Drug Repurposing for Japanese Encephalitis Virus Infection by Systems Biology Methods.

Japanese encephalitis is a zoonotic disease caused by the Japanese encephalitis virus (JEV). It is mainly epidemic in Asia with an estimated 69,000 cases occurring per year. However, no approved agents are available for the treatment of JEV infection, and existing vaccines cannot control various types of JEV strains. Drug repurposing is a new concept for finding new indication of existing drugs, and, recently, the concept has been used to discover new antiviral agents. Identifying host proteins involved in the progress of JEV infection and using these proteins as targets are the center of drug repurposing for JEV infection. In this study, based on the gene expression data of JEV infection and the phenome-wide association study (PheWAS) data, we identified 286 genes that participate in the progress of JEV infection using systems biology methods. The enrichment analysis of these genes suggested that the genes identified by our methods were predominantly related to viral infection pathways and immune response-related pathways. We found that bortezomib, which can target these genes, may have an effect on the treatment of JEV infection. Subsequently, we evaluated the antiviral activity of bortezomib using a JEV-infected mouse model. The results showed that bortezomib can lower JEV-induced lethality in mice, alleviate suffering in JEV-infected mice and reduce the damage in brains caused by JEV infection. This work provides an agent with new indication to treat JEV infection.

The impact of ambient air pollution on suicide mortality: a case-crossover study in Guangzhou, China.

BACKGROUND: Preventing suicide is a global imperative. Although the effects of social and individual risk factors of suicide have been widely investigated, evidence of environmental effects of exposure to air pollution is scarce. We investigated the effects of ambient air pollution on suicide mortality in Guangzhou, China during 2003-2012. METHODS: A conditional logistic regression analysis with a time-stratified case-crossover design was performed to assess the effects of daily exposure to three standard air pollutants, including particulate matter less than 10 µm in aerodynamic diameter (PM10), sulphur dioxide (SO2) and nitrogen dioxide (NO2), on suicide mortality, after adjusting for the confounding effects of daily mean temperature, relative humidity, atmospheric pressure and sunshine duration. Further analyses were stratified by season, gender, age group, educational attainment and suicide type. RESULTS: Between 2003 and 2012, there were a total of 1 550 registered suicide deaths in Guangzhou. A significant increase in suicide risk were associated with interquartile-range increases in the concentration of air pollutant, with an odds ratio of 1.13 (95 % confidence interval (CI): 1.01, 1.27) and 1.15 (95 % CI: 1.03, 1.28) for PM10 and NO2 at lag 02, and 1.12 (95 % CI: 1.02, 1.23) for SO2 at lag 01, respectively. The suicide risks related to air pollution for males and people with high education level were higher than for females and those with low education level, respectively. Significant air pollution effects were found on violent suicide mortality and in cool season but not on non-violent suicide mortality or in warm season. CONCLUSIONS: Suicide risk was positively associated with ambient air pollution levels. This finding would provide important information for the health impact assessment of air pollution and for the development of effective strategies and interventions for the prevention of suicide.

Identification of Interleukin-9 Producing Immune Cells in Endometrial Carcinoma and Establishment of a Prognostic Nomogram.

