ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Impact of GADD45A on Radiation Biodosimetry Using Mouse Peripheral Blood.

High-dose-radiation exposure in a short period of time leads to radiation syndromes characterized by severe acute and delayed organ-specific injury accompanied by elevated organismal morbidity and mortality. Radiation biodosimetry based on gene expression analysis of peripheral blood is a valuable tool to detect exposure to radiation after a radiological/nuclear incident and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation, can potentially obscure the predictive power of the method. GADD45A (Growth arrest and DNA damage-inducible gene a) plays important roles in cell growth control, differentiation, DNA repair, and apoptosis. GADD45A-deficient mice develop an autoimmune disease, similar to human systemic lupus erythematosus, characterized by severe hematological disorders, kidney disease, and premature death. The goal of this study was to elucidate how pre-existing inflammation in mice, induced by GADD45A ablation, can affect radiation biodosimetry. We exposed wild-type and GADD45A knockout male C57BL/6J mice to 7 Gy of X rays and 24 h later RNA was isolated from whole blood and subjected to whole genome microarray and gene ontology analyses. Dose reconstruction analysis using a gene signature trained on gene expression data from irradiated wild-type male mice showed accurate reconstruction of either a 0 Gy or 7 Gy dose with root mean square error of ± 1.05 Gy (R^2 = 1.00) in GADD45A knockout mice. Gene ontology analysis revealed that irradiation of both wild-type and GADD45A-null mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. However, based on their z-score, these pathways were predicted to be more significantly overrepresented in GADD45A-null mice, implying that GADD45A deletion may exacerbate the deleterious effects of radiation on blood cells. Numerous immune cell functions and quantities were predicted to be underrepresented in both genotypes; however, differentially expressed genes from irradiated GADD45A knockout mice predicted an increased deterioration in the numbers of T lymphocytes, as well as myeloid cells, compared with wild-type mice. Furthermore, an overrepresentation of genes associated with radiation-induced hematological malignancies was associated with GADD45A knockout mice, whereas hematopoietic and progenitor cell functions were predicted to be downregulated in irradiated GADD45A knockout mice. In conclusion, despite the significant differences in gene expression between wild-type and GADD45A knockout mice, it is still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of pre-existing inflammation status.

Circulating cancer giant cells with unique characteristics frequently found in patients with myelodysplastic syndromes (MDS).

Myelodysplastic syndromes (MDS) are incurable diseases characterized by dysplastic hematopoietic cells, cytopenias in the blood and an inherent tendency for transformation to secondary acute myeloid leukemia (AML). Since most therapies fail to prevent rapid clonal evolution and disease resistance, new and non-invasive predictive markers are needed to monitor patients and adapt the therapeutic strategy. By using ISET, a very sensitive approach to isolate cells larger than mature leukocytes from peripheral blood samples, we looked for cellular markers in 99 patients (158 samples) with MDS and 66 healthy individuals (76 samples) used as controls. We found a total of 680 Giant Cells, defined as cells having a size of 40 microns or larger in 46 MDS patients (80 samples) and 28 Giant Cells in 11 healthy individuals (11 samples). In order to understand if we had enriched from peripheral blood atypical cells of the megakaryocyte line, we studied the Giant Cells using immunolabeling with megakaryocytes and tumor-specific markers. We report that the Giant Cells we found in the peripheral blood of MDS patients primarily express tumor markers. Our results show that Polyploid Giant Cancer Cells (PGCC), similar to those described in solid tumors, are found in the peripheral blood of patients with MDS and suggest the working hypothesis that they could play a role in hematological malignancies.

Technological advancements in valorisation of industrial effluents employing hydrothermal liquefaction of biomass: Strategic innovations, barriers and perspectives.

