Jak2K915X), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2+/-). Timed pregnancy experiments revealed that Jak2K915X/K915X and Jak2-/- displayed embryonic lethality; however, Jak2K915X/K915X embryos were viable an additional two days compared to Jak2-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 ( Cytokines play an integral role in hematopoiesis by providing growth signals to progenitor and committed cells that promote mitogenesis, survival and in some cases differentiation. Once bound to their cognate receptors, cytokines mediate downstream signaling through activation of components of the Jak-Stat signaling pathway. Thrombopoietin (Tpo) is the principal cytokine regulator of megakaryopoiesis, through binding to its cognate receptor Mpl. Tpo activates the Jak2 and Tyk2 tyrosine kinases  as well While screening ENU mutagenized mice for dominant hematopoietic defects, we identified a mouse with thrombocythemia and determined that the mutation resulted in a truncated allele of Jak2 that lacked catalytic activity. Analysis of this mutation has uncovered a novel function of Jak2 in the megakaryocyte/platelet lineage.C57Bl6/J (B6) and 129S1/SvImJ (129) mice were purchased from The Jackson Laboratory. Jak2+/- mice (on the B6 genetic background) were provided by Dr. James Ihle, Memphis, TN. All mice were maintained in specific-pathogen free facilities at the Toronto Centre for Phenogenomics or Ontario Cancer Institute. Animal protocols were approved by the OCI Animal Care Committee (Permit Number 1517). All efforts were made to reduce animal suffering.129 mice (mutagenized strain) [129;B6) was produced by out-crossing ENU-mutagenized males to B6 (mapping strain) females – pups from this breeding were designated generation 1 (G1). G1 mice were screened to detect dominant traits deviating from normal homeostatic venous blood parameters by at least two standard deviations from ‘normal’ G1 parameters. Affected mice with elevated platelets were sequentially back-crossed to B6 mice for genetic mapping. The Jak2K915X allele was maintained on a B6 background by intercrossing heterozygous or wild type (WT) mice. Timed matings were performed on G9 animals and peripheral blood analysis was completed on G10 mice.To induce random mutations, one intraperitoneal injection of 150mg/kg ENU was administered to male  strain) . The F1 Jak2+/+ mice were littermate controls of Jak2+/- animals. Jak2Control mice are littermate controls of Jak2K915X G10 breedings. Bone marrow sections were prepared from femurs of 12-week old mice. Femurs were fixed in 10% formaldehyde and then sectioned (4 µm) and stained with Hematoxylin and Eosin (H&E) at the CMHD pathology core (http://www.cmhd.ca/enu_mutagenesis/pathology.html). Affected mice were sequentially bred to B6 to confirm heritability and to genetically map the mutation using microsatellite base genome scan and single-nucleotide polymorphism markers differentiating 129 and B6 alleles [Peripheral blood from 6-8 week old mice was collected by saphenous venipuncture. Complete blood counts (CBC) were performed using a Coulter Ac-T Differential Hematology Analyzer.  alleles . Once th alleles ,15.Jak2K915X and Jak2+/- mice using genomic DNA prepared from tail or biopsy tissue [K915X allele (T 3056nt).Multiplex PCR was used to genotype y tissue . All JakCFU-C, CFU-E and CFU-Mk assays were performed as previously described ,18.Six to eight-week old mice were injected with 5-fluorouracil (5FU) or Phenylhydrazine (PHZ), as previously described ,19. Brie293T cells (ATCC) were transfected with HA-tagged Jak2 or HA-Jak2 K915X. Alternatively, Jak2 constructs were generated that expressed the 3’ UTR or had the 3’ UTR removed. Thirty-six hr after transfection, cells were washed, lysed as described . Lysate Membranes were blocked in optimal blocking agent , 150 mM NaCl, 0.1% Tween 20 (TBST)) for 1 hr. Primary antibody incubations were performed for 1 hr, followed by 30 min washing in TBST. Secondary incubations were performed with HRP-Sheep anti-mouse IgG  or HRP-Protein A  for 30 min. After washing, membranes were developed by ECL.Anti-Jak2 and β-tubulin antibodies were purchased from Cell Signaling Technology  and Millipore . The HA antibody was from Covance . Phosphorylation-specific pSer-523  and pTyrMegakaryopoiesis was assessed by microscopic examination of bone marrow of femurs on histology sections. Megakaryocytes are identified by their characteristic morphology of large size, lobulated nuclei and abundant cytoplasm and quantified by counting the number of megakaryocytes per microscopic field under a 40x objective.7254, with elevated platelets. Back-crossing on to the B6 strain and SNP-based mapping resulted in the identification of a 6.7 Mb heritable region on chromosome 19 as the interval encoding the responsible mutation. We noted that Jak2 was within this interval and hypothesized that the mutation underlying the 7254 phenotype may be in Jak2. We performed genomic DNA sequencing which identified an A3056T mutation in exon 19 of the Jak2 locus. This mutation leads to a K915X premature stop codon in the functional JH1 kinase domain of Jak2 .Western blotting of splenocytes isolated from Recent evidence has suggested that the Jak2 JH2 domain possesses weak intrinsic kinase activity ,24. ConsThe possibility of nonsense-mediated decay occurring was eliminated by expression of Jak2 cDNAs that lacked or contained the Jak2 3’ untranslated region. The presence of the Jak2 3’ UTR resulted in comparable protein expression . Representative sections from wild type and Jak2K915X/+ mice illustrate increased megakaryocytes in mutant mice  CFU-MK frequency in the bone marrow grown in the presence or absence of TPO. (B) Bone marrow CFU-E frequency grown with or without EPO. (C) Splenic CFU-E frequency grown in the presence or absence of EPO. All CFU-E and CFU-Mk were derived from Jak2K915X/-and Jak2+/- and littermate controls at 12-14 wks of age. Data are presented as ± SEM; n=5-8.(TIF)Click here for additional data file.Figure S2Jak2K915X/-and Jak2+/- does not disrupt CFU-C frequency in the bone barrow or spleen.Functional loss of Jak2 in (A) Total CFU-C frequency in the bone marrow. (B) The frequency of CFU-C in the spleen. (C) CFU-C differential count of bone marrow derived colonies included: CFU-G (granulocyte), CFU-M (monocyte), CFU-GEMM , CFU-GM (granulocyte and monocyte) and BFU-E (erythroid). (D) Splenic CFU-C differential. All CFU-C were derived from Jak2K915X/-and Jak2+/- and littermate controls at 12-14wks of age. Data are presented as ± SEM; n=5-8.(TIF)Click here for additional data file.Figure S3Jak2K915X/- and Jak2+/- results in similar recovery.5FU hematopoietic challenge of The recovery curves for 5FU induced hematopoietic stress in Jak2K915X/-  and Jak2+/- . The recovery curves for red blood cells (A and B), platelets (C and D) and white blood cells (E and F). Data are presented as ± S.D. and n=9-11.(TIF)Click here for additional data file.Figure S4Jak2K915X/- and Jak2+/-.PHZ challenge of erythropoiesis in Red blood cell recovery curves of PHZ challenged of Jak2+/- (A) and Jak2K915X/- (B). The data are presented as ± S.D.; n=8-9.(TIF)Click here for additional data file.


G93A). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1G93A in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1G93A forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1G93A model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1G93A action on mitochondrial dynamics, indicating SOD1G93A likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1G93A inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may actively promote ALS progression. Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1 ALS is an adult onset and fatal neuromuscular disease characterized by the progressive loss of motor neuron (MN) and skeletal muscle atrophy. Most cases of ALS are sporadic (SALS), with about 10% being familial (FALS) .G93A). Both mouse models showed muscle degeneration [The common pathological hallmark of ALS is the death of motor neuron. However, defects present in other cell types may also actively contribute to the disease progression . Motor nneration , but onlneration . Interesneration . This reneration . In addineration . TherefoG93A (G93A) and found that a portion of muscle fibers had depolarized mitochondria near NMJ [The high energy demand of muscle contraction is met by a large endowment of mitochondria that occupy 10–15% of muscle fiber volume . Morpholnear NMJ ,20. ThisG93A alters mitochondrial dynamics in skeletal muscle of normal mice, where there is no motor neuron degeneration. Remarkably, only the mutant SOD1G93A (not the wild type SOD1) overexpression results in formation of protein aggregates inside mitochondria, mitochondrial fragmentation and abnormal dynamics. The abnormal mitochondrial dynamics evoked by SOD1G93A overexpression is associated with depolarization of mitochondrial membrane potential and augmented mitochondrial fission process. Overall, our data indicate the SOD1G93A mutation directly causes abnormal mitochondrial dynamics in skeletal muscle even in the absence of motor neuron degeneration. Thus, abnormal mitochondrial dynamics in muscle may contribute to SOD1 mutation-associated ALS disease onset and progression. Mitochondria are morphologically highly dynamic organelles that are constantly remolded by fusion and fission processes . This ph2 inhalation, and the flexor digitorum brevis (FDB) muscles were removed for functional studies. All experiments were carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the IACUC of Rush University. The injection and electroporation procedures were performed under the anesthesia using constant isoflurane inhalation. All efforts were made to minimize suffering.G93A  and age 2+) plus 0.2% type I collagenase for 55 min at 37°C. Following collagenase treatment, muscle fibers were stored in an enzyme-free Krebs solution at 4°C, and used for imaging studies within 24 hours. Individual muscle fibers were isolated following the protocol used previously ,20. BrieG93A were amplified from pBluescript-SOD1 and pBluescript-SOD1G93A . To construct SOD1-GFP and SOD1G93A-GFP, the amplified SOD1 and SOD1G93A were subcloned into pEGFP-N1 (Clontech) at sites of BamH I and Nhe I. To make mt-SOD1-Dendra and mt-SOD1G93A-Dendra, the cDNAs of SOD1 or SOD1G93A was inserted into pDendra2-N (Evrogen) at sites of BamH I and Hind III. The mitochondrial targeting sequence consisting of 29 amino acids amplified from pCMV/myc/mito (invitrogen) was subsequently fused at the 5’ end of SOD1-Dendra and SOD1G93A-Dendra constructs. All final constructs were confirmed by sequencing. mt-PAGFP was purchased from Addgene (#23348).The cDNAs of SOD1 and SOD1FDB muscle fibers were incubated with 50 nM tetramethylrhodamine ethyl ester (TMRE) for 10 min at 25 °C for visualization of mitochondrial membrane potential. Photoactivation with 364 nm laser light was used to activate PAGFP to show GFP green fluorescence. GFP and TMRE images were simultaneously recorded. GFP was excited at 488 nm and its emitted fluorescence was collected at 490-540. TMRE was excited at 543 and its emitted fluorescence was collected at 560-620. Before photoactivation Dendra is a green fluorescent protein. Photoactivation with 364 nm laser light converts Dendra from a green to a red fluorescent protein. Thus after photoactivation Dendra was excited at 543 and its emitted fluorescence was collected at 560-620. A confocal microscope  capable of line-interleaving images excited with different lasers, was used. TMRE was purchased from Invitrogen.The