Background: Interleukin-9 (IL9) plays a critical role in immunity and the pathogenesis of endometrial cancer (EC), especially endometrioid EC (EEC). This study aimed to identify the IL9+ immune cell subsets and their pleiotropic functions and establish an optimized prognostic nomogram towards the promotion of personalized treatment of EEC. Methods: 1,417 EC patients were involved in the present study. 143 patients from the tertiary gynecology centers in Shanghai between 2013 and 2019 were recruited, and the study protocol was approved by the Institutional Review Board (IRB) of Shanghai First Maternity and Infant Hospital. The genomic data of the other 1,274 patients were extracted from the TCGA and the MSKCC datasets, respectively. Immune and stromal scores were calculated using the ESTIMATE R tool, and the tumor infiltration of immune cells was analyzed using the TIMER platform. Metascape and GEPIA datasets were used for bioinformatic analysis. P < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism and R studio. Results: 552 genes that were correlated with leukocyte infiltration, lymphocyte activation, and regulation of innate immune response were up-regulated in the high immune score group. More IL9+ cell infiltration was detected in the highly and moderately differentiated EC (p = 0.04). High IL9+ lymphocyte infiltration was related to a better overall survival (p = 0.0027). IL9 positive cell clusters included ILC2s, Vδ2 γδT cells, mast cells, macrophages, and Th9 cells. Parameters such as FIGO stage, IL9 score, Vδ2 + γδT cell infiltration, classification of differentiation, and diabetes mellitus were assigned a weighted number of points in the nomogram for a specific predicted 3-, 5- and 10-year overall survival (OS). IL9-IL9R axis played a vital role in EEC, IL9R positive cell subgroups were also identified, and the related function was analyzed in the present study. Additionally, PR (Progesterone Receptor, or PGR) expression was relevant to a higher density of IL9+ lymphocyte infiltration. However, PGRMC1 (Progesterone Receptor Membrane Component 1) was negatively relevant to IL9R (p = 4.26e-8). Conclusion: We observed a significant infiltration of IL9+ cells and the overrepresentation of IL-9R in tissue specimens of patients in EC cases. The nomogram incorporating the IL9 could accurately predict individualized survival probability in EEC. Additionally, this study not only established a prognostic nomogram but also assist in the firmer understanding of the relevance of the IL9-IL9R axis and IL9-producing cells in EC immunity.

Effects of ambient temperature on ambulance emergency call-outs in the subtropical city of Shenzhen, China.

The associations between meteorological factors and mortality have been well documented worldwide, but limited evidence is available for the non-fatal health impacts of ambient temperature, particularly there are few population-based investigations on the impacts of emergency ambulance dispatches in Asia. In this study, based on 809,906 ambulance emergency call-outs (AECOs) for the total population from 2010-2016 in the subtropical city of Shenzhen, China, a Poisson regression combined with a distributed lag nonlinear model was used to simultaneously assess the nonlinear and lag effects of daily mean temperature on AECOs. Stratified analyses by age and sex were performed to identify vulnerable subpopulations. A U-shaped relationship was found between temperature and AECOs. Cold effects were delayed and persisted for 3-4 weeks, with a cumulative relative risk (RR) and 95% confidence interval (CI) of 1.23 (1.10-1.38) and 1.25 (1.16-1.35) over lag 0-28 when comparing the 1st and 5th percentile of the temperature distribution to the optimal (i.e. minimum AECOs) temperature, respectively. Hot effects were immediate and diminished quickly in 5 days, with an increase of 19% (RR = 1.19, 95%CI: 1.14-1.23) and 21% (RR = 1.21, 95%CI: 1.16-1.26) in AECOs over lag 0-5 when comparing the 95th and 99th percentile of temperature to the optimal temperature. Children and the elderly were more vulnerable to cold effects. The youth and middle-aged people suffered more from high temperature. The effects of temperature were similar between males and females. In summary, significant increases were observed in the frequency of AECOs during cold and hot days, and the weather-associated increases in AECOs are different among age groups. This information has valuable implications in ambulance demand prediction and service provision planning.

The burden of COPD mortality due to ambient air pollution in Guangzhou, China.

Few studies have investigated the chronic obstructive pulmonary disease (COPD) mortality fraction attributable to air pollution and modification by individual characteristics of air pollution effects. We applied distributed lag non-linear models to assess the associations between air pollution and COPD mortality in 2007-2011 in Guangzhou, China, and the total COPD mortality fraction attributable to air pollution was calculated as well. We found that an increase of 10 µg/m(3) in particulate matter with an aerodynamic diameter of 10 µm or less (PM10), sulfur dioxide (SO2) and nitrogen dioxide (NO2) was associated with a 1.58% (95% confidence interval (CI): 0.12-3.06%), 3.45% (95% CI: 1.30-5.66%) and 2.35% (95% CI: 0.42-4.32%) increase of COPD mortality over a lag of 0-15 days, respectively. Greater air pollution effects were observed in the elderly, males and residents with low educational attainment. The results showed 10.91% (95% CI: 1.02-9.58%), 12.71% (95% CI: 5.03-19.85%) and 13.38% (95% CI: 2.67-22.84%) COPD mortality was attributable to current PM10, SO2 and NO2 exposure, respectively. In conclusion, the associations between air pollution and COPD mortality differed by individual characteristics. There were remarkable COPD mortality burdens attributable to air pollution in Guangzhou.