Hydrothermal liquefaction (HTL) is identified as a promising thermochemical technique to recover biofuels and bioenergy from waste biomass containing low energy and high moisture content. The wastewater generated during the HTL process (HTWW) are rich in nutrients and organics. The release of the nutrients and organics enriched HTWW would not only contaminate the water bodies but also lead to the loss of valued bioenergy sources, especially in the present time of the energy crisis. Thus, biotechnological as well as physicochemical treatment of HTWW for simultaneous extraction of valuable resources along with reduction in polluting substances has gained significant attention in recent times. Therefore, the treatment of wastewater generated during the HTL of biomass for reduced environmental emission and possible bioenergy recovery is highlighted in this paper. Various technologies for treatment and valorisation of HTWW are reviewed, including anaerobic digestion, microbial fuel cells (MFC), microbial electrolysis cell (MEC), and supercritical water gasification (SCWG). This review paper illustrates that the characteristics of biomass play a pivotal role in the selection process of appropriate technology for the treatment of HTWW. Several HTWW treatment technologies are weighed in terms of their benefits and drawbacks and are thoroughly examined. The integration of these technologies is also discussed. Overall, this study suggests that integrating different methods, techno-economic analysis, and nutrient recovery approaches would be advantageous to researchers in finding way for maximising HTWW valorisation along with reduced environmental pollution.

Axin1: A novel scaffold protein joins the antiviral network of interferon.

Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.

Mutation in SF3B1 gene promotes formation of polyploid giant cells in Leukemia cells.

Giant cells with polyploidy, termed polyploid giant cells, have been observed during normal growth, development, and pathologic states, such as solid cancer progression and resistance to therapy. Functional studies of polyploidal giant cancer cells (PGCC) provided evidence that they arise when normal diploid cells are stressed, show stem cell-like properties, and give rise to tumors. In the present study, we report in K562 leukemia cell line that introduction of the hotspot K700E mutation in the gene SF3B1 using CRISPR/Cas9 method results in an increased frequency of multinucleated polyploid giant cells resistant to chemotherapeutic agent and serum starvation stress. These giant cells with higher ploidy are distinct from multinucleated megakaryocytes, are proliferative, and are characterized by increased accumulation of mitochondria. PGCC have been previously documented in solid tumors. This is the first report describing PGCCs in a cell line derived from a liquid cancer where increased frequency of PGCCs is linked to a specific genetic event. Since SF3B1 mutations are predominantly seen in MDS and other hematologic malignancies, our current findings will have significant clinical implications.

Effect of the p38 Mitogen-Activated Protein Kinase Signaling Cascade on Radiation Biodosimetry.

Radiation biodosimetry based on transcriptomic analysis of peripheral blood is a valuable tool to detect radiation exposure after a radiological/nuclear event and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation or immune suppression, can potentially obscure the predictive power of the method. Members of the p38 mitogen-activated protein kinase (MAPK) family respond to pro-inflammatory signals and environmental stresses, whereas genetic ablation of the p38 signaling pathway in mice leads to reduced susceptibility to collagen-induced arthritis and experimental autoimmune encephalomyelitis that model human rheumatoid arthritis and multiple sclerosis, respectively. p38 is normally regulated by the MAP3K-MAP2K pathway in mammalian cells. However, in T cells there is an alternative pathway for p38 activation that plays an important role in antigen-receptor-activated T cells and participates in immune and inflammatory responses. To examine the role of p38 in response to radiation, we used two mouse models expressing either a p38α dominant negative (DN) mutation that globally suppresses p38 signaling or a p38αß double-knock-in (DKI) mutant, which inhibits specifically T-cell receptor activation. We exposed p38 wild-type (p38WT) and mutant male mice to 7 Gy X rays and 24 h later whole blood was isolated subjected to whole-genome microarray and gene ontology analysis. Irradiation of p38WT mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. In contrast, these pathways were significantly underrepresented in p38DN and p38DKI mutant mice, suggesting that p38 attenuation may protect blood cells from the deleterious effects of radiation. Furthermore, radiation exposure in p38 mutant mice resulted in an enrichment of phagocytosis-related pathways, suggesting a role for p38 signaling in restricting phagocytosis of apoptotic cells after irradiation. Finally, despite the significant changes in gene expression, it was still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of p38 status.

Development of an integrated fingerstick blood self-collection device for radiation countermeasures.

