of representative Ontario isolates were compared with A/Perth/16/2009 and constructed using related crystal structures  using This study was exempt from The University of Toronto's Health Sciences Research Ethics Board review as it involved deidentified respiratory tract samples that were tested as part of a clinical virology service provided by Public Health Ontario Laboratories. All test-positive samples and a proportion of test-negative samples are stored for possible further laboratory-based surveillance work. Samples and isolates included in this study were analyzed as part of the routine respiratory viral molecular surveillance program that supports Ontario's Ministry of Health and Long-Term Care.Complete HA and NA sequences of the 41 Ontario H3N2 samples were analyzed and compared against vaccine strains and were shown to be most closely related to the 2010–2011 seasonal vaccine H3N2 strain, A/Perth/16/2009, with 97·8–99·0% and 98·0–98·7% identity at the nucleotide level, respectively. Identity at the AA level was 97·7–98·5% and 98·7–99·5%, respectively.Comparative analysis with A/Perth/16/2009 revealed a mean of 8·7 (range 5–11) mutations across all antigenic sites. Thirty-six (88%) Ontario isolates had eight AA antigenic site substitutions in common. These included K144N at antigenic site A, D53N and E280A at site C, T212A, S214I, and I230V at site D, and K62E and Y94H at site E. In addition, 15 (37%) Ontario isolates had a unique substitution of isoleucine by methionine at residue 140 in antigenic site A, not observed among other Canadian or global isolates. Several other mutations at antigenic sites were observed among Ontario's strains such as T48A, E50K/G, N312S, and A304T (site C); S146G and M168I (site A); R208K V213A, I242K (site D); I260M, R261Q, Q75H, and K92R (site E); and S199A, F159Y, and I192T (site B). Mutations outside of antigenic sites also documented were as follows: T10P, P162S, R220K, N225D, D291E, and V323I. AA residues at the HA receptor binding sites remained conserved among all isolates except two, A/Ontario/C728447/2011 and A/Ontario/C76206/2011, harboring N133D and N225D, respectively . When thThe HA phylogenetic tree showed that 38 (93%) isolates fell within the recently emerged Victoria/208/2009 clade, and the remaining 3 (7%) belong to the Perth/16/2009 clade Figure .The NA gene phylogenetic tree showed the same distribution pattern as HA. Compared with the NA gene of A/Perth/16/2009, 38 (92%) of Ontario's isolates carried the substitution of serine (S) by asparagine (N) at position 367 (S367N), lysine (K) by threonine (T) at position 369 (K369T), and isoleucine (I) by leucine (L) at position 464 (I464L). Less frequent non-synonymous mutations that include E41N, M34V I62M, D127N, T138A, I176V, R210K, I307M, L338F, N342D, I381M, N402D, K415R, and R430S were observed among 2–4% of isolates. No mutations were observed at any of eight catalytic residues, which contact the NA inhibitor, oseltamivir, directly or among the 11 framework residues responsible for stabilizing the active binding site.20The nine putative N-glycosylation sites (Asn-Xaa-Ser/Thr) predicted at AA positions 8, 22, 38, 63, 126, 133, 165, 246, and 285 in A/Perth/16/2009 were identified in all of Ontario's 2010–2011 isolates. In addition, 38 (93%) isolates were shown to possess an additional glycosylation site (NNS) due to a K144N substitution. This K144N substitution was also observed among 72·2% of global isolates [range 42% to 97% of global isolates ].Eighteen representative H3N2 isolates were submitted for HI assay; all were antigenically related to the A/Perth/16/2009. Only one strain, A/Ontario/U10611/2010, showed eightfold reduction in HI titer (HI titer 80) when compared with A/Perth/16/2009 (HI titer 640). This strain exhibited four mutations at antigenic sites A (AA 144), D (AA 212 and 214), and E (AA 62). Three additional HA mutations, D291, K468T, and D489N, were exclusive to this isolate; there were no significant neuraminidase mutations.The remaining 17 isolates were antigenically similar to A/Perth/16/2009. Eleven percent and 17% of isolates exhibited non-significant fourfold and twofold reductions, respectively. Three of the 10 isolates with a mutation (I192T) at antigenic site B were tested by HI; all showed non-significant twofold to fourfold HI titer reductions. The remaining seven I192T isolates were not of adequate HI titer to characterize. No correlation was observed between the presence of 10AA mutations across four antigenic sites  and HI titers  and I192T  when compared with global isolates; these mutations were only present in 11 (0·96%) and 37 (3·2%) global isolates analyzed, respectively. Five  of 11 positively selected sites were conserved in the isolates from the Americas (North and South America). The only substitution conserved among all global isolates irrespective of their origin was S214I .In this study, we have characterized seasonal clusters of Influenza A(H3N2) circulating during the 2010–2011 winter season in Ontario. Nucleotide analysis of the complete coding region of these isolates showed close similarity to the vaccine strain, A/Perth/16/2009. HA nucleotide identity was higher than the NA identity when compared with A/Perth/16/2009. However, HA had a lower AA identity with the A/Perth/16/2009. This implies that nucleotide mutations in HA are more likely to be non-synonymous than those occurring in NA, in keeping with the propensity of this protein to evolve at a higher rate.Co-circulation of variants distinguished by specific AA substitutions was seen among Ontario's H3N2 strain. Phylogenetic trees of the HA and NA genes show that Ontario's 2010–11 isolates fell into two distinct genetic clades represented by A/Perth/16/2009 and A/Victoria/208/2009, with the majority (93%) belonging to Victoria 208 clade. Several phylogenetic subgroups within the A/Victoria/208/2009 clade were identified, with the majority (86%) of Ontario's isolates falling within the newly emerged subclade, A/Hong Kong/2121/2010. Due to the emergence of the Victoria 208 clade of A/Perth/16/2009 strain, WHO recommended a change to the H3N2 strain component of the 2012–2013 influenza vaccine, to A/Victoria/361/2011-like virus.21Evolutionary analysis of the HA of Ontario's 2010–2011 H3N2 isolates revealed strong evolutionary selection pressure (dN/dS = 6·8), resulting in 11 PSS compared to A/Perth/16/2009 . Three oAll of Ontario's 2010–2011 isolates possessed the same nine glycosylation sites identified in A/Perth/16/2009. Thirty-eight (93%) of Ontario's viruses were shown to possess an additional glycosylation site (NNS) due to a K144N substitution, which also resulted in an AA change in antigenic site A. This mutation was also common among global isolates . A posit5A substitution from isoleucine to methionine at position 140 was observed in 37% of Ontario's 2010–2011 isolates, but was only found in 0·96% of other global 2010 to 2011 isolates. I140M was previously detected in six A/Brisbane/10/2007-like H3N2 isolates collected in the USA in 2008  and 2009 . Substitutions D53N (in antigenic site C) and I140M (in antigenic site A) were found to decrease affinity of H3N2-specific antibodies based on microneutralization (MN) assay with A/Perth/16/2009 (H3N2) antiserum; however, their impact on immune recognition is not known.27Despite the presence of up to 11 mutations at four antigenic sites, considerable evidence for antigenic drift was not observed based on the data obtained by HI assay. This is in contrast to the previously held idea that four mutations across two or more antigenic sites would predict a propensity for antigenic drift.30The majority of the antigenic site mutations identified in this study appear at the surface structures of antigenic sites that are accessible to antibodies and resulted in conformational changes Figure . Such stIn conclusion, the presence of up to 11 positively selected antigenic site mutations with high frequency among 2010–2011 influenza season H3N2 isolates confirms the ongoing adaptive evolution of circulating H3N2 strains. Further work is needed to better understand the clinical relevance of mutations at antigenic sites that do not result in drift in HI assays. In addition, further investigation is needed to better understand the extent to which viral sequencing should influence vaccine strain selection, even when there is no associated drift in HI assays. Relying on detection of antigenic drift in HI assays before altering candidate vaccine strains may not be sensitive enough to detect clinically relevant changes in vaccine effectiveness.