mitochondrial fission inhibitor, Mdivi-1  was applTibialis anterior muscles from normal and G93A mice were collected and homogenized in protein extraction buffer containing protease inhibitor cocktail (Thermo Scientific) using motorized homogenizer (Wheaton). Protein concentrations were determined by BCA protein assay (Thermo Scientific). Protein samples ~30 µg) were subject to 8% SDS-polyacrylamide gel electrophoresis. The protein was transferred into an Immobilon PVDF membrane . The primary antibodies used to probe the protein samples are anti-Mfn1 (1:2500) and anti-Mfn2 (1:5000) from Abcam; anti-Drp1 (1:5000) from Cell Signaling, and anti-tubulin (1:10000) from Santa Cruz Biotechnology. The C-SOD1 antibody was a generous gift from Dr. Han-Xiang Deng [ µg were IDL 7.0  was used for image processing. Sigmaplot 11.0 and Microsoft Excel were used for data analysis. Data are represented as mean ± S.E.M. Statistical significance was determined by Student’s t test.The ALS mouse model (G93A) is asymptomatic until after 3 months of age . To examMitochondrial dynamics were evaluated by examining the time-dependent migration of mt-PAGFP out of the original photoactivation area. Representative control and G93A images are shown in G93A in skeletal muscle directly (i.e. neuron independent) alters mitochondrial dynamics in skeletal muscle of G93A mice before ALS onset. The reduced mitochondrial dynamics observed in skeletal muscle of G93A mice could be a consequence of the motor neuron withdrawal from muscle. However, two-month old G93A mice are asymptomatic and have no significant motor axonal withdrawal . Thus, wG93A in skeletal muscle of the G93A mouse model by immunoblot assay. The C-SOD1 antibody used here recognizes both human and mouse SOD1. Since human SOD1 is larger than mouse SOD1, these two proteins are easily differentiated in the blot [G93A at all disease stages. The muscle from a control normal mouse (Lane 1) had no human SOD1G93A expression. We then determined whether mutant SOD1G93A is associated with mitochondria in skeletal muscle. SOD1 is a cytosolic protein that can be transported into mitochondria in some cell types [G93A-GFP fusion proteins. Both plasmids were transfected into the FDB muscle of normal mice respectively. Seven days after transfection, muscles were collected and single muscle fibers were isolated. Fibers expressing SOD1-GFP or SOD1G93A-GFP were incubated with TMRE to visualize mitochondria. Overlay of TMRE and SOD1-GFP or SOD1G93A-GFP images  was attached to its C-terminus. Before photoactivation, Dendra2 protein absorbs 488 nm light. After photoactivation by a 364 nm laser light, Dendra2 absorbs 543 nm light. In other words, pohotoactivation converts Dendra2 from a green to a red fluorescent protein. The mt-SOD1G93A-Dendra fusion protein was used to access mitochondrial dynamics and morphology. A plasmid, mt-SOD1-Dendra was also constructed as a negative control. Each of these plasmids was transfected into the FDB muscle of normal mice. Seven days after transfection, FDB muscle fibers expressing mt-SOD1-Dendra or mt-SOD1G93A-Dendra were isolated and incubated with TMRE. G93A-Dendra fusion proteins were preferentially expressed in mitochondria. Remarkably, 90% (n=73) of muscle fibers expressing mt-SOD1G93A-Dendra showed aggregated fluorescent protein inside mitochondria. In contrast, not a single fiber expressing mt-SOD1-Dendra had aggregated fluorescent protein (n=36). In addition, 63% of fibers expressing mt-SOD1G93A-Dendra showed fragmented mitochondria compared to only 11% of fibers expressing mt-SOD1-Dendra  while mt-SOD1G93A-Dendra migrated just 3.5±0.6 m-steps in the same period . The migration rates were determined by linear regression fits  show reduced TMRE staining (a1.1 and a2.2), which is indicative of mitochondrial inner membrane depolarization. The correlation between depolarization and aggregated fluorescent protein is also evident in G93A mutation) altered mitochondrial dynamics. We found that incubation of normal muscle fibers with 200 nM FCCP (20 min) partially depolarized the inner membrane potential evoking a reduction of TMRE fluorescence intensity of about 19% (data not shown). To evaluate mitochondrial dynamics, the same FCCP challenge was applied to fibers expressing wild type SOD1 fusion protein (mt-SOD1-Dendra). After 20-min FCCP incubation, a small fiber region was photoactivated and the migration of red fluorescent protein from this region was measured. G93A. Two groups of normal mice (3 in each) were transfected with mt-SOD1G93A-Dendra at the site of FDB muscle. In the next 7 days, one group received intraperitoneal injection of Mdivi-1, a specific inhibitor of Drp1 as described in Method section. The control group received the intraperitoneal injection of same amount of DMSO (vehicle). After the 7-day period, the FDB muscles were removed and mitochondrial dynamics were determined. Representative images are shown in G93A-Dendra migrated 10.2±1.4 m-steps in 18 min (n=8). In the control group, mt-SOD1G93A-Dendra migrated just 4.4±1.0 m-steps in the same period . The migration rates were determined by linear regression fits  and dynamin-related protein 1 (Drp1) respectively (reviewed in ). We exaion fits 6C and rG93A mutation does not significantly alter the protein expression level of these key proteins, but may enhance the function of Drp1 to promote mitochondrial fission events.We also conducted Western blot analysis to determine the expression level of key proteins involved in mitochondrial fusion and fission processes. Blots were prepared using skeletal muscle of young G93A mice at the same disease stage, a stage known to have altered mitochondrial dynamics (n=5 G93A mice and 5 age-matched control mice). G93A protein in skeletal muscle of normal mice. Our data suggest that SOD1G93A mutation drives ALS-associated muscle pathology in the absence of motor neuron degeneration.In this study, we discovered that skeletal muscle of an ALS mouse model G93A has abnormal mitochondrial dynamics at the age of two months when there is no significant axonal withdrawal and disease symptoms. We further demonstrated that similar abnormality of mitochondrial dynamics can be directly induced by overexpressing mutant SOD1Mitochondrial dynamics involves mitochondrial fission and fusion processes . In skelG93A in skeletal muscle. At two months of age G93A mice are asymptomatic and have no significant axonal withdrawal, but have altered mitochondrial dynamics in skeletal muscle. This implies that altered mitochondrial dynamics may be not due to axonal withdrawal. To verify this, we overexpressed SOD1G93A in skeletal muscle of normal mice and reported that mutant SOD1G93A targets to mitochondria and directly affects mitochondrial dynamics in skeletal muscle in the absence of motor neuron degeneration. Abnormal mitochondrial dynamics observed in G93A skeletal muscle could be 1) a consequence of the motor neuron degeneration or 2) directly caused by expression of the mutant ALS-causing protein SOD1G93A-GFP plasmids and overexpressed these fusion fluorescent proteins in skeletal muscle of normal mice. Our results are the first evidence that both wild type SOD1 and mutant SOD1G93A accumulate inside muscle mitochondria . These results provide strong evidence that accumulation of mutant SOD1G93A inside mitochondria is able to disrupt the homeostasis of mitochondrial fission and fusion dynamics in skeletal muscle. Further, these results show that the disruption occurs in the absence of motor neuron degeneration. The migration rate (during the first 2 min) in fibers expressing mt-SOD1G93A-Dendra was reduced 1.6-fold, which is identical to that observed in the G93A muscle  reasonably reproduce the phonotype of G93A muscle (Expression of wild type SOD1 (mt-SOD1-Dendra) inside mitochondria did not alter normal mitochondrial dynamics see 1. The mA muscle 1. This A muscle 1. G93A protein [G93A mouse model has systematic overexpression of mutant SOD1 protein . It is p protein ,26. HereG93A (mt-SOD1G93A-dendra). Remarkably, inhibition of Drp1 restored mitochondrial network and the migration rate of the fluorescent protein. The results indicate that mutant SOD1G93A promotes Drp1-based mitochondrial fission in skeletal muscle. We also examined expression levels of Drp1 and the mitochondrial fusion promoters (Mfn1/2)  in sin cells  independc muscle . Thus, tc events , which mG93A-Dendra  [G93A inside mitochondria initially promotes partial depolarization of mitochondrial inner membrane potential and this enhances Drp1 function, which promotes the fission process. As a result, G93A muscle has less connected mitochondrial network and less exchange of mitochondrial contents. As observed in our previous study [2+ activity. Mitochondria with defective dynamics may be more susceptible to Ca2+ overload at the site of NMJ and thus those mitochondria near NMJ are first to be completely depolarized. A subset of G93A muscle fibers with depolarized mitochondria near NMJ may be fibers that are further along in the disease process. Abnormal mitochondrial dynamics in G93A muscle may be associated with mitochondrial membrane depolarization. Indeed, we found that partially depolarization of mitochondria by FCCP slowed the content exchange between mitochondria in normal muscle fibers. Localized mitochondrial depolarization was also evident in muscle fibers expressing mt-SOD1A-Dendra 4 at theon (NMJ) ,20. Remaus study , mitochoG93A inside mitochondria and can occur independent of motor neuron degeneration. Our data provide direct evidence that mitochondria in skeletal muscle are primary points of pathological failure caused by an ALS-causing mutation SOD1G93A. The altered mitochondrial dynamics in G93A muscle likely contributes to overall disease progression. Future studies will be required to define the causal link between the defective muscle mitochondrial dynamics and the NMJ defect in ALS muscle as well as identify specific downstream pathways responsible for the defective mitochondrial dynamics and mitochondrial degeneration in ALS muscle. Importantly, this study suggests that development of agents that target and restore the homeostasis of mitochondrial dynamics could have therapeutic value in alleviating the muscle dysfunction associated with SOD1 mutation-related ALS.In summary, this study is the first to show that mitochondrial dynamics is disrupted in G93A skeletal muscle. This dysfunction is associated with accumulation of mutant SOD1