We report the development of system for packaging critical components of the traditional collection kit to make an integrated fingerstick blood collector for self-collecting blood samples of 100 µl or more for radiation countermeasures. A miniaturized vacuum tube system (VacuStor system) has been developed to facilitate liquid reagent storage, simple operation and reduced sample contamination. Vacuum shelf life of the VacuStor tube has been analyzed by the ideal gas law and gas permeation theory, and multiple ways to extend vacuum shelf life beyond one year have been demonstrated, including low temperature storage, Parylene barrier coating and container vacuum bag sealing. Self-collection was also demonstrated by healthy donors without any previous fingerstick collection experience. The collected blood samples showed similar behavior in terms of gene expression and cytogenetic biodosimetry assays comparing to the traditionally collected samples. The integrated collector may alleviate the sample collection bottleneck for radiation countermeasures following a large-scale nuclear event, and may be useful in other applications with its self-collection and liquid reagent sample preprocessing capabilities.

Human Transcriptomic Response to Mixed Neutron-Photon Exposures Relevant to an Improvised Nuclear Device.

In the possible event of a detonation of an improvised nuclear device (IND), the immediate radiation would consist of both photons (gamma rays) and neutrons. Since neutrons generally have a high relative biological effectiveness (RBE) for most physiological end points, it is important to understand the effect that neutrons would have on the biodosimetry methods that are being developed for medical triage purposes. We previously compared the transcriptomic response in human blood after neutron and photon irradiation. In this study, we analyzed the effect of mixed-field-neutron-photon radiation on gene expression responses in human peripheral blood, to elucidate the neutron contribution in the setting of a radiation exposure from an IND detonation. We used four combinations of mixed neutron-photon exposures, with increasing percentages of neutrons, to a cumulative dose of 3 Gy. The mixed-field exposures consisted of 0%, 5%, 15% and 25% of neutrons, where 0% corresponds to 3 Gy of pure X rays. A maximum neutron exposure, corresponding to 83% neutrons (0.75 Gy) was also used in the study. Increases were observed in both the number and expression level of genes, with increasing percentages of neutrons from 0% to 25% in the mixed-field exposures. Gene ontology analysis showed an overall predominance of TP53 signaling among upregulated genes across all exposures. Some TP53 regulated genes, such as EDA2R, GDF15 and VWCE, demonstrated increased expression with increasing neutron percentages in mixed-field exposures. Immune response, specifically natural-killer-cell mediated signaling, was the most significant biological process associated with downregulated genes. We observed significant suppression of T-cell-mediated signaling in mixed-field exposures, which was absent in the response to pure photons. In this first study investigating gene expression in human blood cells exposed to mixed neutron-photon fields similar to an actual IND explosion, we have identified a number of genes responding to the 3 Gy dose that showed increasing expression as the neutron percentage increased. Such genes may serve as better indicators of the expected biological damage than a report of total physical dose, and thus provide more relevant information for treating physicians.

Impact of inflammatory signaling on radiation biodosimetry: mouse model of inflammatory bowel disease.

BACKGROUND: Ionizing Radiation (IR) is a known pro-inflammatory agent and in the process of development of biomarkers for radiation biodosimetry, a chronic inflammatory disease condition could act as a confounding factor. Hence, it is important to develop radiation signatures that can distinguish between IR-induced inflammatory responses and pre-existing disease. In this study, we compared the gene expression response of a genetically modified mouse model of inflammatory bowel disease (Il10-/-) with that of a normal wild-type mouse to potentially develop transcriptomics-based biodosimetry markers that can predict radiation exposure in individuals regardless of pre-existing inflammatory condition. RESULTS: Wild-type (WT) and Il10-/- mice were exposed to whole body irradiation of 7 Gy X-rays. Gene expression responses were studied using high throughput whole genome microarrays in peripheral blood 24 h post-irradiation. Analysis resulted in identification of 1962 and 1844 genes differentially expressed (p < 0.001, FDR < 10%) after radiation exposure in Il10-/- and WT mice respectively. A set of 155 genes was also identified as differentially expressed between WT and Il10-/- mice at the baseline pre-irradiation level. Gene ontology analysis revealed that the 155 baseline differentially expressed genes were mainly involved in inflammatory response, glutathione metabolism and collagen deposition. Analysis of radiation responsive genes revealed that innate immune response and p53 signaling processes were strongly associated with up-regulated genes, whereas B-cell development process was found to be significant amongst downregulated genes in the two genotypes. However, specific immune response pathways like MHC based antigen presentation, interferon signaling and hepatic fibrosis were associated with radiation responsive genes in Il10-/- mice but not WT mice. Further analysis using the IPA prediction tool revealed significant differences in the predicted activation status of T-cell mediated signaling as well as regulators of inflammation between WT and Il10-/- after irradiation. CONCLUSIONS: Using a mouse model we established that an inflammatory disease condition could affect the expression of many radiation responsive genes. Nevertheless, we identified a panel of genes that, regardless of disease condition, could predict radiation exposure. Our results highlight the need for consideration of pre-existing conditions in the population in the process of development of reliable biodosimetry markers.

Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells.

PB1-F2 is a multifunctional protein and contributes to the pathogenicity of influenza A viruses. PB1-F2 is known to have strain and cell specific functions. In this study we have investigated the apoptotic and inflammatory responses of PB1-F2 protein from influenza viruses of diverse pathogenicities in A549 lung epithelial cells. Overexpression of PB1-F2 resulted in apoptosis and heightened inflammatory response in A549 cells. Comparison revealed that the response varied with each subtype. PB1-F2 protein from highly pathogenic H5N1 virus induced least apoptosis but maximum inflammatory response. Results indicated that apoptosis was mediated through death receptor ligands TNFα and TRAIL via Caspase 8 activation. Significant induction of cytokines/chemokines CXCL10, CCL5, CCL2, IFNα, and IL-6 was noted in A549 cells transfected with PB1-F2 gene construct of 2008 West Bengal H5N1 virus (H5N1-WB). On the contrary, PB1-F2 construct from 2007 highly pathogenic H5N1 isolate (H5N1-M) with truncated N-terminal region did not evoke as exuberant inflammatory response as the other H5N1-WB with full length PB1-F2, signifying the importance of N-terminal region of PB1-F2. Sequence analysis revealed that PB1-F2 proteins derived from different influenza viruses varied at multiple amino acid positions. The secondary structure prediction showed each of the PB1-F2 proteins had distinct helix-loop-helix structure. Thus, our data substantiate the notion that the contribution of PB1-F2 to influenza pathogenicity is greatly strain specific and involves multiple host factors. This data demonstrates that PB1-F2 protein of influenza A virus, when expressed independently is minimally apoptotic and strongly influences the early host response in A549 cells.

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia.

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

A Prospective Study to Evaluate Vaginal Insertion and Intra-Cesarean Insertion of Post-Partum Intrauterine Contraceptive Device.

OBJECTIVES: Evaluation and comparison of safety and efficacy of vaginal and intra-cesarean insertion of Post-Partum Intrauterine Contraceptive device (PPIUCD). METHODS: An interventional prospective study conducted in the Department of Obstetrics and Gynaecology at NRS Medical College, Kolkata. PPIUCD were inserted in 263 mothers in 1-year study period. Among them, first 100 mothers who delivered vaginally and the first 100 who underwent cesarean section were regarded as study groups and were followed up for 1 year. RESULTS: Both modes of PPIUCD insertion were found to have very low rates of expulsion, vaginal bleeding, infection, missing strings, and also effective as contraceptive. Expulsion rate was 4 % in the vaginal group and 2 % in intra-cesarean group. Strings of PPIUCD were less visible after cesarean insertion than vaginal insertion (p = 0.028). CONCLUSION: PPIUCD is an appealing approach and may become the best choice as post-partum contraception after vaginal as well as cesarean delivery.

Gene expression profile of high-fat diet-fed C57BL/6J mice: in search of potential role of azelaic acid.