We have previously reported that increased glucose levels were associated with higher serum nitric oxide (NO) levels in fructose-fed insulin resistant rats. However, the relationship between hyperglycemia and serum NO level was not clear. Therefore, the present study was designed to find the association between hyperglycemia and serum NO levels in Type 2 diabetic (T2DM) patients and T2DM with cardiovascular complication.Endothelial cells (HUVEC) were treated with of D-glucose (10-100mM), and NO levels and NOS gene expression was measured. Hyperglycaemia was induced in Sprague-Dawley rats, and serum NO levels were measured after 8 weeks. For clinical evaluation, five groups of patients were recruited: Control , Type 2 diabetes , T2DM with hypertension , Coronary artery diseases  and T2DM with coronary artery diseases . NO (nitrite + nitrate) levels were measured from human serum.We found a significant (p<0.05) and dose-dependent increase in NO levels in HUVEC cells after 4 hours of high glucose exposure. eNOS and iNOS gene expression was increased in HUVEC cells after different concentrations and time periods of glucose treatment. We also observed significant  increase in serum NO levels in hyperglycaemic rats compared to control (76.6±13.2μM). Serum NO level was significantly higher in T2DM (111.8 μM (81.7-122.4), p<0.001) and DMCD patients ((129.4 μM (121.2-143.5), p <0.001) but not in CAD patients (76.4 μM (70.5-87)), as compared to control (68.2 μM (56.4-82.3)). We found significantly lower NO levels (83.5 μM (60.5-122.9)) in subjects suffering from diabetes since more than 5 years, compared to subjects (115.3 μM (75.2-127.1), p<0.001) with less than 5 years.In conclusion, high NO levels were observed in South Indian diabetic patients. Higher glucose levels in serum might be responsible for activation of endothelial cells to enhance NO levels. Metabolic syndrome is a serious health concern. In United States, 35% of adults are at high risk of developing cardiovascular diseases, diabetes, stroke, atherosclerosis and coronary artery disease . The gloDiabetes is a metabolic disorder characterised by chronic hyperglycaemia. The long-term effects of diabetes mellitus include cellular injury, inflammation and failure of various organs . The comNitric oxide is a gaseous molecule secreted by the endothelium and a major modulator of endothelial function . NO is sTherefore, the present study was designed to understand the alteration in nitric oxide levels with T2DM and T2DM with cardiovascular complications in South Indian patients and to find whether hyperglycaemia can induce NO production in endothelial cells. Our study showed an increased level of nitric oxide in Indian T2DM patients, but not in patients with coronary artery disease (CAD) alone. Similarly, diabetic rats with hyperglycaemia also showed higher NO levels as compared to controls. Our study indicated that hyperglycaemia is responsible in generation of high levels of NO from HUVEC cells through induction of iNOS and eNOS gene expression.Sample of 189 patients, including men and women aged 35–65 years, was taken from the Mediciti Hospital, Hyderabad, a city from southern part of India.Group 1: Control (CT) subjects (n = 50)Group 2: Type 2 diabetes mellitus (T2DM) subjects (n = 26)Group 3: Type 2 diabetes with hypertension (DMHT) subjects (n = 46)Group 4: Coronary artery disease (CAD) subjects (n = 29)Group 5: Coronary artery disease with Type 2 diabetes mellitus (DMCD) subjects (n = 38).Subjects were allocated into five study groups mentioned below:The study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Mediciti Ethics Committee  Hyderabad. All patients were given detailed information of the study and they gave written consent before enrolling into the study.Group 1 Control (CT) subjects had no prior history of T2DM, hypertension, coronary artery diseases or any other cardiovascular diseases, and were not taking medication for any chronic medical condition. Fasting blood glucose, HbA1c and blood chemistry were normal. Group 2 (T2DM) included subjects with HbA1c levels ≥ 6.5% as per American Diabetes Association (ADA) guidelines with proven history of T2DM but no other complications. Group 3 (DMHT) included subjects with HbA1c levels ≥6.5% and systolic blood pressure ≥140 mm of Hg and diastolic blood pressure ≥90 mm of Hg, with history of T2DM as well as hypertension. Group 4 (CAD) included subjects with narrowing or blockage of one or more epicardial coronary artery with greater than 25% stenosis shown in coronary angiography and diagnosed by cardiologists. This group had no prior history of T2DM. Group 5 (DMCD) included subjects with coronary artery disease as defined for group 4 but patient had HbA1c levels ≥6.5% and prior history of T2DM. Exclusion criteria defined for this study were clinical or laboratory evidence of liver failure, renal failure (plasma creatinine levels > 1.5 mg/dl), Type 1 diabetes, cancer, thyroid disease and pregnancy.Coronary artery disease patients were identified in the Cardiac Catheterization Unit of Mediciti hospital. After coronary angiogram, all patients were evaluated by cardiologists in the inpatient setting. If patients were found to have any evidence of CAD, demographic, clinical, and angiographic data were collected from all such patients. Fasting sample were collected prior to the percutaneous coronary intervention or coronary artery bypass graft.Clinical history and complete physical examination including measurements of blood pressure, was collected and conducted for all the participants. Blood samples were collected by venipuncture after an overnight fast, using Becton Dickison Vacutainer Red colour coded Tubes and blood was allowed to clot by leaving at room temperature for 15 min. After centrifugation at 1500 g for 15 min at 4°C, serum was collected in 1.2 ml cryo vials and immediately stored at -80°C until testing.2)]. Fasting blood glucose levels were measured by the FreeStyle optimum glucometer . HbA1c levels were measured by the Bayer A1C Now+ Multi test A1C system . Creatinine (Siemens #DF33A) and uric acid (Siemens #BA4007) were measured by Siemens automated analyser (Dimension Xpand plus), estimated Glomerular Filtration Rate (eGFR) calculated from creatinine by using Modification of Diet in Renal Disease (MDRD) formula. Serum Insulin and C-peptide levels were measured by Bio-Rad Multiplex assay kit . Assay was performed as per the manufacturer instructions.Height and weight were obtained using standardised techniques and instruments. The body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters  NO detection by fluorescent dye DAF 2 DA in HUVEC cells after treated with D-Glucose  for 4 hrs (20X magnification). B] NO levels produced from HUVEC cells after 4hrs treatment of D-Glucose . Data were represented as ± SEM. * p<0.05,** p<0.01,*** p<0.001 vs Control (CT).(DOCX)Click here for additional data file.S2 FigA] NO detection by fluorescent dye DAF 2 DA in HUVEC cells after treated with D-Glucose  for 8 hrs (20X magnification). B] NO levels produced from HUVEC cells after 8hrs treatment of D-Glucose . Data were represented as mean ± SEM. * p<0.05, *** p< 0.001 vs Control (CT).(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity.Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N Is79A3  to reduce unis Nm2 , have geunis Nm2 . Further. Is79A3  and N. ueae Nm10 . Both Niammonium . In contammonium .N. europaea, a eutrophic strain, showed that both hydroxylamine (NH2OH) oxidation and 2O  determine whether gene content, trophic state, and/or phylogeny of these diverse AOB were predictive of their capacity to metabolize and/or emit NO or N2O.Due to the lack of comparative information on nitrogen oxide metabolism in AOB, five strains representing different phylogenies and trophic states and with closed genomes were selected for this study. Our main objectives were to: (i) compare NO and NN. europaea ATCC 19718T, N. communis strain Nm2T, Nitrosomonas sp. Is79A3, N. ureae Nm10T, and N. multiformis ATCC 25196T. All strains have closed genomes and grow under similar cultivation conditions to allow for proper comparisons across phylotypes and trophic status. Furthermore, an AOB strain was selected from each cluster in the Betaproteobacteria with a cultured representative, 3, 6, 7, and 8 2SO4 for the meso- and eutrophic strains , or 2.5 mM (NH4)2SO4 for the oligotrophic strains , and rinsed three times with NH3-free HK media . Previous testing revealed that all strains could tolerate up to 250 μM NH2OH  with the exception of N. communis which was unable to tolerate more than 100 μM NH2OH  per injection (data not shown). Chamber O2 was determined by an O2 electrode , N2O concentration was measured using an N2O-500 N2O minisensor electrode with 500 μm tip diameter , and NO was measured using an ami-600 NO sensor with 600 μm tip diameter . The availability of O2 in the MR chamber, a closed system, was ca. 243 μM O2 based on equilibrium O2 concentration at operating temperatures and medium salinities for experiments performed without N2-sparged medium.Instantaneous microrespirometry (MR) experiments of AOB are described in detail elsewhere . BrieflyHK media . Ca. 1 ×2O from reactivity of NH2OH with media + 10 total cells) with media + NH2OH. Chemical controls used N2-sparged medium (to achieve 0–3% O2 saturation in liquid phase) containing 250 μM NaNO2 and then adding 250 μM NH2OH  to reflect conditions in the chamber when testing for 2OH as the electron donor. Cells for control experiments were heat-killed by boiling for 30 min. The heat-killed cell controls involved addition of 250 μM NH2OH to the MR-chamber containing N2-sparged media with 1 × 1010 total heat-killed cells of each AOB strain. N2O was measured as described above.Chemical controls were performed to determine the production of NBetaproteobacteria AOB chosen for physiological analysis in the present study separated into individual clades . The separation of each AOB into a unique branch, using 400 core protein markers from available complete genome sequences to form a high-resolution tree, shows a clearer and greater separation than currently available 16S rRNA or amoA single gene sequence phylogenies . In agreement with previous analyses of AMO gene clusters in betaproteobacterial AOB (amoCABED cluster encoding ammonia-monooxygenase (Table 1). All strains encoded at least one monocistronic copy of the amoC gene with the exception of N. communis (Table 1), a feature shared in common with the gammaproteobacerial AOB  copies of the haoAB-cycAB cluster (N. ureae represents the first sequenced AOB to harbor 4 complete copies of the Hydroxylamine dehydrogenase (HAO) gene cluster (Table 1). Knockouts of one or two haoA gene copies from N. europaea did not result in a significant phenotype , encoding cytochrome Cm552 (Nitrosomonas sp. Is79A3 (Table 1) proposed to be involved in the NH3-oxidation pathway as a redox sensitive electron carrier . This is interesting as nirK is present in all published genomes of ammonia-oxidizing Thaumarchaeota , features shared by the closely related N. eutropha C-91 strain . All the Nitrosomonas strains, but not N. multiformis, encode the NO-responsive NnrS transcriptional regulator. Two strains, Nitrosomonas sp. Is79A3 and N. ureae, both within the Cluster 6 AOB, lack annotated operons for cytochrome c nitric oxide reductases (Table 1). The genome of the closely related Nitrosomonas sp. AL212  does encode norCBQD but lacks genes for the other NOR frequently found in AOB genomes, norSY-senC-orf1  whereas N. communis has two copies (Table 1), a feature shared with Nitrosomonas sp. AL212 . Although each AOB had a unique and dynamic NO production profile, making comparative rate calculations impractical, all strains produced>50 nM NO  prior to anoxia in the MR chamber. N. europaea produced the least amount of NO compared to the other strains during active oxidation and prior to anoxia  and both N. europaea and N. communis re-consumed a small amount of NO following anoxia in the MR-chamber . Interestingly, either immediately upon O2 depletion in the case of Nitrosomonas sp. Is79A3  or ca. 5 min. post-anoxia for N. ureae, these two strains released massive quantities of NO outside the limit for measurement by the ami-600 NO microsensor . Unlike the other AOB strains, neither Nitrosomonas sp. Is79A3 nor N. ureae re-consumed NO during active NH3-oxidation or following anoxia in the MR-chamber.Measurement of NO or NN. europaea . It is known, however, that the thaumarchaeotal ammonia-oxidizers (AOA) also produce NO during NH3-oxidation . It should be noted that the Km for the copper-containing nitrite reductase, NirK, has not been tested for AOB and therefore it is not known whether ca. 162 or 243 μM 3 or NH2OH oxidation, respectively, in the chamber is at saturation for NirK.Following Ofication . This wa3-oxidation, N2O was produced by all strains in the MR-chamber . A greater delay of ca. 3 min in measureable N2O was seen from traces with both N. communis  and N. ureae . The lowest concentrations and slowest rates of N2O came from N. communis and N. multiformis , N. communis , and N. multiformis  was produced in a linear fashion directly following anoxia suggesting enzymatic reduction of available 2O and thus nitrifier denitrification. However, in the case of both Nitrosomonas sp. Is79A3  and N. ureae  the majority of N2O was measured during active NH2OH-oxidation with production in both traces slowing upon complete O2-depletion. Furthermore, the quantity of N2O measured from both Nitrosomonas sp. Is79A3 and N. ureae during active NH2OH-oxidation was much greater overall than that produced from any other AOB, suggesting a greater overall release of NO, or other reactive intermediates, during this process .With NH process  . The linearity of N2O formation with NH2OH as substrate suggests that there is an enzymatic pathway for N2O formation under anoxic conditions, but this pathway is not active when NH3 is provided as substrate. This observation provides insight into the function of unidentified enzymology that links direct NH2OH oxidation to N2O production in N. communis that requires further investigation. Similarly, an N. europaea NirK deficient mutant was also able to reduce 2O  are congruent with a rapid and abundant release of NO  being abiotically reduced to N2O, a characteristic trait observed in the AOA N. viennensis  is the observation that the majority of N2O was measured during active oxidation of NH2OH. Accumulation of NH2OH can lead to NO and N2O production at the active site of the HAO , especially under environmental conditions that facilitate NO or NH2OH release (2O release to the environment.The present study highlights many new findings in the comparative phylogeny and nitrogen oxide metabolism of betaproteobaterial AOB. First, the data support the previous study of activity . Second, release . This ob release . Third, JK and LS conceived the project; JK designed and performed all experiments, KK performed a PhyloPhlAn analysis and created the phylogenetic tree; JK, KK, and LS analyzed the data, JK and LS wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma brucei and a phenylalanine at the equivalent position to E. coli HslU is found in T. brucei HslU1. Unexpectedly, we found that an F441Y mutation in HslU enhanced the peptidase and caseinolytic activity of HslV in E. coli but it showed partially reduced ATPase and SulA degradation activity. Previously, only the C-terminal tail of HslU has been the focus of HslV activation studies. However, the Pro315 residue interacting with Phe441 in free HslU has also been found to be critical for HslV activation. Hence, our current biochemical analyses explore the importance of the loop region just before Pro315 