Intestinal epithelium is essential for maintaining normal intestinal homeostasis; its breakdown leads to chronic inflammatory pathologies, such as inflammatory bowel diseases (IBDs). Although high concentrations of S100A9 protein and interleukin-6 (IL-6) are found in patients with IBD, the expression mechanism of S100A9 in colonic epithelial cells (CECs) remains elusive. We investigated the role of IL-6 in S100A9 expression in CECs using a colitis model.IL-6 and S100A9 expression, signal transducer and activator of transcription 3 (STAT3) phosphorylation, and infiltration of immune cells were analyzed in mice with dextran sulfate sodium (DSS)-induced colitis. The effects of soluble gp130-Fc protein (sgp130Fc) and S100A9 small interfering (si) RNA (si-S100A9) on DSS-induced colitis were evaluated. The molecular mechanism of S100A9 expression was investigated in an IL-6-treated Caco-2 cell line using chromatin immunoprecipitation assays.S100A9 promoter. S100A9 may recruit immune cells into inflamed colon tissues.IL-6 concentrations increased significantly in the colon tissues of DSS-treated mice. sgp130Fc or si-S100A9 administration to DSS-treated mice reduced granulocyte infiltration in CECs and induced the down-regulation of S100A9 and colitis disease activity. Treatment with STAT3 inhibitors upon IL-6 stimulation in the Caco-2 cell line demonstrated that IL-6 mediated S100A9 expression through STAT3 activation. Moreover, we found that phospho-STAT3 binds directly to the Elevated S100A9 expression in CECs mediated by an IL-6/STAT3 signaling cascade may play an important role in the development of colitis. Crohn's disease (CD) and ulcerative colitis (UC), which develop as the result of chronic inflammatory reactions in the gastrointestinal tract and are defined collectively as inflammatory bowel disease (IBD), are among the most common autoimmune diseases worldwide Immune cells, which infiltrate the inflamed guts of patients with IBD, produce various cytokines and chemokines that trigger inflammatory responses STAT3 region and clinical phenotypes of CD and UC, as well as those of multiple sclerosis (MS) IL-6 plays an important role in enhancing T-cell survival and apoptosis resistance in the lamina propria at the inflamed site S100A8  and S100A9 (MRP14) are members of the S100 family of calcium-binding proteins and exist mainly as a S100A8/S100A9 heterodimer  in the extracellular milieu The S100A8/S100A9 heterodimer as well as S100A9 induce neutrophil adhesion to fibrinogen by activating the β2 integrin Mac-1 and adhesion molecules , as well as proinflammatory chemokines Apart from their known functions under inflammatory conditions, little has been proven about the expression mechanism of S100A9 in intestinal epithelial cells (IECs), which are important in intestinal homeostasis We established DSS-induced colitis shown a loss of approximately 20% body weight on day 11A, left. To characterize the expression of the effector cytokine IL-6 during DSS-induced colitis, the mRNA expression of IL-6 was evaluated by conventional PCR. We found that IL-6 concentrations increased significantly in the colon tissue of DSS-treated mice , althougPY705 by immunofluorescence and immunoblotting. STAT3 was highly activated in the CEC regions of colon tissues after DSS exposure for 6 days, whereas it was not expressed in control mice . Sustained STAT3 activation was observed until 60 min  cell line at concentrations varying from 32 to 1,024 ng/ml. Cell migration was induced directional migration, depending on concentration of S100A9 , inducing the differentiation of adoptive immune cells , and enhancing T-cell survival. This evidence provides insight into IL-6 function in multiple steps of UC generation and suggests a rationale for therapeutic approaches using IL-6 blockades, which could be effective treatment modalities for patients with UC.Our results provide supporting evidence for the crucial role of IL-6 in the generation of colonic inflammation, as IL-6 blockade delayed disease onset and attenuated colitis-associated symptoms in DSS-treated mice . This fiXL and cyclin D However, IL-6 has a protective effect on enterocytes and IECs There are several possible explanations for the difference between our findings and the results of previous studies of the blockade of IL-6 signaling or STAT3 activation. It could stem from the use of knockout mice, the administration of the soluble receptor blocker (sgp130Fc), or different methods of targeting the S100A9 molecule. In intestinal inflammation, S100A9 is an effector molecule that enhances TLR signaling In conclusion, our results suggest the existence of an immune-triggering mechanism mediated by S100A9 released by CECs that helps to scavenge invading microorganisms when epithelial barriers are impaired. This mechanism may also induce further undesirable destruction in epithelial linings, leading to the exacerbation of colitis symptoms. Further studies should investigate how this mechanism can be controlled without interfering with immune surveillance in CECs. Our findings suggest that drugs inhibiting STAT3 signals may be good candidates for controlling disease activities in patients with UC while avoiding CAC development.A Caco-2 human IEC line was obtained from the Korean Cell Line Bank . Human embryonic kidney cell line 293T and human colon cancer cell line HCT116 were purchased from the American Type Culture Collection . The cells were cultured in appropriate media with 10% heat-inactivated fetal bovine serum .DSS  was used to induce experimental colitis in mice. IL-6  was used to stimulate Caco-2 cells. The following inhibitors were used to treat Caco-2 cells throughout IL-6 stimulation: S3I and STAT3 inhibitor peptide (Merck) to block phosphorylation by targeting a tyrosine residue and to dimerize STAT3, respectively To prepare small interfering (si) RNA-incorporated chitosan nanoparticles (CH-NPs), siRNAs for STAT3 and S100A9 were purchased from Bioneer Corporation . siRNA-incorporated CH-NPs were prepared according to a previously described method Six-week-old male C57BL/6 mice were purchased from Joongang Laboratory Animal Co.  and maintained under specific pathogen-free conditions. As described previously 2+- and Mg2+-free Hanks Balanced Salt Solution  containing 2% calf serum at 37°C for 30 min while stirring. After collecting the supernatant, remaining tissue was resuspended in HBSS with 3 mM ethylenediaminetetraacetic acid (EDTA). The tissues were incubated while stirring at 37°C for 30 min. The supernatant was then collected and centrifuged at 300×g for 10 min.CECs were isolated using a modified version of previously described methods PY705) PY705 immunoglobulin G  and stained with 3,3-diaminobenzidine substrate. Image acquisition and processing were performed using a Leica microscope and the Leica Application Suite .Large intestine specimens were fixed in 10% neutralized formalin for 24 h, embedded in paraffin, and sectioned at 4 µm. Colon sections were stained with hematoxylin and eosin (H&E) for histopathological analysis, and stained immunohistochemically as described previously to investigate the expression of phospho-STAT3  mounting medium  and analyzed under a confocal laser scanning microscope . Images were taken under a confocal microscope using a 40× objective.To perform immunofluorescence analyses, mouse large intestine specimens were embedded in optimal cutting temperature compound  and sectioned at 10 µm by cryostats (Leica Microsystems). The sections were incubated with a combination of anti-Gr-1-biotin and purified anti-CD11c antibodies (BD Biosciences) or with rabbit anti-STAT3PY705 and visualized using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific).Cells were harvested in a lysis solution  in the presence of a protease inhibitor cocktail  and phosphatase inhibitor (Santa Cruz Biotechnology). After incubation for 30 min on ice, insoluble debris was removed by centrifugation for 15 min at 4°C. Total protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The membranes were then probed with antibodies against α-tubulin , STAT3 , or STAT3t quantification AccuPower PCR Premix (Bioneer). The specific primers for genes used in this study were obtained from QIAGEN.Total RNA was isolated from colon tissues and cDNA was synthesized using a QuantiTech Reverse Transcription Kit , then mixed with QuantiFast SYBR Green polymerase chain reaction (PCR) master mix (QIAGEN) and specific primers. Quantitative reverse-transcription PCR (qRT-PCR) was performed with an Applied Biosystems 7300 Real-Time PCR System  and analyzed by comparative CCell culture supernatants were analyzed for S100A9 levels using an S100A9 ELISA kit  according to the manufacturer's instructions.PY705 IgG or rabbit IgG. Cross-links were reversed by incubating at 65°C. Precipitated DNA was amplified by conventional PCR using specific primers covering the canonical STAT3 binding motif within the S100A9 promoter: forward, 5′-ACACATCTTGCCCACCAGGAGGCTA-3′; reverse, 5′-AAATCCTGCTCCCAGCAAGGGTTCC-3′.Chromatin immunoprecipitation (ChIP) assays were performed using a Chromatin Immunoprecipitation Assay Kit  according to the manufacturer's instructions. Briefly, Caco-2 cells were fixed with formaldehyde for 10 min at room temperature. After sonication (30 s on/1 min off for three cycles), the fragmented soluble chromatin was immunoprecipitated with anti-STAT3P values <0.05 were considered statistically significant. Two-tailed Student's t-tests were conducted using the GraphPad Prism software .Data are presented as means ± standard deviations (SDs). Figure S1Changes in interleukin-6 (IL-6) expression in the colons of an experimental ulcerative colitis mouse model established by dextran sulfate sodium (DSS) treatment. (A) The difference (%) between baseline and subsequent body weight was measured daily for 11 days (left). Changes in clinical symptoms are represented by the disease activity index . Data are presented as means ± standard deviations of values from six mice per group. *p<0.01, **p<0.001 vs. control mice (two-tailed Student's t-test). PBS, phosphate-buffered saline. (B) Colon histology was examined in control and DSS-treated mice at 6 or 12 days after DSS administration by hematoxylin and eosin (H&E) staining of paraffin-embedded sections. Scale bars = 100 µm. Data are representative of three independent experiments. (C) In each of three groups, colon tissues were homogenized using a tissue homogenizer. IL-6 and GAPDH mRNA levels in each group of colonic tissue samples was analyzed by conventional reverse-transcription polymerase chain reaction assays. Data are representative of three independent experiments.(EPS)Click here for additional data file.Figure S2The purity of the isolated colonic epithelial cells (CECs) from a mouse model of dextran sulfate sodium (DSS)-induced colitis. (A) Epithelial cells were isolated from colon tissue specimens from DSS-treated or control mice, and the purity of the isolated CECs was confirmed by staining with fluorescein isothiocyanate-labeled antibodies against E-cadherin  by flow cytometry. Numbers indicate the percentage of positive cells in the histogram. Data are representative of three independent experiments. (B) CD45 and villin mRNA expression was analyzed by conventional reverse-transcription polymerase chain reaction (RT-PCR) for isolated CECs and intraepithelial lymphocyte (IEL), which is control cell expressing CD45.(EPS)Click here for additional data file.Figure S3Effect of soluble gp130-Fc (sgp130Fc) injection on the expression of CXCL10 in colonic epithelial cells (CECs) from mice with dextran sulfate sodium (DSS)-induced colitis. CECs from mice treated with or without 3% DSS for 8 days and a subsequent sgp130Fc or PBS injection were prepared, and CXCL10 mRNA expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Data are presented as means ± standard deviations of values from six mice per group. P values were obtained using the two-tailed Student's t-test.(EPS)Click here for additional data file.Figure S4S100A9 is potent chemotactic factors for human promyelocytic leukemia (HL-60) in vitro. Chemotaxis assays were performed using CytoSelect Cell migratory Assay Kit . Media (control) or increasing concentration of S100A9  were added to the lower chamber. HL-60 cells were added to the upper chamber (5×103 cells) and allowed to migrate through the membrane (5 µm pore size) for 6 h. Migrated cells were stained with trypan blue and counted with hemocytometer. Results are the mean % of migrated cells. Data are presented as means ± standard deviations of values from triplicate experiments. P values were obtained using the two-tailed Student's t-test.(EPS)Click here for additional data file.