[Construction of eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 and their expressions in human microvascular endothelial cells].

OBJECTIVE: To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) and study their expressions in human microvascular endothelial cells (HMVECs). METHODS: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1(+)-HIF-1alpha-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1alpha mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting. RESULTS: DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1+-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one. CONCLUSION: The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.

[C-reactive protein inhibits the expression of hypoxia-inducible factor-1alpha in human umbilical vein endothelial cells in hypoxic condition].

OBJECTIVE: To investigate the effect of C-reactive protein (CRP) on expression of hypoxia-inducible factor-1alpha (HIF-1alpha). METHODS: Using CoCl(2) (100 micromol/L) to simulate hypoxic condition for culturing human umbilical vein endothelial cells (HUVECs), we examined the effects of CRP on expression of HIF-1alpha. The proteins were extracted from the cells after a 24-hour exposure of the cells to CRP of varied concentrations in the presence of CoCl(2), and Western blotting was performed for quantification of HIF-1alpha expression, the results were analyzed statistically with SPSS software. RESULTS: CRP at the concentration of 5 microg/ml decreased the expression of HIF-1alpha (P<0.001), producing the maximum inhibitory effect at the concentration of 100 microg/ml, an effect exhibiting dose-dependence. CONCLUSION: CRP inhibits the expression of HIF-1alpha in HUVECs subjected to hypoxic condition, which serves as an important evidence for the inhibitory effect of CRP on angiogenesis in hypoxic condition.

Excess winter mortality and cold temperatures in a subtropical city, Guangzhou, China.

BACKGROUND: A significant increase in mortality was observed during cold winters in many temperate regions. However, there is a lack of evidence from tropical and subtropical regions, and the influence of ambient temperatures on seasonal variation of mortality was not well documented. METHODS: This study included 213,737 registered deaths from January 2003 to December 2011 in Guangzhou, a subtropical city in Southern China. Excess winter mortality was calculated by the excess percentage of monthly mortality in winters over that of non-winter months. A generalized linear model with a quasi-Poisson distribution was applied to analyze the association between monthly mean temperature and mortality, after controlling for other meteorological measures and air pollution. RESULTS: The mortality rate in the winter was 26% higher than the average rate in other seasons. On average, there were 1,848 excess winter deaths annually, with around half (52%) from cardiovascular diseases and a quarter (24%) from respiratory diseases. Excess winter mortality was higher in the elderly, females and those with low education level than the young, males and those with high education level, respectively. A much larger winter increase was observed in out-of-hospital mortality compared to in-hospital mortality (45% vs. 17%). We found a significant negative correlation of annual excess winter mortality with average winter temperature (rs=-0.738, P=0.037), but not with air pollution levels. A 1 °C decrease in monthly mean temperature was associated with an increase of 1.38% (95% CI:0.34%-2.40%) and 0.88% (95% CI:0.11%-1.64%) in monthly mortality at lags of 0-1 month, respectively. CONCLUSION: Similar to temperate regions, a subtropical city Guangzhou showed a clear seasonal pattern in mortality, with a sharper spike in winter. Our results highlight the role of cold temperature on the winter mortality even in warm climate. Precautionary measures should be strengthened to mitigate cold-related mortality for people living in warm climate.

[Roles of N-glycosylation in immunity of prME and NS1 gene of JEV].

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.

Evolutionary characterization of influenza virus A/duck/Hubei/W1/2004 (H9N2) isolated from central China.

Full-length eight gene segments of avian influenza virus A/duck/Hubei/W1/2004(H9N2) (Dk/Hub/W1/04) were amplified by RT-PCR and completely sequenced. Phylogenetic analysis revealed that Dk/Hub/W1/04 was derived from A/Duck/HongKong/Y280/97, not displaying direct evolutional relationship with A/Quail/HongKong/G1/97 or Hubei H5N1 viruses. Meanwhile, Dk/Hub/W1/04 was found highly related to recent three chicken isolates. The connecting peptide of HA and the deletion in NA stalk were consistent with three chicken isolates, and the number of potential glycosylation site on the HA and NA also was similar or identical to the three chicken isolates. These findings suggested that Dk/Hub/W1/04 is likely to transmit back to ducks from chickens.

[Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1].

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.