for HslVU complex functionality. The proline and phenylalanine pair in prokaryotic HslU was replaced with the threonine and tyrosine pair from the functional eukaryotic HslU2. Sequence comparisons between multiple HslUs from three different biological kingdoms in combination with biochemical analysis of E. coli mutants have uncovered important new insights into the molecular evolutionary pathway of HslU.The ATP-dependent HslVU complexes are found in all three biological kingdoms. A single HslV protease exists in each species of prokaryotes, archaea, and eukaryotes, but two HslUs (HslU1 and HslU2) are present in the mitochondria of eukaryotes. Previously, a tyrosine residue at the C-terminal tail of HslU2 has been identified as a key determinant of HslV activation in  HslVU is a two-component ATP-dependent protease in which the ATPase HslU unfolds and translocates protein substrates and the HslV protease degrades the unfolded proteins T. brucei (TbU1 and TbU2) activates the HslV protease in this species (TbV) E. coli) located at the C-terminal tail which activates TbV. In most prokaryotic systems, the equivalent residue for this Tyr494 is a strictly conserved phenylalanine and eukaryotic HslU1s also possess a conserved phenylalanine at this position (E. coli (EcV) with a synthetic peptide containing tyrosine at the position 441. Surprisingly, the peptidase activity of EcV was markedly enhanced and we further performed a biochemical characterization of an F441Y mutant EcU. There is a proline residue at position 315 near Phe441 in the free HslU structure. This proline locates at the end of a loop (residues 312–314) facing HslV, which seems to be important for HslV interaction Recently, we reported that only one HslU (HslU2) of the two HslUs present in position . To diss5′-GAAGAAGATATACATATGACAATAGTAAGCGTACGCCGTAA-3′; 5′-TTACGGCGTACGCTTACTATTGTCATATGTATATCTCCTTC-3′), for P315T or P315T/F441Y  and for F441Y . Mutagenesis was confirmed by DNA sequencing. The expression and purification of ΔT1_EcV, P315T, F441Y, and P315T/F441Y was carried out as for wild-type EcV and EcU.The cloning, expression, and purification of EcV and EcU were performed as described previously E. coli BL21(DE3) ΔhslU::amp, ΔhslV:cm) using Red/ET Recombination methods and the pRedET vector E. coli ΔEcVU cells for overproduction of MBP-SulA in cultures grown at 25°C. The purification of MBP-SulA has been described previously For the efficient expression of MBP-SulA , we constructed an ΔEcVU strain (YIL).C-terminal octapeptides were synthesized by JPT Peptide Technologies GmbH and included wild-type (EDLSRFIL) and mutant forms  dimethylformamide. When EcU or its mutant enzymes and not a peptide were added as an activator for EcV, ATP was added in the reaction mixture. The release of 7-amido-4-methyl-coumarin (AMC), which is dependent on peptidase activity, was monitored as reported previously Peptidase was assayed using the chromogenic peptide, carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin  as a substrate Haemophilus influenzae as a template A molecular model of EcV-activation of EcU was generated using the structure of HslU in the HslVU complex from http://www.uniprot.org/). Sequence alignment and phylogenetic analyses were performed at standard settings using the Clustal Omega website . Tree reconstruction was obtained using the Neighbor-joining method and the tree visualization results from TreeView . The protein sequences used were as follows: UniProt ID P0A6H5 (Escherichia coli HslU), E7SX86 (Shigella boydii HslU), P0A6H7 (Shigella flexneri HslU), D4DZ19 (Serratia odorifera HslU), P43773 (Haemophilus influenza HslU), I3DSM1 (Haemophilus haemolyticus HslU), C9PP45 (Pasteurella dagmatis HslU), H8IG73 (Pasteurella multocida HslU), E8VAJ1 (Bacillus subtilis HslU), D3FT45 (Bacillus pseudofirmus HslU), Q9WYZ2 (Thermotoga maritime HslU), D2C619 (Thermotoga naphthophila HslU), F7YXR0 (Thermotoga thermarum HslU), H9UA01 (Fervidobacterium pennivorans HslU), A7HK26 (Fervidobacterium nodosum HslU), O66574 (Aquifex aeolicus HslU), C1DWL5 (Sulfurihydrogenibium azorense HslU), Q01Q20 (Solibacter usitatus HslU), A4H7X9 (Leishmania braziliensis HslU1), A4H5J8 (Leishmania braziliensis HslU2), E9BC50 (Leishmania donovani HslU1), E9BS7 (Leishmania donovani HslU2), S0CVC5 (Leishmania guyanensis HslU1), S0CS82 (Leishmania guyanensis HslU2), A4HWA6 (Leishmania infantum HslU1), Q8I0E1 (Leishmania infantum HslU2), Q4QFH5 (Leishmania major HslU1), Q4QI03 (Leishmania major HslU2), E9AQ06 (Leishmania Mexicana HslU1), E9AMM2 (Leishmania mexicana HslU2), Q57VB1 (Trypanasoma brucei HslU1), Q382V8 (Trypanosoma brucei HslU2), G0UMN2 (Trypanasoma congolense HslU1), G0V2B6 (Trypanasoma congolense HslU2), Q4DEP1 (Trypanasoma cruzi HslU1), and Q4DRN5 (Trypanasoma cruzi HslU2).BLAST searches were carried out on the UniProt website , has been reported to possess a dynamic nature There is an analogy here with the ClpXP system Trypanosoma brucei is known and this HslU2 forms a functional complex with HslV The ATP-dependent two-component HslVU proteases exist in all three biological kingdoms. The eukaryotic HslUs are classified into two groups, HslU1 and HslU2. Interestingly, the C-terminal tails of the HslU1s possess a phenylalanine at the equivalent position to prokaryotic HslUs. The importance of the tyrosine residue at this position in HslU2 from the mitochondria of an eukaryote,


Auto-inflammatory syndromes now include polygenic diseases, such as Behcet's syndrome and Still's disease; however, the best characterized auto-inflammatory diseases are relatively rare, but florid conditions arising from mutations in single genes. The prevalence of each auto-inflammatory disease varies depending on ethnic background.Auto-inflammatory syndromes are defined as conditions caused by an exaggerated innate immune system response, resulting in episodes of spontaneous inflammation affecting multiple organs. The prototypical auto-inflammatory disorders are associated with periodic febrile episodes. To analyze the prevalence of polymorphisms of the genes responsible for auto-inflammatory diseases managed in a single rheumatology institute in Japan.A total of 202 individuals < 40 years of age with recurrent febrile episodes were enrolled in this study. Recurrent fever was defined as > 2 episodes of fever > 38.5 degrees Celsius lasting > 3 days in a year. Infections, autoimmune disorders, and malignancies were excluded as causes of fever prior to enrollment. Genomic DNA was isolated from the patients’ peripheral blood and a polymerase chain reaction (PCR) was used to amplify the indicated exons of 10 genes , which have been reported as the genes responsible for auto-inflammatory diseases. After cleaning the PCR products, cycle sequencing was carried out using the Big Dye® Terminator v3.1 kit and analyzed with an ABI 3130xl Prism Genetic Analyzer. For the most frequently reported 4 genes, genetic polymorphisms within MEFV (exons 1-10), TNFRSF1A (exons 2-4), MVK (exons 9-11), and NLRP3 (exon 3) were examined. With respect to the other 6 genes, the existence of polymorphisms was also determined within NOD2 (from L248R to P727L), LI1RN (from N52KfsX25 to C91F), IL36RN (from R10X to G141Mfs*29), PSMB8 (from T75M to G201V), NALP12 (from T260M to F402L and R1016X), and PSTPIP1 (from A230T to E277D), with reference to the INFEVERS database, an evolving mutation database for auto-inflammatory syndromes (http://fmf.igh.cnrs.fr/ISSAID/infevers/index.php).Gene polymorphisms in the targeted genes were identified in 116 of the 202 patients (57%) based on INFEVERS. One hundred four of the 116 polymorphisms (90.0%) were associated with MEFV genes. Other polymorphisms were identified in TNFRSF1A (n=7), NLRP3 (n=5), NOD2 (n=4), MVK (n=2), and PSTPIP1 (n=1).Polymorphisms in MEFV were most frequently identified among Japanese patients with recurrent fevers.None declared.