TNF-α and TNFRSF1B genes that alter gene expression are likely to be associated with risk and clinical outcomes of cancers. However, few reported studies have investigated the association between potentially functional SNPs in both TNF-α and TNFRSF1B and prognosis of NSCLC patients treated with chemoradiotherapy.The dysregulation of gene expression in the TNF-TNFR superfamily has been involved in various human cancers including non-small cell lung cancer (NSCLC). Furthermore, functional polymorphisms in TNF-α and TNFRSF1B genes  in 225 NSCLC patients treated with chemoradiotherapy or radiotherapy alone. Kaplan-Meier survival analysis, log-rank tests and Cox proportional hazard models were used to evaluate associations between these variants and NSCLC overall survival (OS).We genotyped five potentially functional polymorphisms of TNFRSF1B +676 GG genotype was associated with a significantly better OS of NSCLC . Further stepwise multivariate Cox regression analysis showed that the TNFRSF1B +676 GG was an independent prognosis predictor in this NSCLC cohort , in the presence of node status  and tumor stage .We found that the TNFRSF1B +676 T>G (rs1061622) in the prognosis of NSCLC. Further large and functional studies are needed to confirm our findings.Although the exact biological function for this SNP remains to be explored, our findings suggest a possible role of  Lung cancer is the most common tobacco-induced cancer and the leading cause of cancer-related deaths worldwide, with an estimated 1.61 million new cases and 1.38 million deaths in 2008 ; howeverTNF and TNFRSF1B may play a role in patients' treatment response, toxicity, and survival. Thus, genetic variations in TNF and TNFRSF1B that alter gene expression and/or protein production may be potential candidates for prognosis predictors of NSCLC patients.Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine produced by activated macrophages and exerts its action through binding to its two cognate cell surface receptors, TNFRSF1A/TNFR1 p55/60) and TNFRSF1B/TNFR2 (p75/80). It is well known that TNF and its superfamily members have both beneficial and harmful activities, playing a role as a "double-edged sword" 5/60 and . For exaTNF-α and TNFRSF1B genes are highly polymorphic, and several functional single nucleotide polymorphisms (SNPs) in these two genes have been identified, which may contribute to differences in expression levels of the genes or protein products [TNF-α SNPs (SNP -308 G>A and -1031 T>C in the promoter region) and one TNFRSF1B SNP (+676 T>G in exon 6), which have been widely investigated for their associations with susceptibility to and progression and prognosis of various cancers [TNF-α, and +676 T>G) as well as the other two potentially functional SNPs (-1709A>T and +1663A>G in TNFRSF1B) are associated with overall survival of NSCLC.products -20. Of a cancers -37. HoweEpidemiological and clinical data were available from a larger dataset of 576 NSCLC patients who were treated with definitive radiation at The University of Texas M.D. Anderson Cancer Center  between 1998 and 2006. The detailed information about these subjects has been described elsewhere . After thttp://www.ncbi.nlm.nih.gov/, build 131) and Hapmap database  for common, potentially functional SNPs with a minor allele frequency (MAF) ≥ 0.05 in CEU populations, i.e., those SNPs located in the 5' near gene, 5'- and 3'-untranslated regions and coding region of TNF-α and TNFRSF1B. Meanwhile, linkage disequilibrium (LD) analysis and bioinformatics prediction by SNPinfo (http://snpinfo.niehs.nih.gov/) [TNF-α -308 G>A, TNF-α -1031 T>C, and TNFRSF1B +676 T>G, TNFRSF1B -1709A>T and TNFRSF1B+1663A>G) were selected for genotyping. Among these, three SNPs are located in the promoter regions of the genes , which are predicted to affect the binding of some transcription factors. One SNP (TNFRSF1B +676 T>G) causes a missense change and the other (TNFRSF1B +1663A>G) is located in the 3'-untranslated region, which may affect miRNA binding.We screened the NCBI dbSNP database (ih.gov/)  were alsTNF-α, and -1709A>T in TNFRSF1B ) were determined by the primer-introduced restriction analysis (PIRA)-PCR assay. The primers used for TNF-α -308 G >A, TNF-α -1031 T>C and TNFRSF1B -1709A>T were 5'-GCAATAGGTTTTGAGGGCCATG-3' (sense) and 5'-TTTGGAAAGTTGGGGACACACA-3'(anti-sense); 5'-GGGAAGCAAAGGAGAAGCTGAGAACA-3' (sense) and 5'-GGGGGGTCCCCATACTCGACTT-3' (anti-sense); and 5'-CCACACACCTCAAGACCAATGAG-3' (sense) and 5'-CTTGAATTCGTTCCCAGGATGG-3' (anti-sense), respectively. The mismatched G, C and A were respectively introduced into the sense primers at 6 bp, 2 bp and 2 bp from these three polymorphic sites to create NcoI, NlaIII and MlyI restriction sites. Additionally, TNFRSF1B +676T>G and +1663A>G were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with the primers 5'-CTCCTCCTCCAGCTGTAACG-3' (sense) and 5'-CCAACTGGAAGAGCGAAGTC-3' (anti-sense), and 5'-AGGCCCCCACCACTAGGACTCT-3' (sense) and 5'-GTTGTGGAAAGCCTCTGCTGC-3' (anti-sense), respectively. The following PCR conditions were performed: 5 min of initial denaturation at 95°C followed by 35 cycles of 30 s at 95°C; 45 s at 64°C (TNF-α -308 G >A) or 59°C (TNF-α -1031 C>T) or 64°C (TNFRSF1B +676 T>G) or 59°C (TNFRSF1B -1709A>T) or 62°C (TNFRSF1B +1663A>G), respectively; 45 s at 72°C; and a final 10-min step at 72°C for final extension.Genomic DNA was isolated from the buffy coat fraction of each blood sample with a Blood Mini Kit  according to the manufacturer's instructions. Genotypes of the three selected SNPs  and MspA1I for TNFRSF1B (+1663A>G), and the products were separated in 3% MetaPhor agarose gel. After completion of gel electrophoresis, the TNF-α -308 GG genotype produced two bands (127- and 18-bp), AA produced a single band (145-bp), and AG displayed all three bands   , GG produced a single band (248-bp), and TG displayed all three bands     Figure ; TNFRSF1) Figure ; and TNF) Figure Genotyping was performed without knowing the subjects' disease status. Two research assistants independently read the gel pictures and performed the repeated assays, if they did not reach a consensus on the tested genotype. About 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant.2 tests were performed to determine any statistically significant differences in the distributions of the TNF-α and TNFRSF1B genotypes by demographic variables and clinical features. Median survival time (MST) was calculated, and mean survival time was presented when the MST could not be calculated. Kaplan-Meier estimates were used to estimate overall survival (OS) among four genotype groups, and the log-rank test was used to test for equality of the survival distributions. Univariate analysis and multivariate Cox proportional hazards models were conducted to estimate the effect of each genotype on survival with or without the presence of known prognostic factors. Cox stepwise hazards regression model was also conducted to determine independently predictors of NSCLC prognosis, with a significance level of 0.050 for entering and 0.051 for removing the respective explanatory variables. Haplotype frequencies and individual haplotypes based on the observed genotypes were generated by using SAS PROC HAPLOTYPE. Analyses were performed by using SAS statistical package version 9.1 .The two-sided χth edition stage grouping criteria [P = 0.117), ethnicity (P = 0.827), smoking status (P = 0.457), histology (P = 0.092), Karnofsky's performance scores , chemotherapy status (P = 0.407) and radiotherapy dose (P = 0.706). However, sex, tumor stage, and node status were significantly associated with MST of NSCLC . Because these variables may be confounding factors for the effect of the genotypes on OS, they were further adjusted in the multivariable analysis.As shown in Table criteria , 33 caseTNF-α and TNFRSF1B and their associations with OS are summarized in Table TNFRSF1B +676 G>T (rs1061622) were statistically significantly associated with OS , but this association was not observed for the other four SNPs investigated in this study.The genotype distributions of the five SNPs in TNFRSF1B +676 T>G (rs1061622) on NSCLC survival. Three variables  were identified in the regression model with a significance level of 0.050 for entering and 0.051 for removing a variable   genotypes and selected factors including age, ethnicity, tumor stage and node status. The longest survival was observed in subgroups with both rs1061622 GG and age≤62 years (MST = 44.0 months), or white ethnicity (MST = 48.3 months), or T0-2 tumors (MST = 48.3 months), or N2-3 tumors (MST = 44.0 months), compared with the poorest survival in subgroups with both rs1061622 GT/TT and age>62 years (MST = 20.7 months), or non-white (MST = 21.0 months), or T3-4 tumors (MST = 18.2 months), or N0-1 tumors (MST = 29.2 months); however, small sample size in these subgroups might have led to a suboptimal statistical power to detect a statistical difference in the survival time. We also conducted the stratification analysis by histology and different treatments, but the numbers of patients in subgroups were too small, and no significant results were found in every stratum (data not shown).As shown in Figure TNF-α -308G/-1031T (GT) and TNFRSF1B +676T/+1663A/-1709A (TAA) were the most common haplotypes in the patients with the frequencies of 63.3% and 40.2%, respectively. However, we did not found significant associations between other haplotypes and OS of NSCLC patients, compared to the most common haplotypes (data not shown), which is likely due to limited study power.In addition, we also evaluated the combined effect of these polymorphisms on NSCLC survival by using the haplotype analysis. The results showed that TNF-α and TNFRSF1B on survival of NSCLC patients treated with chemoradiotherapy or radiotherapy alone. We found that the TNFRSF1B +676 GG (rs1061622) variant homozygous genotype was associated with a significantly improved survival of NSCLC in this non-Hispanic patient population. Such an effect was not observed for other SPNs under investigation.In the present study, we examined the effect of five selected polymorphisms in TNF-α is mostly regulated at the transcriptional level, and polymorphisms within the TNF-α promoter have been related to TNF-α levels [TNF-α-308 G/A in the promoter of TNF-α has been reported to be associated with higher expression levels of TNF-α [TNF-α-308 G/A and another SNP in the promoter of TNF-α  were significantly associated with prognosis of bladder cancer and breast cancer [TNF-α-308 G/A or TNF-α-1031 T>C and the prognosis of several other cancers, including gastric cancer [Tumor necrosis factor-alpha (TNF-α) executes multiple functions in immunity, inflammation, differentiation, control of cell proliferation, and apoptosis through distinct receptors known as TNF receptor type I (TNFR1) and type II (TNFR2) . Expressα levels ,23. For of TNF-α  and thusof TNF-α , breastsof TNF-α , oral caof TNF-α ,32, bladof TNF-α  and lungof TNF-α . Additiot cancer ,29,36, ct cancer . Howeverc cancer , colorecc cancer , Hodgkinc cancer , and brec cancer . Such a TNFRSF1B rs1061622 GG might be an independent predictor for survival of NSCLC patients treated with chemoradiotherapy or radiotherapy alone. The TNFRSF1B is the principal TNF receptor found on circulating T lymphocytes, which functions both as a TNF antagonist by neutralizing it and as an agonist by facilitating interaction between TNF and TNFR1 at the cell surface [TNFRSF1B (M196R) that causes a functional amino acid change from methionine (M) to arginine (R) [Interestingly, logistic and stepwise regression analysis in our study indicated