High-fat diet (HFD) elevates circulatory fatty acids and influences glucose and fat metabolism. Azelaic acid (AzA), a naturally occurring α,ω-dicarboxylic acid in wheat, rye, barley, oat seeds and sorghum, has been reported to exert antidiabetic effects in HFD-induced type 2 diabetes mellitus (T2DM) C57BL/6J mice. The present study was undertaken to identify the genes that are differentially modulated by treatment with AzA in HFD-fed mice. Mice were fed HFD for 10 weeks and subjected to intragastric administration of 80 mg/kg body weight (BW) of AzA daily along with HFD from 11 to 15 weeks. Lipid profile, adipokines and cytokines were examined in the plasma/liver of mice. Whole genome profiling was performed in the liver of mice using microarray and validated by qRT-PCR, Western blot and immunohistochemical analyses. HFD intake resulted in significantly elevated lipids (except high-density lipoproteins), resistin, tumour necrosis factor alpha and interleukin-6 with marked reduction in adiponectin. Administration of AzA to HFD-fed mice significantly restored the lipids, adipokines and cytokines to near normal. Transcript profiling revealed that HFD intake activated the genes involved in stress response, cell cycle regulation and apoptosis. Treatment with AzA caused increased expression of genes involved in reactive oxygen species (ROS) scavenging, receptor-mediated signalling, transcription, protein modification and insulin signal transduction. AzA activates insulin signal molecules leading to insulin sensitivity. The ability of AzA to modulate the expression of these genes supports the notion that AzA is a promising drug candidate for the treatment of insulin resistance associated with T2DM.

Comparative Assessment of Gasification Based Coal Power Plants with Various CO2 Capture Technologies Producing Electricity and Hydrogen.

Seven different types of gasification-based coal conversion processes for producing mainly electricity and in some cases hydrogen (H2), with and without carbon dioxide (CO2) capture, were compared on a consistent basis through simulation studies. The flowsheet for each process was developed in a chemical process simulation tool "Aspen Plus". The pressure swing adsorption (PSA), physical absorption (Selexol), and chemical looping combustion (CLC) technologies were separately analyzed for processes with CO2 capture. The performances of the above three capture technologies were compared with respect to energetic and exergetic efficiencies, and the level of CO2 emission. The effect of air separation unit (ASU) and gas turbine (GT) integration on the power output of all the CO2 capture cases is assessed. Sensitivity analysis was carried out for the CLC process (electricity-only case) to examine the effect of temperature and water-cooling of the air reactor on the overall efficiency of the process. The results show that, when only electricity production in considered, the case using CLC technology has an electrical efficiency 1.3% and 2.3% higher than the PSA and Selexol based cases, respectively. The CLC based process achieves an overall CO2 capture efficiency of 99.9% in contrast to 89.9% for PSA and 93.5% for Selexol based processes. The overall efficiency of the CLC case for combined electricity and H2 production is marginally higher (by 0.3%) than Selexol and lower (by 0.6%) than PSA cases. The integration between the ASU and GT units benefits all three technologies in terms of electrical efficiency. Furthermore, our results suggest that it is favorable to operate the air reactor of the CLC process at higher temperatures with excess air supply in order to achieve higher power efficiency.

F1 hybrids of BALB/c and C57BL/6 mouse strains respond differently to low-dose ionizing radiation exposure.

There are evidences to show that response to ionizing radiations have genetic influence. To investigate this further, reciprocal F1 hybrids were generated by crossbreeding the radiation-susceptible BALB/c mouse strain with resistant C57BL/6 in a sex-specific manner (BALB/c♂ x C57BL/6♀ = B6BcF1; C57BL/6♂ x BALB/c♂ =BcB6F1). These hybrids were compared with each other and to the parental strains with respect to transcriptional responses to low-dose ionizing radiation exposure (LDIR). The two F1 hybrids showed drastic differences in their gene expression profiles to ionizing radiation exposure particularly in case of the genes involved in DNA damage response and repair process. Also, the inheritance pattern of the gene expression was found to be complex and could not be explained solely on the basis of parental expression pattern. It was concluded that there is a differential transmission of susceptible trait alleles from the parents to F1 progeny which is dependent on the sex of the parent mouse strain used to set up the crosses and other environmental factors.

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes.

BACKGROUND: Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. OBJECTIVE: The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. METHODS: A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. RESULTS: We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. CONCLUSION: Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells.

BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quite complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2653/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-ß mediated innate immune responses, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.