T17M rhodopsin expression in rod photoreceptors leads to severe retinal degeneration and is associated with the activation of ER stress related Unfolded Protein Response (UPR) signaling. Here, we show a novel role of a UPR transcription factor, ATF4, in photoreceptor cellular pathology. We demonstrated a pro-death role for ATF4 overexpression during autosomal dominant retinitis pigmentosa (ADRP). Based on our results in ATF4 knockout mice and adeno-associated viral (AAV) delivery of ATF4 to the retina, we validated a novel therapeutic approach targeting ATF4 over the course of retinal degeneration. In T17M rhodopsin retinas, we observed ATF4 overexpression concomitantly with reduction of p62 and elevation of p53 levels. These molecular alterations, together with increased CHOP and caspase-3/7 activity, possibly contributed to the mechanism of photoreceptor cell loss. Conversely, ATF4 knockdown retarded retinal degeneration in 1-month-old T17M Rhodopsin mice and promoted photoreceptor survival, as measured by scotopic and photopic ERGs and photoreceptor nuclei row counts. Similarly, ATF4 knockdown also markedly delayed retinal degeneration in 3-month-old ADRP animals. This delay was accompanied by a dramatic decrease in UPR signaling, the launching of anti-oxidant defense, initiation of autophagy, and improvement of rhodopsin biosynthesis which together perhaps combat the cellular stress associated with T17M rhodopsin. Our data indicate that augmented ATF4 signals during retinal degeneration plays a cytotoxic role by triggering photoreceptor cell death. Future ADRP therapy regulating ATF4 expression can be developed to treat retinal degenerative disorders associated with activated UPR. RHO) gene, have become a valuable murine model for studying autosomal dominant retinitis pigmentosa (ADRP). Severe retinal degeneration is detected by one month in these animals, resulting in a significant loss of scotopic a- and b-wave ERG amplitudes and in compromised retinal integrity [RHO photoreceptors [RHO gene [T17M rhodopsin mice, carrying a mutant human rhodopsin  and T17M RHO+/- ATF4+/- (T17M ATF4+/-) carrying mutant and endogenous mouse rhodopsins. These mice were compared with control groups of C57BL6/J and ATF4+/- mice, expressing two copies of endogenous mouse rhodopsin. All the mice had the same genetic background as C57BL/6J mice. ATF4+/- mice were purchased from Jackson  and have a normal appearance, organ morphology and eye development.T17M RHO mice were created as described [All experiments with mice followed the animal protocol approved by the Animal Care and Use Committee of the University of Alabama at Birmingham, conforming to the Association for Research in Vision and Ophthalmology guidelines. All experimental procedures were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) protocol . Transgenic mice expressing human T17M rhodopsin, C57BL6/J, RHOescribed . All mic+/- mice was performed using forward primers: ATATTGCTGAAGAGCTTGGCGGC for Neo allele and AGCAAAACAAGACAGCAGCCACTA for wild type allele and a common reverse primer GTTTCTACAGCTTCCTCCACTCTT for both alleles. The T17M genotyping was performed as previously described [ER stress Activating Inducer) mice were used as previously described [The genotyping of ATF4escribed .13 genome particle per ml for both viruses) driven by the CMV enhancer-chicken β-actin (CBA) promoter in their right eye and GFP in their left eye, as previously described [Sub-retinal injections were performed in pups at postnatal day 15 with 1 μl of either AA2/5 virus (serotype 5) expressing the mouse ATF4 cDNA or GFP  and xylazine (10 mg/kg), and their pupils were dilated in dim red light with 2.5% phenylephrine hydrochloride ophthalmic solution . Scotopic ERGs were recorded using a wire contacting the corneal surface with 2.5% hypromellose ophthalmic demulcent solution (Akorn.Inc). ERG was performed at different light intensities (0 db (2.5 cd*s/m2), 5 db (7.91 cd*s/m2), 10 db (25 cd*s/m2), and 15 db (79.1 cd*s/ m2). Five scans were performed and averaged at different light intensities. Photopic, cone-mediated responses were performed following 10 min light adaptation on the background light intensity of 30 cd*s/m2. Recordings were obtained at the light intensity of 25 cd*s/m2. Fifteen waveforms from each animal were recorded and the values were averaged. The a-wave amplitudes were measured from the baseline to the peak in the cornea-negative direction, and the b-wave amplitudes were determined from the cornea-negative peak to the major cornea-positive peak. The signal was amplified, digitized, and stored using the LKC UTAS-3000 Diagnostic System .SD-OCT was performed in P30, P60 and P90 animals using the Spectral Domain Ophthalmic Imaging System (SDOIS) (Bioptigen). The mice were anesthetized. Horizontal volume scans through the area dorso-temporal from the optic nerve (superior retina) and the area ventro-temporal from the optic nerve (inferior retina) were used to evaluate the thickness of the ONL. For measuring the thickness of the ONL, six calibrated calipers were placed in each region of the superior and inferior hemispheres of retinas within 100, 200, 300 and 400 μm of the optic nerve head. The thickness of the ONL was determined by averaging six measurements.Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally, and the pupils were dilated using 2.5% phenylephrine hydrochloride ophthalmic solution . The fundus was examined using the Micron IV camera  with StreamPix 5 software in fluorescent modes. GFP fluorescence was detectable using green fluorescent barrier filters.Mouse eyes were enucleated at 1 and 3 months of age and were fixed overnight in 4% paraformaldehyde freshly made in phosphate-buffered saline (PBS) as previously described . DigitalFor immunohistochemistry retinal cryosections were rinsed in PBS and blocked in 2% normal goat serum, 0.3% Triton X-100 in 0.01% BSA in PBS for 1 hour at room temperature. The sections were then stained with primary anti-rhodopsin antibodies  which were diluted in PBS with 0.1% Triton X-100 and 1% BSA and incubated overnight at 4°C. The Cy2-labeled anti-IgG secondary antibody  diluted 1:500 in PBS was applied at room temperature for 1 hour. Sections were mounted with Vectashield Mounting Medium (Vector Lab) and cover slipped. Images were taken using a confocal microscope .For staining retinas for reactive oxygen species, 12-micron sections were incubated with a general oxidative stress indicator, CM-H2DCFDA, Invitrogen (10 μM/L in PBS) in the dark for 1 hour. Sections were washed three times and were mounted and coverslip with Vectashield Mounting Medium. The 485nM fluorescence filter was used to take images. Images were converted to black and white to improve contrast.+/-, T17M and T17M ATF4+/-) by surgical excision. Total RNA was extracted using the QIAGEN RNeasy Mini Kit. One μg of purified RNA was reverse transcribed into cDNA using iScriptTM Reverse Transcription Supermix (BioRad). Integrity of the RNA samples as well as efficiency of cDNA reaction was verified prior to the qRT-PCR. TaqMan Gene Expression Assay kits (Applied Biosystems) were used to measure gene expression . Quantitative real-time PCR was performed with the Step One PlusTM Real-Time PCR System (Applied Biosystems) based on the relative standard curve method. Reactions were performed at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Results were expressed as cycle threshold time (Ct) and were normalized to Ct times for the housekeeping gene GAPDH. The replicated RQ (Relative Quantity) values for each biological sample were averaged. Biological samples from each strain were used for qRT-PCR analyses.For RNA extraction, whole retinas were isolated from 1- month-old mice  by surgical excision. Total protein was extracted via sonication in a protein extraction buffer containing 25 mM sucrose, 100 mM Tris-HCl, pH = 7.8, and a mixture of protease inhibitors . Protein concentrations were determined using BioRad Protein Assays based on the Bradford method for protein quantitation. Proteins (30–40 μg) were separated in 4–20% Criterion Precast gels and 5% polyacrylamide gels (BioRad), transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (BioRad) and incubated with primary antibodies overnight at 4°C under agitation. After washing, the membranes were incubated for 1.5 h with respective  Fluorescence or HRP conjugated secondary antibody (LI-COR Odyssey). For HRP tagged antibody blots were developed with enhanced chemiluminescence (ECL) according to manufacturer’s instructions (Amersham Bioscience). β-actin was used as a gel loading control and was detected using an anti-β-actin antibody . The developed membrane was imaged using the LI-COR Odyssey Quantitative Fluorescence Imaging System. For list of primary antibodies and dilutions see For protein extraction, whole retinas were isolated from 1- month-old mice  kit in accordance with the manufacturer's recommendations. Luciferase signal was read by a luminescent plate reader  and used to compare the activation of caspase 3/7 in C57BL6/J, ATF4P-value lower than 0.05 was considered significant . Data are represented using mean ± SEM.Two-way ANOVA comparisons were used to calculate differences in the a- and b-wave ERG amplitudes and in the average ONL thickness of inferior and superior retinas in 1-, 2- and 3-month-old