that  surface . rs10616nine (R) , which inine (R) ,48.TNFRSF1B +676 T>G (rs1061622) with susceptibility and survival of breast cancer patients [TNFRSF1B +676 T>G (rs1061622) and lung cancer survival, and the exact biological mechanism underlying this association in our study remains to be investigated. It is possible that this SNP affects the function of TNFRSF1B and enhances radiotherapy or chemotherapy efficacy through inhibiting tumor therapy resistance. Another possibility is that this SNP may change the expression level of TNFRSF1B and influence the character of immunological response to infection in lung cancer patients, finally resulting in the difference of survival in NSCLC [TNFRSF1B +676 T>G (rs1061622) genotypes and selected factors including age, ethnicity, tumor stage and node status. Although our sample size was relatively small in these subgroups, it provided a clue that effect of this SNP on prognosis of NSCLC may be dependent on patients' characteristics and clinical features as well. However, the exact functional relevance of this SNP in the prognosis of NSCLC patients treated with chemoradiotherapy remains to be explored.However, the results from published studies on associations between rs1061622 and prognosis of cancers were controversial rather than conclusive. For example, a Tunisian study showed significant associations of patients , but anopatients . Up to nin NSCLC . FurtherTNF and TNFRSF1B genes in NSCLC patients, several potential limitations should be taken into consideration. Firstly, our sample size is relatively small with a limited statistical power to evaluate interactions between the studied polymorphisms and clinical factors including tumor stage and node status. For the same reason, the results from our subgroups by TNFRSF1B +676 T>G (rs1061622) genotypes and selected factors need validation by larger studies. Secondly, only five potentially functional SNPs in TNF and TNFRSF1B were included in our study, which is far from comprehensive. Indeed, both genes are highly polymorphic, and it is possible that some important SNPs may have been missed or the observed association may have been due to other polymorphisms in LD with the studied ones. Again, much larger studies would have the statistical power to confirm the associations of more SNPs with OS in such a patient population.Although our study reported some new findings about polymorphisms of TNFRSF1B +676 T>G in the prognosis of NSCLC. However, replication studies with diverse ethnic groups, larger sample sizes and functional characterizations are warranted. Currently, we have ongoing projects that help continue recruiting similar patients for additional analysis in the future.In summary, in this cohort of NSCLC patients, we identified a possible role of TNF-α: Tumor necrosis factor-alpha; TNFR2: tumor necrosis factor receptor super family 2; PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism; HR: hazard ratio; CI: confidence interval; SNP: single nucleotide polymorphism; NSCLC: non-small cell lung cancer; OS: overall survival.The authors declare that they have no competing interests.XXG, QYW and ZXL designed the study and prepared the manuscript. XXG performed the experiments with the assistance from ZSL and JQ. XXG and HXM conducted the analysis with assistance from LEW. XLY, DG and RK helped to collect and organize the clinical data. HXM, QYW and LEW helped to finalize the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/447/prepub


We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009–2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness. Sporadic transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses from birds to humans has been documented since 1997 (Genetic and antigenic divergence of HPAI (H5N1) viruses among poultry challenges development of effective vaccines for poultry and to pandemic preparedness and development of antiviral drugs for humans. Assessment of drug susceptibility has become an integral part of subtype H5N1 virus surveillance. To assist laboratories worldwide in their surveillance and pandemic preparedness efforts, the Influenza Division of the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, along with other partners, developed the H5N1 Genetic Changes Inventory that includes established and potential markers of drug resistance  viruses isolated from poultry specimens collected in Vietnam during 2009–2011. The antiviral drugs tested included FDA-approved medications and investigational antiviral agents. We report the detection of an oseltamivir-resistant virus with H275Y mutation from the expanding clade 2.3.2.1.Viruses collected from poultry on farms, in backyard flocks, and in live-poultry markets in Vietnam during 2009–2011 were identified as HPAI A(H5N1) at the National Center for Veterinary Diagnostics (NCVD), Vietnam, by using the World Health Organization (WHO) protocol (www.megasoftware.net) neighbor-joining methods implemented with 1,000 bootstrap replicates. Phylogenetic data for the strain A/goose/Guangdong/1/1996 (clade 0) were used as a reference for tree rooting and numbering, and trees were annotated according to the WHO/World Organisation for Animal Health/Food and Agriculture Organization of the United Nations criteria  and assembled by using Sequencher 5.0 . Phylogenetic trees were generated by using MEGA version 5.0 (50) values, the drug concentration needed to inhibit virus NA activity by 50%, were determined by using a CDC in-house program, the JASPR v1.2 curve-fitting software  to identify outliers, using a statistical cutoff value U = Q3+3.0*(interquartile range). The interquartile range was determined as Q3–Q1; Q1 or Q3 denoted 25th or 75th percentile, respectively. The resulting value was applied for clade and drug. Mild outliers were defined as viruses that had IC50 values >U and <10 times the median, and extreme outliers as having IC50values >U and ≥10 times the median. SigmaPlot 12  was used to generate box-and-whisker plots to visualize outliers. The median/mean IC50 values among virus clades were analyzed by using the Kruskal-Wallis 1-way analysis of variance and the Dunn’s multiple comparison test, respectively.Susceptibility to the drugs zanamivir , oseltamivir , peramivir , and laninamivir  was assessed by fluorescent neuraminidase inhibition (NI) assay, by using inhibitor concentrations ranging from 0.03 nmol/L to 1,000 nmol/L (50/mL) in MDCK-SIAT1 cells. The 90% effective concentration (EC90) of drug (drug concentration that reduces the infectious virus yield by 90%) was determined by using the 4-parameter logistic nonlinear regression model equation in GraphPad Prism 5 software .Susceptibilities to the M2 blocker amantadine and to investigational agents DAS181 and favipiravir (T705) were assessed in a virus yield reduction assay on Madin-Darby canine kidney (MDCK) SIAT1 cells (50/mL) were determined by titrating the supernatants on MDCK-SIAT1 cells (6 TCID50 of either WT or H275Y virus. Virus titers (TCID50/mL) were measured in the nasal washes collected postinoculation on days 1–3, 5, 7, and 9.Comparison of replicative capacity of WT and oseltamivir-resistant H275Y mutant virus was achieved by inoculating MDCK-SIAT1 cells at a multiplicity of infection of 0.0001 PFU/cell in 24-well plates. Cells were incubated at 37°C, and supernatants were collected every 12 hours until 72 hours postinoculation. At each time point, the infectious virus yields , NA, and membrane protein, or M genes, is required for interpretation of drug susceptibility data and for uncovering new trends. According to the HA gene phylogeny, viruses isolated from poultry in Vietnam during 2009–2011 were assigned to 3 clades: 1.1 (n = 25), 2.3.2.1 (n = 70), and 2.3.4 (n = 30) , panel BThe M tree was similar to the HA tree and showed evidence of reassortment for a single clade 2.3.4.2 virus that clustered with M genes from subclade 2.3.4.1 , panel C50 values were calculated for each virus and drug. The median IC50 values were similar for oseltamivir and zanamivir  and ≈2-fold lower for peramivir , whereas the range was much narrower for zanamivir (145-fold) and intermediate for peramivir . Analysis of IC50 values was further achieved by individual clade to enable better correlation with NA sequences , we performed the NI assay using oseltamivir, zanamivir, and peramivir. ICeramivir , Table. equences . The 2 r50 value for clade 1.1 was lower than that of clades 2.3.2.1 and 2.3.4, by 7- to 27-fold, respectively. In clade 1.1, virus A/chicken/Vietnam/NCVD-780/2011 was identified as an extreme outlier and showed a 12-fold increased oseltamivir IC50 value  that carried the V424I change were identified within the same clade. In clade 2.3.4 viruses, 4 outliers for oseltamivir were detected, 3 of which possessed I223T, which conferred a 6–7-fold increase in IC50 values. The fourth virus had a V147R substitution and exhibited a 4-fold increase in IC50  matched those of clade 2.3.2.1 viruses  tested with laninamivir, the virus that carried the R430W mutation showed a 29-fold increase, and the virus that had the H275Y mutation showed a 6-fold increase in IC50 values.The virus A/duck/Vietnam/NCVD-664/2010 that carried the H275Y mutation was predictably identified as an extreme outlier for peramivir with a 415-fold increase in IC50 values of the test virus and a reference IC50 value (such as median IC50); different criteria are set for seasonal type A and type B viruses. The reporting for H5N1subtypes is not specified; therefore, we followed the criteria as outlined for seasonal type A viruses, but grouped the IC50 values by clade , which is consistent with the M2 blocker–sensitive genotype. Inoculation of cells with DAS181 before incubation was equally effective in inhibiting replication of the virus with H275Y mutation and the WT virus . Both vi6 µmol/L . For risnfection , panel Anfection , panel BIn this study, we identified a single oseltamivir-resistant virus among 125 HPAI A(H5N1) viruses isolated from poultry in Vietnam during 2009–2011. It was recovered from a domestic duck in Ninh Binh Province, northern Vietnam. This virus belonged to the rapidly expanding clade 2.3.2.1 and contained the H275Y mutation, which is the principal marker of oseltamivir resistance in N1-subtype viruses , similar to previous findings  IC50 value  viruses on susceptibility to NA inhibitors in humans. The range of H5N1 subtype oseltamivir IC50 values of clade 1.1 viruses were 7–27-fold lower than those of clade 2 viruses; this finding is in accord with previous reports , possessing N295S  compared with values of clade 2.3.4 viruses (1.62 nmol/L) and, thus, would be reported as exhibiting normal inhibition. Like the H275Y-mutated viruses previously identified, this variant has also been isolated from an oseltamivir-treated patient pdm09 virus. Acquired additional changes in the NA  may confer a higher level of resistance to the NA-inhibitor class of drugs among these viruses  viruses resistant to M2 blockers are prevalent among poultry throughout Asia, including Vietnam  viruses sampled from poultry on informing antiviral stockpiling decisions for pandemic preparedness. Because 15 countries have reported human cases of HPAI (H5N1) virus infection to date, these findings also emphasize the need to enhance the armamentarium of available anti-influenza drugs worldwide for treatment of subtype H5N1-infected patients, including agents with diverse mechanisms of action, which could enable combination treatment (Drug susceptibility and accession data for highly pathogenic avian influenza A(H5N1) viruses.Analysis of highly pathogenic avian influenza A(H5N1) viruses.


However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca2+ binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons  that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca2+ sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca2+-binding ability in C2A or C2B in SACs. Subsequent live Ca2+ imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca2+ transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca2+ transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca2+ transients.Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca2+ binding to C2A or C2B, the Ca2+ sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.Through Ca Accordingly, the intracellular Ca2+ concentrations exhibit spontaneous and correlated increases, producing wave-like Ca2+ oscillations across the RGC layer 2+ transients in RGCs has been widely used to elucidate the synaptic mechanisms underlying retinal waves Binocular visual circuit refinement requires a robust patterned spontaneous activity in the developing retina . Retinal waves are spontaneous, correlated action potentials propagating through the retinal ganglion cell (RGC) layer during a developmental critical period prior to visual experience. In particular, retinal waves are mediated via synaptic transmission with a periodicity on the order of minutes. In the developing mammalian retina, three different stages of retinal waves have been classified, of which the stage-II waves are the best studied 2+-regulated exocytosis. Members of the Ca2+ sensor protein family synaptotagmin (Syt) transduce the Ca2+ signal to trigger fusion between vesicle and plasma membranes 2+ through their two Ca2+-binding C2 domains (C2A and C2B), with three binding to C2A and two binding to C2B 2+ binding, Syts interact with distinct effectors, such as lipids, SNARE (soluble NSF attachment protein receptor) proteins, assembled SNARE complex, and other Syt molecules to trigger exocytosis Synaptic transmission is initiated by neurotransmitter release via Ca2+ binding to distinct C2 domains in Syt controls the choice of exocytotic modes 2+ binding to C2A reduces full fusion by limiting fusion pore dilation and preventing release of some vesicle content. By contrast, weakened Ca2+ binding to C2B abolishes both full fusion and kiss-and-run 2+ binding to C2A or C2B in Syt I 2+ binding to various C2 domains of Syt confers a repertoire of functions in controlling neurotransmitter release and synaptic transmission Previous studies revealed that preferential Ca2+ spikes and Ca2+-dependent afterhypolarizations 2+-dependent. Borges et al. reported that a small number of primed vesicles, very likely one per release site, can be quickly exocytosed upon Ca2+ influx into SACs 2+ influx into SACs may cause Ca2+ binding to its sensor Syt to drive exocytosis during stage-II waves.Many studies have characterized the role of SACs in regulating wave dynamics during the stage-II period. However, the release mechanism in SACs has not been well understood. Immunolabeling and confocal microscopy suggested that several SNARE proteins are expressed in SACs, such as syntaxin 1 and 2 2+ imaging to investigate the expression of Syt I in SACs and determine the roles of C2AB domains in regulating stage-II waves during the development of neural circuits.Although the human genome encodes more than seventeen Syt isoforms 2+ sensor in triggering neurotransmitter release during stage-II waves.Syt I is the major Syt isoform found in many parts of the nervous system 2+ imaging to measure wave-associated Ca2+ transients in both acutely isolated retinas and retinal explants after 3–4 day in vitro culture. The characteristics of wave-associated Ca2+ transients were not significantly changed after in vitro culture   . These d2+ transients  promoter has been shown to target SACs specifically To manipulate neurotransmitter release from SACs during stage-II waves, we aimed to transfect SACs with Syt I mutants with the weakened CaThe immunoreactivities from retinal cross-sections were used to evaluate our transfection in SACs. The HA/EGFP immunoreactivity represented all transfected cells , whereas ChAT immunoreactivity (in red) indicated SACs. Therefore the transfected SACs would display yellow color . We first determined our transfection efficiency in SACs, which was calculated as the number of cells with yellow signal divided by the number of cells with red signal . The transfection efficiency with the mGluR2 promoter was 57.14±4.56% , compared to 30.25±3.45%  with the CMV promoter.right insets). With the mGluR2 promoter, the promoter specificity for SACs was 83.87±1.61% , compared to 8.79±1.22% (mean ± S.E.M.) with the CMV promoter (N = 6 sections). Thus, the mGluR2 promoter achieved a significantly higher SAC specificity compared to the CMV promoter . This indicates that the mGluR2 promoter can be used to target SACs for molecular perturbation in the developing rat retina.We then calculated the promoter specificity, which was defined as the number of cells with yellow signal divided by the number of cells with green signal  , compared to relatively large RGCs (∼20 µm) stained by the Ca2+ indicator fura-2 , whereas D363N confers a Ca2+-binding site mutation in the C2B domain of Syt I (designated Syt I-C2B*). Each mutated residue abolishes the first ligand of the third loop in the Ca2+-binding pocket of the respective Syt C2 domain 2+ transient frequency in the developing rat retina, live Ca2+ imaging was conducted in the retinal explants after transfection. Spontaneous, correlated Ca2+ transients in individual cells revealed retinal waves in the RGC layer of explants transfected with control vector, wild-type Syt I, Syt I-C2A* or Syt I-C2B*  and the fraction of cells displaying Ca2+ transients . In contrast, both Syt I mutants reduced the mean frequency of Ca2+ transients by 2-fold compared to wild-type Syt I . Thus, weakening Ca2+ binding to either the C2A or C2B domain of Syt I reduced Ca2+ transient frequency. In addition, the cumulative probability for Ca2+ transient frequency was constructed from all recorded cells for control, Syt I, Syt I-C2A* and Syt I-C2B*  was 0.58 for control, 0.55 for Syt I, 0.35 for Syt I-C2A*, and 0.35 for Syt I-C2B* . The mean fraction of cells with Ca2+ transients in control was indistinguishable from intact, untransfected culture retinas . Wild-type Syt I left the fraction of cells with Ca2+ transients identical to that seen in control cells . The mean fraction of cells with Ca2+ transients was significantly reduced in both Syt I-C2A* and Syt I-C2B* compared to control and wild-type Syt I . Hence, weakened Ca2+ binding to either C2 domain in Syt I reduced the fraction of cells with Ca2+ transients. With fewer cells displaying Ca2+ transients and less frequent Ca2+ transients in most cells, these results suggest that weakened Ca2+ binding to the C2AB domains of Syt I may reduce the frequency of stage-II waves.Second, to determine whether Syt I mutants change the fraction of cells participating in waves, we calculated the fraction of cells with Ca2+ transients associated with waves can last for tens of seconds following relatively brief spontaneous depolarizations 2+ transients in an unbiased way . Moreover, cumulative probability distributions were constructed to get the median Ca2+ transient durations   , and the retinas . The curpe Syt I . Thus, w2+ transient amplitude, we compared the mean amplitude from different transfection groups. Similar to the effects on Ca2+ transient duration, wild-type Syt I did not change the Ca2+ transient amplitude compared to control (p>0.05). Both Syt I-C2A* and Syt I-C2B* significantly reduced Ca2+ transient amplitude compared to wild-type Syt I and control  were also reduced in Syt I-C2A* and Syt I-C2B* . To assess wave correlation structure across cells, we computed cross-correlograms over cell pairs Both Syt I-C2A* and Syt I-C2B* dampened wave-associated Ca2+ binding to the C2AB domain of Syt I may not alter the spatial correlation structure of stage-II waves.To further quantify the strength of the correlations, the correlation index (C.I.) 2+ binding in the C2AB domains of Syt I, we tested the effects of presynaptic perturbation on wave-associated Ca2+ transients. Our Ca2+ imaging results provide the first demonstration that Ca2+ binding to C2A or C2B of Syt I regulates the temporal properties of stage-II waves. In contrast, Ca2+ binding to C2AB in Syt I has relatively minor effects on the spatial properties of stage-II retinal waves. These results suggest that the C2AB domains of Syt I regulate the dynamics of patterned spontaneous activity during the development of neural circuits.In this study, we showed that Syt I is strongly expressed in cholinergic