mice. A one-way ANOVA was used to calculate a fold change of mRNA expressions and a level of normalized proteins in P30 retinas. For all experiments, To demonstrate the effect of ATF4 overexpression in ADRP retinas, we injected P15 T17M and C57BL6 pups with AAV2/5- ATF4 in the right and AAV2/5-GFP in the left eyes. This serotype has been shown to preferentially transduce photoreceptors in the retina , 22 and Accelerated loss of function is usually accompanied by a greater rate of cell death. Our examination of H&E-stained T17M retinal cryostat sections and counts of the number of photoreceptor rows  revealedIt is known that the induction of ATF4 may result in increases in downstream targets . Therefo+/-Rho+/+ ATF4+/- mice with Rho-/- mice to obtain T17M+/-Rho+/-ATF4+/- mice (henceforth referred to as T17M ATF4+/-or ADRP ATF4 deficient mice) and T17M+/-Rho+/- (henceforth referred to as T17M or ADRP) animals. These mice were compared to C57BL6 (Rho+/+) and ATF4+/- (Rho+/+) mice. The ATF4-/- mice develop severe microphthalmia with no recognizable lens, so they were unsuitable for this experiment. However, ATF4+/-Rho+/+ mice (henceforth referred to as ATF4+/- mice) appear to develop normally and have normal vision.We tested the hypothesis that a reduction in ATF4 would preserve visual function and prevent photoreceptor cell death in T17M retinas. For this we created an ADRP animal model with reduced ATF4 by crossing T17M+/- animals demonstrated a functional protection of ADRP photoreceptors. No difference was observed between T17M ATF4+/- and C57BL6 animals at this age. However, by 2 months of age, when T17M mice exhibited significant decreases in their ERG responses, T17M ATF4+/- mice did show a slight drop in a-wave amplitudes, with b-wave amplitudes remaining unchanged relative to C57BL6 controls. At 3 months of age, the drop in ERG amplitudes for T17M ATF4+/- mice became more noticeable. Significantly, despite the initial degeneration, the scotopic a- and b-wave ERG amplitudes were 2.15- and 1.3-fold greater, respectively, in T17M ATF4+/- mice when compared to T17M animals  ERG amplitude which was already diminished in 1-month-old T17M mice  showed a+/- mice. Representative spidergrams of the distribution of ONL thicknesses across the retina at 1, 2, and 3 months of age are shown in +/- ONL thickness in the superior and inferior hemispheres were both appreciably preserved when compared to T17M mice and were 83% of the ONL thickness found in C57BL6 mice  and Chop (1.65-fold) mRNAs, whereas Hsp90 mRNA was downregulated (1.92-fold) in T17M retinas compared to wild type retinas, suggesting an augmented ER stress response. In T17M retinas deficient in ATF4, however, the Hsp90 and Chop mRNA levels were restored to the level of wild type retinas. Both ADRP strain retinas showed activation of the IRE1 arm marker, with Xbp1 mRNA splicing at P15, suggesting that UPR activation occurred earlier than at P30  transgenic mice carrying a human XBP1 and a Venus (a variant of green fluorescent protein) fusion gene under control of a CAG promoter [XBP1 mRNA resulted in GFP expression in the ONL of both T17M and T17M ATF4+/- retinas at P15, suggesting activation of IRE1 UPR signaling in both ADRP groups.To show +/- mice , by crospromoter . The spl+/- retinas: ATF4 was up-regulated 1.8-fold in T17M retinas as compared to C57BL6 retinas and almost 2-fold downregulated in T17M ATF4+/- retinas as compared to T17M retinas. The ATF4 level in T17M ATF4+/- retinas was correspondingly compatible and higher when compared to C57BL6 and ATF4+/- retinas.We also confirmed UPR activation by measuring the expression of UPR-induced cleavage of pATF6, pPERK, peIF2, ATF4, CHOP, and GADD34 proteins . Protein+/- retinas, while its production was elevated 1.7-fold in T17M retinas when compared to C57BL6 mice. Upregulation of CHOP is known to control expression of GADD34 [+/- mice continued to show increased GADD34 protein (3-fold increase in GADD34 protein). This result demonstrates that the ATF4 deficient T17M photoreceptors are able to transduce partially the signal for the sustained activation of GADD34 via CHOP-independent mechanism [+/- mice. This unexpected response was not due to reduced eIF2a expression. In western blot analysis, we found no differences in the eIF2a level among all four groups   , p53 is Therefore, our results demonstrated that limiting ATF4 expression in T17M mice dramatically reduces overall ER stress response by attenuating the activation of the PERK and ATF6 arms and abolishing p53-dependent responses.Nrf2 mRNA was significantly up-regulated in the P30 T17M retina, NRF2 protein level was similar to that found in the C57BL6 mice. In T17M ATF4+/- retinas, western blot analysis revealed an increase in NRF2 protein at P30 (P<0.01) as compared to both the C57BL6 and T17M retinas. This was consistent with observed 1.5- and 3-fold up-regulation of the HO-1 mRNA and protein, respectively in T17M ATF4+/- retinas . This data indicated a possible role of antioxidants in restoring a cellular homeostasis in the T17M ATF4+/- retinas.Knowing that NRF2 is a direct PERK substrate and that PERK-mediated activation of NRF2 can contribute to cell survival , 29, we P<0.01). This would suggest the induction of an inhibitory signal that prevented the elevated expression of NRF2 between P15 and P30. In addition to NRF2, we also observed a dramatic reduction in p62 levels in T17M retinas  in retinal cryostat sections using a chloromethyl derivative of H2DCFDA fluorescence probe . The T17+/- mice was observed (P<0.05 and P<0.01)  .+/- retinas as compared to T17M  increase in expression of human and mouse Rho mRNAs  of eIF2α affecting translational initiation step is known to undergo dynamic changes due to persistently activated ATF4-CHOP-GADD34 negative feedback loop leading to dephosphorylation of peIF2a. Perhaps, because of this fact, we did not catch changes in peIF2a in T17M retinas at P30, while our previous study at P15 suggested changes in peIF2a [We showed, in a separate experiment, that ATF4 over-expression was cytotoxic for photoreceptors and induced severe retinal degeneration. Accelerated photoreceptor cell death and activation of cell death markers occur, respectively in T17M and the wild type mice following two weeks after AAV-mediated ATF4 over-expression. The decline in ERG amplitudes in naive T17M mice is due to a dramatic loss of photoreceptors and is associated with elevations of CHOP, p53 and activation of caspase3/7. We also found in these mice that a portion of total RHO was trapped in the ONL suggesting a role malfunctioned autophagosome-lysosome system in RHO clearance.Therefore, persistent UPR, failure to augment antioxidant defenses, and autophagy occurring during stress appear to contribute to the pathogenesis of retinal degeneration in T17M mice. During ADRP progression, T17M mice experience a progressive loss of a- and b-wave scotopic ERG, which culminates in only 5 rows of photoreceptors remaining by 3 months of age.+/- retinas. The ATF4 level, as expected, is reduced in these animals and is undistinguishable from the level in C57BL6 mice. However, this level might be sufficient to overcome oxidative stress in degenerating retinas together with elevated NRF2 protein [+/- retinas, despite the Nrf2 mRNA level comparable to the C57BL6, the rise in NRF2 protein is most likely mediated by restoration of p62 levels. No significant differences in the mRNA copy number of Nrf2 between the C57BL6 and T17M ATF4+/- and remarkable elevation in the NRF2 protein levels perhaps indicate that posttranslational factors underpin the different levels of expression of NRF2 protein in the wild type and the ADRP retinas[+/- as compared to T17M mice, may contribute to an NRF2-induced antioxidant defense.The major contribution of ATF4 knockdown to the mechanism of retinal degeneration is likely through a reduction of overall ER stress response, induction of an antioxidant program, and restoring of autophagy, which together result in a delay of apoptotic photoreceptor cell death. The dramatic reduction in pPERK, pATF6, peIF2a, and CHOP proteins indicates a significantly lower degree of UPR activation in T17M ATF4 protein . We specP retinas. Thus, sP retinas, and JunP retinas. The lasin vivo [p62 is one of the best known selective substrate of autophagy degradation. However, analysis of p62 and LC3-II conversion in both ADRP retinas suggests that the mechanism of p62 degradation is not strictly dependent on autophagy. Most likely the ubiquitin proteasomal system contributes to p62 degradation as well. Thus, the Parkin E3 ligase that ubiquitinates and degrades a diverse array of substrates, has been shown to degrade the p62 in vivo . Moreovein vivo . AltogetHsp90 at P30 when compared to the wild type. This reduction could perhaps be responsible for the pp53 overexpression observed in T17M retinas since inhibition of HSP90 expression induces p53 over-expression [+/- mice show increases in Hsp90 associated with the restoration of p53 to normal levels is a predictable outcome. This observation could perhaps relate to the proposed switch of ATF4-mediated apoptosis and autophagy during stress, since p53 appears to hold a core position in transducing the p38-promoted signal to either eIF2a or eIF4E [Hsp90 and p62, elevation of p53, reduction of peIF4E, and elevation of CHOP protein. Taken together, these findings support the notion that preferential activation of apoptosis vs. autophagy in T17M retinas, and alternatively, autophagy vs. apoptosis in T17M ATF4+/- mice are important event in retinal degeneration. In support of this hypothesis, the UPR reduction due to knockdown of ATF4 in T17M mice correlates with normal levels of activated caspase-3/7 at P30. Perhaps, similar to targeting caspase-7 [Targeting of HSP90 during retinal degeneration has been proposed as a therapeutic strategy , 46; howpression . Therefoor eIF4E . Consequor eIF4E . Conversaspase-7 , this ap+/- retinas. While GADD34 increase during CHOP downregulation in retinas requires further detailed study, the recent findings of a feedback loop provided by GADD34, canonical UPR signaling, and the enhancement of autophagy, known to be associated with GADD34 over-expression could in part explain our observation [Reduction in CHOP is associated with a paradoxical increase in GADD34, which, in turn, correlates with downregulation of peIF2a protein in T17M ATF4ervation –51.