neurons (SACs) and synapses (the IPL) in retinas exhibiting stage-II waves. We further established a molecular perturbation method targeted to presynaptic SACs. By weakening Ca2+ transients via Ca2+ binding to its C2AB domains. Together with the previous findings 2+ oscillations in SACs 2+ binding to its sensor Syt I to trigger exocytosis While the importance of Syt I in synaptic transmission has been recognized for a couple of years 2+-dependent manner 2+ binding to Syt I warrants further investigation, especially in the form of kiss-and-run or full fusion.Developing SACs corelease ACh and GABA in a CaManipulations that alter the spatiotemporal properties of stage-II waves result in defective retinogeniculate and retinocollicular projections The roles of the C2AB domains in Syt I have been intensively investigated at the molecular and functional levels. Biochemical analysis showed that both Syt I-C2A* and Syt I-C2B* greatly reduce their binding to t-SNARE heterodimers (corresponding to target proteins on plasma membrane) and PS-containing liposomes (corresponding to vesicle membrane constituent) 2+ dynamics in SACs and thus reduce exocytosis. However, several in vitro and in vivo studies have demonstrated that overexpression of Syt I with Ca2+-binding site mutations can not alter whole-cell Ca2+ current or Ca2+ dynamics 2+ channels or dynamics in SACs.Alternative interpretation for the effects of Syt I mutations on wave dynamics is that overexpression of Syt I-C2A* and Syt I-C2B* might alter cytoplasmic Ca2+ transients in nearby RGCs. We observed that Syt I-C2A* and Syt I-C2B* dampen the temporal properties of stage-II waves  promoter was used for SAC-specific expression  imaging . For Wes imaging , the pCM imaging , the pCMad libitum access to food and water. Rat pups (P0–P2) were deeply anesthetized with ice before decapitation with all efforts to minimize suffering.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for all animal experiments was approved by the Institutional Animal Care and Use Committee of National Taiwan University (Permit Number: 97-27 and 99-82). Neonatal (P0–P2) Sprague-Dawley rats  were used in the study. All rat pups were housed with their own mothers in the individually ventilated cages with well-controlled conditions  and 3, pH 7.35]. The retinal pieces were attached onto nitrocellulose membranes (Millipore) with the RGC layer facing up. Retinal explants were cultured at 35°C in a 5% CO2 humidified incubator, and daily supplied with fresh Retinal Serum-Free Culture Medium (SFCM-A), containing Neurobasal-A (GIBCO #10888), 0.6% Glucose, 2 mM L-Glutamine (Sigma #G6392), 1X B-27 (GIBCO #17504-044), 10 mM HEPES, 1 mM Sodium Pyruvate (GIBCO #11360-070), 2.5 µg/mL Insulin (Sigma #I1882), 100 µg/mL Penicillin/100 µnits/mL Streptomycin (GIBCO #15140-122), and 6 µM Forskolin The retina was isolated and cut into thirds in dissection buffer  for 1 hr. After fixation, the explants were blocked at RT for 1 hr in 3% donkey-serum blocking solution (DBS), containing 3% donkey serum (Jackson Lab #017000121), 0.5% Triton X-100, and 0.1% sodium azide in PBS. The explants were first incubated with primary antibodies in 1% DBS [goat polyclonal anti-ChAT  and mouse anti-HA ] at RT for 2 days, and then washed with PBS. Secondary antibodies in 1% DBS [donkey-anti-goat IgG conjugated with Alexa Fluor 568  and donkey-anti-mouse IgG conjugated with Alexa Fluor 488 ] were further added at RT for 1 day, and then washed out with PBS. The anti-fade reagent Fluoromount G (Electron Microscopy Sciences #17984-25) was added to retinal explants on the slides. The slides were sealed by coverslips. Fluorescent images were acquired by confocal microscopy .For whole-mount retina staining, the transfected retinal explants were placed on poly-lysine-coated slides, fixed with 4% paraformaldehyde (PFA) at RT for 30 min, and washed with phosphate buffered saline  at 4°C overnight. Membranes were washed three times with TBST and then incubated with secondary antibody in 5% milk in TBST at RT for 1 hr. Membranes were washed three times with TBST, and the signals were visualized by enhanced chemiluminescence followed by autoradiography.The P0–P2 rat retinal explants transfected with pCMV-HA or pCMV-HA-Syt I were spun down in dissection buffer at ∼2,000 2+ imaging, retinal explants were placed in the forskolin-free SFCM-A containing 10 µM fura-2-AM (Molecular Probes #F1221), 0.02% pluronic acid (Molecular Probes #P3000MP), and 1% DMSO for 30–60 min. During Ca2+ imaging experiments, retinal explants were perfused continuously with artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 26.2 NaHCO3, 2.5 KCl, 1.0 K2HPO4, 1.3 MgCl2, 2.5 CaCl2, and 11 D-glucose bubbled with 95% O2/5% CO2. The temperature of ACSF was maintained at 30°C with a TC-344B controller (Warner Instruments). Live Ca2+ imaging was performed on an upright Olympus BX51WI, using a 20X water immersion objective (Olympus). The transfected regions in the retinal explants were identified by EGFP fluorescence . The fura-2 fluorescence was excited at 380 nm (Chroma #D380xv2) via a xenon arc lamp  and a 455DCLP dichotic mirror (Chroma). The fura-2 emission fluorescence was collected at 510 nm (Chroma #D510/40 m) and captured by a CCD camera . To measure spontaneous Ca2+ transients associated with retinal waves, the fura-2 fluorescence images were continuously acquired at 1 sec-intervals for 10 min , with 100–150 msec exposure times and two as binning number. One or two transfected regions from each retinal explant were chosen for 10-min recordings. Digitized imaging data were subsequently processed by MetaMorph software. Further data analysis was conducted by Igor Pro (WaveMetrics), Excel and Orgin 5 or 8 (OriginLab).Retinal explants were transferred to forskolin-free SFCM-A overnight before imaging. Prior to Ca2+ imaging data for each transfection group were acquired by the following procedures. First, 49 RGCs were randomly selected from the 7×7 divisions in one imaged region (340×460 µm) using MetaMorph. The wave data from single RGCs were acquired from the fluorescence changes across all the time frames, previously background subtracted for each frame. The fluorescence signals were further corrected for photobleaching by using Igor Pro to set the baseline. Second, to analyze wave characteristics (frequency/duration/amplitude) in an unbiased way, an Igor procedure was written in this laboratory to automatically pick the wave peaks, with their fluorescence intensity 2-fold greater than the RMS noise (∼0.25% ΔF/F). The definitions of the duration and amplitude for individual waves are presented in The Ca2+ waves between nearby cells was calculated from pairs of RGCs in the same imaged region separated by various distances (10–500 µm). The positions of individual cells were calculated using MetaMorph. The intercellular distance was taken as the shortest distance between selected cells. The lag times of the waves in these two nearby cells were acquired by individually subtracting from the presenting time of every wave in the paired cells. All the subtracted times were collected for each cell pair to construct cross-correlograms from one imaged region (t  = 3 sec), as given by the following equation:NAB is the wave number for which cell B fires with a time ± Δt from cell A, NA and NB are the total numbers of waves fired by cells A and B, respectively, during the total recording time (both NA and NB ≥ 3 in 10 min), T is total recording time (600 sec), and Δt is the correlation time interval (3 sec). The cell pairs from one imaged region were grouped according to their intercellular distances with a bin width of 50 µm. The median C.I. values were computed from each distance group out of the same imaged region. The averaged C.I. values were computed from the same distance group out of many imaged regions for each transfection group, and then plotted against the upper limit of intercellular distance for each distance group .All data were presented as mean ± S.E.M. Differences between means of different groups (≥3 groups) were evaluated for statistical significance with One-way ANOVA followed by Figure S12+ transients in acutely isolated retinas (Acute) and retinal explants after 3–4 day Comparison of wave-associated Cain vitro culture (Culture). (A) Ca2+ transient frequency. (B) Fraction of cells with Ca2+ transients. (C) Ca2+ transient duration. (D) Ca2+ transient amplitude. (E) Pairwise C.I. values as a function of intercellular distance. The methods to measure the properties of Ca2+ transients were described in p<0.05, unpaired t-test for comparison at a given distance; n.s., not significantly different from acutely isolated retinas)(TIF)Click here for additional data file.


Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010–2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene.Among the 201 consecutive RSV isolates studied, RSV-A (55.7%) was more commonly observed than RSV-B (42.3%). 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication. Paramyxoviridae family, is an enveloped virus with a negative-sense single-stranded RNA genome which encodes for 11 proteins. Two groups, RSV-A and RSV-B, have been described on the basis of reactions with monoclonal antibodies against the G and F glycoproteins Human Respiratory Syncytial virus (RSV) is the major cause of lower respiratory tract infection (LRTI) in infants and young children, and is also responsible for a significant proportion of RTIs in the elderly. It causes repeated infections throughout life due to limited immune protection from earlier RSV exposure RSV-A and RSV-B evolved separately at different time periods The mature G glycoprotein consists of three unique regions consisting of the cytoplasmic tail (amino acids [AAs] 1–38), transmembrane domain (AA 38–66), and the ectodomain (AA 66–298). The C-terminal ectodomain of G protein is comprised of 2 variable regions flanking the putative receptor binding site, a conserved region of 13 AAs (AA 164–176) situated between them. Although the G protein is highly glycosylated with N- and O-linked sugars, these positions are poorly conserved In this study we evaluated the genetic variability in the G protein gene of RSV-A viruses isolated from clinical samples collected in Ontario, Canada. Phylogenetic analysis was performed to establish the relationships between Ontario's strains and previously described RSV-A genotypes deposited in Genbank. In depth positive selection pressure analysis was also done to examine the replacement behavioural patterns of G protein gene encoded AAs. Further, we tried to derive a possible mechanism for the occurrence of an observed G gene duplication by viral RNA secondary structure analysis.This study was considered exempt from 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.Public Health Ontario performs a large proportion of primary respiratory viral testing for the province of Ontario from a variety of clinical settings including ambulatory, hospital and outbreaks. All consecutive HRSV culture isolates, identified from November 2010 to February 2011 at Public Health Ontario Laboratory – Toronto (PHOL), were selected for this study. . Following the testing algorithm for respiratory specimens, nasopharyngeal swabs were forwarded directly to PHL for respiratory viral testing. All nasopharyngeal swabs (NPS) from ambulatory and hospitalized, non-ICU patients are cultured for virus isolation in two cell lines, 1. either rhesus monkey kidney (RMK) or African green monkey kidney cells (AGMK), along with 2. WI-38 human embryonic lung fibroblast . Cell lines showing cytopathic effect are stained with a blend of murine monoclonal antibodies (MAbs) directed against seven respiratory viruses plus separate DFA Reagents, each consisting of MAb blends directed against a single respiratory virus, including RSV . In addition to viral culture, all NPS from infants under 12 months of age with bronchiolitis or pneumonia, and when requested in children ≤5 years of age, are initially screened by a rapid RSV antigen test . Samples submitted from patients in the outbreak or intensive care unit (ICU) setting undergo multiplex molecular testing for respiratory viruses, but not viral culture, and were not evaluated in this studyTotal nucleic acid was extracted from 250 ul of the supernatant of each cell-cultured sample using the NucliSens easyMAG extraction system  according to manufacturer's instructions. Sub-grouping was undertaken targeting the nucleocapsid (N) gene using a modified duplex version of a previously published method A 900-bp fragment of the G gene and a 500-bp fragment of the F gene of RSV-A was amplified with the OneStep RT-PCR kit (QIAGEN). Primer G267 corresponds to bases 247 to 267 in the G glycoprotein of the A2 strain (Genbank accession number M11486) and F164 primer complementary to bases 164 to 186 in the F protein Express Software . Multiple sequence alignments of the 264 nucleotides in the second hypervariable region of G gene compared to available reference genotypes were performed by the ClustalW algorithm. Phylogenetic analyses using the neighbor-joining method, and the statistical significance of the tree topology tested by bootstrapping  were performed using the MEGA 5.05 software The nucleotide sequences of a fragment of the second hypervariable region of G glycoprotein gene (264 nucleotides corresponding to codon positions 210 to 298) from RSV-A isolates were determined and compared with those of reference strains representing different RSV-A genotypes deposited in Genbank. Sequence editing was performed using Vector NTI® p0, with ω0 = 0, and the remaining sites with proportion p1 (p1 = 1−p0), assuming ω1 = 1. The M2a model facilitates detection of an extra class of sites under positive selection with proportion p2 (where p2 = 1−p1−p0) with ω1>1. The model M7 incorporates a beta distribution (with parameters p and q) to account for variable ω among neutral or negatively selected sites. The model M8, allows positively selected sites with proportion p2, with ω2>1. Likelihood ratio tests (LRT) between nested models (M1a vs. M2a and M7 vs. M8) were conducted by comparing twice the difference in log-likelihood values (2Δl) against a chi-square distribution with two degrees of freedom (d.f.) equal to the difference in the number of parameters between models In order to understand the selection pressure at codon sites, we used the multiple aligned dataset of all Ontario G-gene  sequences including NA1 as a reference sequence and the maximum likelihood (ML) tree as input for the CODEML program of Phylogenetic Analysis by Maximum Likelihood (PAML 4.4 version) Potential N-glycosylation (Asn-Xaa-Ser/Thr) and O-glycosylation sites were predicted using NetNGlyc 1.0 http://www.dbs.umt.edu/research_labs/wrightlab/upload/mfg.htmlRNA secondary structures were predicted using the MFOLD web server Representative sequences of RSV-A isolates obtained in this study have been submitted to Genbank under accession numbers JN257682–JN257692 for G-gene and JN257693–JN257703 for F-gene sequences.Two hundred and three consecutive RSV-positive NPS specimens were identified at PHL between November 2010 and February 2011 from non-ICU, non outbreak patients. Of these, four were positive by the RSV rapid test but were RSV-negative by culture and PCR, and excluded from the study. Among the 199 consecutive RSV isolates included in this study, 47 (23.6%) were obtained from patients reviewed in the emergency room but not hospitalized, 80 (40.2%) collected from hospitalized (non ICU) patients and 21 (10.6%) collected from an ambulatory community setting. There were no data available for the remaining 55 (28%) specimens. RSV-A and B co-infection was identified in 2 samples, which were not evaluated further. One hundred and twelve (55.7%) and 85 (42.3%) of the remaining 197 isolates were identified as subgroup A and B, respectively. Among RSV-A positive specimens, 67 (59.8%) were from infants below 12 months of age, 23 (20.5%) from children 12 to 24 months old, 11 (9.8%) from children 3 to 5 years of age, and 4 (3.6%) were from children of 6 to 10 years of age. Only 2 and 5 isolates were obtained from adults 51 to 66 and 68 to 100 years of age, respectively.GTCAAGAGGAAACCCTCCACTCAACCACCTCCGAAGGCTATCTAAGCCCATCACAAGTCTATACAACATCCG) in the C-terminal end of the G gene. The duplication starts after residue 850 of the G gene (RSV-A2 prototype numbering) and appears to disrupt the codon “GAG” (residue 850–852) coding for E284, switching it to “GGT” and coding for G284, which is followed by a duplication of 23 AAs (QEETLHSTTSEGYLSPSQVYTTS) spanning positions 261–283 and 285–307 , sharing a high homology of 94.2–98.8% at the nucleotide level and 89.5–98.8% at the amino acid level. However, these ratios dropped to 75.4% and 72.7% at the nucleotide and AA levels, respectively, for ON1 novel RSV-A genotype sequences. Ontario's NA1 and ON1 strains displayed an early stop codon at positions 298 and 322, respectively, when compared to the prototype RSV-A2 strain.Homology between members of the novel RSV-A ON1 genotype was between 99–100% and 3 unique substitutions, E232G, T253K and P314L (P290L if the duplication is removed), were noted to be specific for this group and not observed in other isolates. Compared to the reference RSV-A2 strain, 16 G gene amino acid substitutions were identified universally among Ontario's NA1 and ON1 strains including S222P, P226L, E233K, N237D, I244R, L258H, M262E, F265L/P/H, S269T, N273Y, P274L, S280Y, P283S, P286L, P289S, S290P/L, P292S, P293S, P296T, and R297K.The nucleotide sequence of the G gene from the ON1 genotype is translated to a polypeptide of 322 AAs, the largest found so far among RSV-A isolates. The central domain, HFEVFNFVPCSICSNNPTCWAIC , remained conserved among all of Ontario's RSV-A isolates .Only 2 (1.8%) of the RSV-A isolates, ON/RSV89 and ON/RSV181, were closely related to the GA5 genotype, sharing homology of 94.6%–95.4% at the nucleotide level and 91.9% at the amino acid level with the reference GA5 strain TX67951. They contained several unique mutations including N237Y, S270F, V279I, N297D and H298Q.Representative sequences of RSV-A strains circulating in Ontario along with 23 reference strains of RSV-A genotype derived from Genbank were included in the phylogenetic analysis . SequencComparing the F gene phylogeny of the study RSV-A isolates based on a 500 nucleotide partial sequence  respectively, whereas 33±2 sites were potentially O-glycosylated (G scores of 0.5–0.8) in Ontario NA1 isolates . All ON1p2 = 0.1599 with ω2 = 4.4946) to 17.09% (p2 = 0.1709 with ω2 = 4.2356). A total of nineteen positively selected sites were observed with posterior probability greater than 50% , ON1 (ON67-1210A) and rON1  were compared to provide insight into the possible mechanisms of the duplication occurrence. When compared to their respective cRNA structures, higher free energies  were observed with the viral RNA secondary structures of the NA1 reference (−47.82 vs. −5.32), ON1 (−80.83 vs. −23.84) and rON1 (−45.57 vs.−12.49) . DiffereThe ‘mfg’ program reports the most stable SLS for a given window size of nucleotides and assumes that each SLS has to be initiated from an unpaired base. In this study we analyzed G and F genes of 112 RSV-A isolates from clinical samples tested during winter 2010–2011 in Ontario, Canada. All sequences were analyzed using various bioinformatics methods in order to better understand the genotype variability, molecular epidemiology and evolutionary adaptability of circulating strains. We documented circulation of two genotypes of NA1 (89% of RSV-A isolates) and GA5 (1.8% of RSV-A isolates) in Ontario during winter 2010–2011. In addition, 11 (10%) of the RSV-A isolates belonged to a novel genotype, ON1, characterized by a 72nt duplication in the C terminal third of the G gene. Our findings differ from an earlier Canadian study, which documented a high prevalence of GA5 and GA7 genotypes among Winnipeg isolates in 2000, with each accounting for 30% of circulating RSV-A isolates at that time We identified a novel genotype of RSV-A (ON1), with a 72 nucleotide duplication in the C terminal third of the G gene. This duplication resulted in codon disruption and lengthening of the subsequent predicted polypeptide by 24 AAs, including 23 duplicated AAs. Nucleotide duplications have rarely been reported in wild type populations of RSV Although the sequence composition among the novel RSV-A ON1 genotype remained conserved, three unique substitutions  were noted to be specific for ON1, and not observed in other Ontario isolates. In addition, two amino acid mutations (T253K and N273Y) which were positively selected sites also resulted in loss of two potential N-glycosylation sites identified previously in the NA1 reference strain (AB470478). Such changes in N-glycosylation sites of the G protein might alter the antigenicity between the genotypes and facilitate binding of circulating antibodies High homology among wild type NA1 genotypes in Ontario and isolates from Genbank reveals the global distribution of this genotype. On the contrary, the presence of ON1 genotype with a 72 nucleotide insertion in Ontario might suggest the effect of geographical and temporal factors on the genetic evolution of RSV-A, as previously speculated for RSV-B Site-specific evolutionary analysis of the C-terminal hypervariable region of the G protein among Ontario NA1 isolates revealed strong evolutionary selection pressure (dN/dS = 4.4), resulting in 19 positively selected sites compared to the NA1 reference genotype . The higin vitro studies also demonstrates one form of RNA polymerase pausing, called backtracking, where after encountering an obstruction such as a secondary structure, the RNA polymerase reverses its direction and relocates itself upstream Theoretical mechanisms have been proposed for duplication events during replication and transcription processes The novel RSV-A genotype (ON1) is of considerable interest because of its 72nt duplication in the G gene C-terminal one-third region, which is the largest duplication described to date in this genus. This area is the target for strain specific neutralizing antibodies and such changes in structure might alter the immunogenicity and pathogenicity of the virus. However, further detailed studies should be undertaken to explore pathogenicity, transmissibility and the replication pattern of this new variant.The results of this study emphasize the importance of early detection and characterization of newly emerging genotypes. Understanding the effect of the novel RSV-A ON1 genotype 72nt G gene duplication on fitness, virulence and transmissibility could help predict changes in viral phenotype and immunogenicity. It will also provide insight into vaccine potential of the G gene protein. Continued genotyping and molecular epidemiological surveillance of RSV are essential to further understanding RSV evolution and transmission in communities and healthcare settings.