+/- retinas revealed that both scotopic and photopic ERG amplitudes were preserved, and that cone photoreceptor function was well-maintained in 1-month-old animals. This may be due to the presence of normally functioning rod photoreceptors as reduction in ATF4 is known to reprogram the ER stress signaling network in ADRP retina. These results are of great importance as they support the notion that inherited retinal degeneration can be delayed. To the best of our knowledge, this is the first report to demonstrate rescue of rapidly degenerating ADRP retinas at P30 and a significant delay afterwards. The SD-OCT imaging shows a difference in average superior and inferior ONL thicknesses in the T17M ATF4+/- mice, but this difference would appear to be due to altered photoreceptor morphology rather than a change in the number of photoreceptors. The H&E staining revealed no observable difference between C57BL6 and T17M ATF4+/- mice, suggesting that no photoreceptor cell death occurred at P30.The analysis of T17M ATF4+/- retinas demonstrated elevated expression of both the mouse and human RHO mRNAs and increased RHO protein levels. Total RHO is also elevated in T17M ATF4+/- retinas and reached the level about half that of RHO measured in C57BL6 mice (monomer). Moreover, this level corresponds to that found in RHO+/- mice with normal vision that express 50% of the wild type RHO. Unlike the RHO+/- mice, the T17M ATF4+/- mice have both human and mouse RHO and the level of wild type rhodopsin may then be sufficient to maintain the function of photoreceptors during the first month of life. However, later on, the mutant rhodopsin transported to the outer segment could cause problems by reducing the ability to regenerate pigment after light exposure [The T17M ATF4exposure , thereby+/- mice and these may contribute to photoreceptor health at early time points but may not be sufficient to maintain cellular homeostasis at later time-points.Activated autophagy in ATF4-deficent ADRP mice could be responsible for the clearance of partially accumulated RHO in the ONL of P30 retinas . This fiTherefore, our results indicate an essential role for ATF4 in the induction of photoreceptor cell death. We can however propose that increased ATF4 expression during retina degeneration contributes to T17M photoreceptor cell death. Reduced ATF4 expression in degenerating retinas experiencing UPR activation, conversely, plays a pro-survival role, suggesting a therapeutic strategy for intervention in ADRP progression. ATF4 deficiency in the T17M retina under conditions of UPR activation, on the other hand, satisfies a photoreceptor cellular demand by adapting the UPR, overall reducing cellular stress in ADRP photoreceptors and activating autophagy that may clear a trapped rhodopsin in the ONL of ADRP retinas. Therefore, this signaling provides functional and morphological benefits to ADRP photoreceptors. This scenario is perhaps, sufficient to rescue the one-month-old ADRP retina and significantly delay further ADRP progression. Overall this indicates that PERK signaling in general and ATF4 in particular, is a target for delaying retinal degeneration in ADRP patients. Diminishing ATF4-dependent cellular stress leading to reduction of apoptosis and enhancement of autophagy may delay the onset of retinal degeneration. Based on our experimental data, reduction of ATF4 up to 50% is safe in T17M retinas. However, the therapeutic effect of ATF4 deficiency in other ADRP animal models requires further validation. This validation should also optimize the therapeutic dose at which ATF4 plays a protective role in degenerating retinas.S1 Fig(A) Micron IV fluorescence image obtained from uninjected (left) and injected (right) with AAV2/5-GFP T17M retinas with fluorescence filter. Untransduced area in AAV2/5-GFP injected eye is shown with asterisk. (B) Analysis of H&E stained T17M retinal sections demonstrated dramatic photoreceptor cell loss that occurs between P15 and P30 . (C) Representative images of P15 C57BL6 and P15 and P30 T17M cryostat-sectioned retinas stained with H&E are shown on the side. (D) Expression of NRF2 in P15 T17M retinas. A western blot image is shown on a side. The data are presented as mean ± SEM.(TIF)Click here for additional data file.S2 Fig(A) Representative images of the scotopic and photopic ERG amplitudes registered at 25 cd*s/m2 for all four groups of animals. Please take in account that the scales (μV) for photopic ERG amplitudes are varied in all four groups of animals. For C57BL6 and ATF4 mice, the max amplitude is 300 and 200 μV respectively; for T17M is 150 μV, for T17M ATF4+/- are 250 μV. (B) Representative SD-OCT images of retinas for all four groups of animals.(TIF)Click here for additional data file.S3 FigImages were obtained by running retinal protein extracts from all four animal groups on polyacrylamide gels and probed with antibodies against the indicated proteins.(TIF)Click here for additional data file.S1 TableResults of RNA, western blot, ERG, SD-OCT and histological analyses of the study.(PDF)Click here for additional data file.


Here, we report full-length genome sequences of influenza pH1N1 viruses obtained prior to and after propagation in MDCK cells. Paired comparisons of the genomes showed that each strain acquired 1.0 to 18.8 mutations per genome per replication cycle, which corresponds to 0.5 to 5.8 mutations per virus proteome per replication cycle. Our analysis indicates that pH1N1 viruses accumulated adaptive mutations among others in response to propagation in cell culture. These results could be important for vaccine and drug-sensitivity surveillance studies, as well as for vaccine and antiviral drug development programs where cell cultures are used for influenza propagation. Influenza viruses receive much public attention because they cause major pandemics and epidemics heavily impacting public health and economy. Different surveillance, prevention, and treatment programs are under development to improve control of influenza outbreaks. Such programs usually utilize cell culture for virus propagation.Here, we assess the genome stability of 10 pH1N1 strains prior to and after propagation in MDCK cells. In particular, we sequenced genomes of pH1N1 strains from patient nasopharyngeal aspirates (NPA) and after four passages in MDCK cells as described elsewhere . We founDuring propagation in cell culture at least three viruses acquired an N/D239G mutation in HA, an R12K mutation in M2, and N29S and A89V mutations in NEP. Two viruses acquired a V544I mutation in HA, D151E/N, S95N, N386K, K397N, G398E, N449K mutations in NA, D2N, I18V, E55K mutations in NS1, and a V113I mutation in PB1. One virus acquired an interesting E151K mutation in NA.Mutations E119K and D151E/N in NA have been found previously to be associated with propagation of influenza viruses in cell culture and linked to an oseltamivir-resistant phenotype –4. Mutat–Interestingly, two influenza pH1N1 strains possess unusual C-terminal extensions of the NS1 protein (230 versus 219 amino acids). Moreover, these viruses retain the extension after passaging in MDCK cells. This indicates that the C-terminal extension and truncation .Twenty whole-genome sequences of 10 pH1N1 isolates from Finland (year 2014) have been deposited in Genbank (accession numbers


Genome variation is very high in influenza A viruses. However, viral evolution and spreading is strongly influenced by immunogenic features and capacity to bind host cells, depending in turn on the two major capsidic proteins. Therefore, such viruses are classified based on haemagglutinin and neuraminidase types, e.g. H5N1. Current analyses of viral evolution are based on serological and primary sequence comparison; however, comparative structural analysis of capsidic proteins can provide functional insights on surface regions possibly crucial to antigenicity and cell binding.We performed extensive structural comparison of influenza virus haemagglutinins and of their domains and subregions to investigate type- and/or domain-specific variation. We found that structural closeness and primary sequence similarity are not always tightly related; moreover, type-specific features could be inferred when comparing surface properties of haemagglutinin subregions, monomers and trimers, in terms of electrostatics and hydropathy. Focusing on H5N1, we found that variation at the receptor binding domain surface intriguingly relates to branching of still circulating clades from those ones that are no longer circulating.Evidence from this work suggests that integrating phylogenetic and serological analyses by extensive structural comparison can help in understanding the ‘functional evolution’ of viral surface determinants. In particular, variation in electrostatic and hydropathy patches can provide molecular evolution markers: intriguing surface charge redistribution characterizing the haemagglutinin receptor binding domains from circulating H5N1 clades 2 and 7 might have contributed to antigenic escape hence to their evolutionary success and spreading.The online version of this article (doi:10.1186/s12859-014-0363-5) contains supplementary material, which is available to authorized users. In fact, changes in each specific property can result in such modulation, and this can be independent on variation of other features, or modulation can result from the aggregate or synergistic effect of multiple feature changes. So far, several sequence-based studies on variation could provide valuable phylogenetic evidence; however, such studies are of minor help in inferring variation at protein regions including amino acids that are far each other in the primary sequence and quite close within the 3D protein structure . In practice, while sequence-based investigation can be good in highlighting very evident changes at individual positions of a protein chain, in general they fail in highlighting meaningful ‘group variation’, i.e. in identifying - especially when the overall variation is relevant and spread - relationship of specific multiple changes to variation in conformational epitopes hence in interactions they mediate.Influenza caused by influenza A viruses occurs in both birds and mammals. In humans, influenza A viruses infect hundreds of millions individuals, causing a high number of deaths per year. Indeed, influenza A outbreaks occurred in 1918, 1957 and 1968 resulted in death for ~100 million people worldwide   mutations in sites crucial to protein machinery mediating basic functions (e.g. in motifs relevant to host recognition or cell entrance) are not allowed because they strongly impair viral fitness, and at the same time, (ii) hyper-variability is needed to escape recognition by neutralizing antibodies . Given We started our analyses using six currently available solved HA structures; then, in order to investigate structural variation possibly underlying H5N1 clades evolution and spreading, we also created clade models by homology modeling. The six HA structures solved so far: H1 , H2 12]12], H3 , H5 14]14], H7 , H9 15]15], all Prior to creating models, preliminary analysis of the six available HA structures was performed in order to evaluate intra- and inter-group structural variation by superposition of all structure pairs and computation of their Root Mean Square Deviation (RMSD). Indeed, the RMSD of two superposed structures indicates their ‘structural divergence’ from one another. As both sequence mutation and conformational variation inflate the RMSD, values up to 2 Ångstrom indicate structural similarity . StructuIn addition to superposing structures of HA monomers, also corresponding structures of their Receptor Binding domains (RBDs) were superposed. Results are summarized in Table Available structures were superposed and then tiled using UCSF Chimera 1.8.1 to keep the same orientation and to avoid visual superposition. This way, variation of secondary structure elements among individual structures can be clearly distinguished and viewed. In order to exclude any artifact from modeling, only the six available solved structures were compared. In terms of secondary structure, three subregions can be distinguished within the HA2 stem [see Additional file In order to perform analyses taking into account the influence of ionic strength (I), the spatial distribution of the electrostatic potential was calculated at both I = 0 mM (Coulombic interactions unscreened by counter-ions) and I = 150 mM , assuming +1/-1 charges for the counter-ions. Prior to electrostatic potential calculations, partial charges and van der Waals radii were assigned with PDB2PQR ,26; thene value of H1 and H9 to −15 e of H7. However, similar net charge does not necessarily correspond to similar distribution  of the potential, that can preferentially locate at either the VED-distal stem subregion (left side in figure) or at the VED-proximal one (right side). This is the case for H1 and H9 stem, sharing net charge −8 e, and showing (more evident at I = 0 mM) preferential VED-distal and VED-proximal negative potential, respectively. Such preferential VED-distal location of the negative potential shown by H1 is conserved also in the other two stems from Group 1, in spite of their different net charge (−10 e). Positive potential is more homogeneously distributed along all stems. Heat maps and corresponding density plots  in other groups. At large, the RBD net charge is less negative than stems  or neutral (H5) net charge, which is positive  while being close to 0 for RBDs (−1e to +3e), stresses the total charge balancing by local basic patches in VED and F’ subregions. Once again, peculiar electrostatic features are evident (and SI independent) for H9, characterized by the less negative net charge and forming its own branch at both I = 0 mM and I = 150 mM  for H5. Instead, trimer values are only roughly twofold increased for H2, H7 and H9. Therefore, different orientations of monomers within corresponding trimers results in significant modulation of the trimer surface electrostatic charge and this in turn can be quite relevant to HA interactions. Different HA clustering at I = 0 mM and I = 150 mM may highlight the importance of ionic screening of coulombic interactions [Once the entire haemagglutinin functional unit is analyzed, disagreement with serological and phylogenetic clustering is highlighted again by Group 1 splitting; in particular (and independently on which SI is used) at I = 0 mM, H1 sorts separately from H2 and H5 .Electrostatic features can vary among different types of haemagglutinins (see above). This prompted us to further investigate on differential electrostatic features as a possible fingerprint for monitoring viral evolution, i.e. as a tool to distinguish among circulating/spreading and extinguished H5N1 clades. Table Based on a very high, average % identity (over 90%) of the clade target sequences with the available structural H5 template (PDB: 3S11), structural models for clades 0 to 9 were obtained by homology. Given that distribution of surface charge is strongly influenced by the orientation of side chains, models refinement was performed using a number of tools based on different algorithms: SCWRL ,36, ModRPreliminary comparison at trimer and monomer level showed meaningful variation only at the VED-RBD subregion. In fact, direct comparison of stems did not allow for inferring any clade-specific signature as all clades were found to share - at both I = 0 mM and I = 150 mM - the typical isocontour of the H5 stem  that the same specific differences are found when comparing refined or not refined clade models or models refined using different algorithmic strategies  were used as templates for modeling: H1, PDB 1RUZ, from viral strain A/South Carolina/1/1918(H1N1); H2, PDB 2WR5, from Asian pandemic influenza virus of 1957; H3, PDB 1MQL, from viral strain A/duck/Ukraine/1963 (H3N8); H5, PDB 3S11, from viral strain A/Goose/Guangdong/1/1996 (H5N1); H7, PDB 1TI8, from viral strain A/turkey/Italy/214845/2002(H7N3); H9, PDB 1JSD, from viral strain A/swine/Hong Kong/9/98(H9N2). UniProtKb accession codes (AC) of target sequences modeled by H.M. and corresponding viral strains (VS) are the followings: H4, AC F2NZ53, VS A/duck/Guangxi/912/2008(H4N2); H6, AC H8PBW2, VS A/duck/Fujian/6159/2007(H6N6); H8, AC D4NQL7, VS A/northern pintail/Alaska/44420-106/2008(H8); H10, AC P12581, VS A/Chicken/Germany/n/1949 (H10N7); H11, AC D5LPX8, VS A/turkey/Almaty/535/2004(H11N9); H12, AC E6XYK2, VS A/mallard/Interior Alaska/9BM1907R1/2009(H12); H13, AC P13101, VS A/Gull/Astrakhan/227/1984 (H13N6); H14, AC P26136, VS A/Mallard/Astrakhan/263/1982 (H14N5); H15, AC Q82565, VS A/duck/Australia/341/1983(H15N8); H16, AC Q5DL23, VS A/black-headed gull/Sweden/3/99(H16N3). Given that original UniProtKb sequences indeed correspond to H0 precursors, sequence fragments missing in mature chains were manually removed to avoid improper structural alignment.Structural superpositions were performed and viewed using UCSF Chimera  v. 1.8.1BT/e, ±2kBT/e and ±1kBT/e (RBDs). PDB2PQR was used to assign partial charges and van der Waals radii according to the PARSE force field [p = 2 and εs = 78.5 were chosen for respectively the protein and the solvent [Isopotential contours were calculated using UCSF Chimera 1.8.1: the software utility allows for connecting - through Opal web server - to the Adaptive Poisson-Boltzmann Solver (APBS) server . Isopotece field . Interio solvent ,44,45, T solvent . Rigid-bHydropathy analysis was performed using the Kyte-Doolittle scale implemented in Protein Hydrophobicity Plots  and in P