Objectives To analyze the characteristic of HIV-1 viral infectivity factor (Vif) gene variants isolated from Shanghai. To construct the prokaryotic expression vector of HIV-1 vif gene and understand its immunogenieity. Methods HIV-1 vii genes were amplified and sequenced from 23 serum samples of HIV-1 infected patients in Shanghai and then compared with the international standard HIV-1 strain. Subsequently, these amplified Vif fragments were sub cloned into pETS2b(+)expression vector. The recombinant prokaryotie plasmids pETS2b (+)-HIV-1/Vif were then transferred into BL21DS(Star)cells for expressing and purifying HIV-1 Vif protein. HIV-1 Viff rat polyclonal antibody was then preparing by injecting the purified Vif proteins into the mice. ELISA was used to determine the purity of Vif proteins and the immunogenicity of its polyclonal antibodies. Results The nucleotide acid mutation rate of HIV-1 vif gene in Shanghai AIDS patient was (0. 179±0. 006)% compared with the international standard HIV-1 strain. Some similar mutations in vif gene were found in HIV-1 strains isolated from Shanghai while the amino acid sequence between 151 and 240 in Vif protein was conserved. The construction of HIV-1 Vif prokaryotic expression plamids and the preparation of Vif polyclonal antibodies were successfully done in this study. The reaction between recombinant HIV-1 Vif protein and the serum from HIV-1 infected patient was not significantly different from that between the recombinant protein and healthy control serum(P＞0.05). HIV-1 Vif polyclonal antibody reacted differently with recombined HIV-1 Vif protein compared with healthy control serum samples (t=178.61, P＜0.01). Conclusions The Vif gene mutation rate is high in HIV-1 strains isolated from Shanghai compared with international standard HIV-1 strain. The prokaryotic expression plasmids of HIV-1 vif antigen are successfully constructed and Vif polyclonal antibodies are prepared well.

Primary biliary cholangitis (PBC) is one of common autoimmune liver diseases, and its pathogenesis remains unclear. This article summarizes the role of microRNA in the pathogenesis, diagnosis, and outcome evaluation of PBC and points out that microRNA may be involved in the development and progression of PBC and may thus be used as a potential biomarker for diagnosis and outcome evaluation.

Objective:To understand the status of uncertainty stress and occupational well-being of medical staff in Zhejiang Province, and to analysis the relationship between them, in the context of a pandemic of novel coronavirus pneumonia (corona virus disease 2019, COVID-19).Methods:From May to June 2021, 1 028 medical staff were selected from 8 Class Ⅲ hospitals in Zhejiang Province by convenience sampling. The cross-sectional survey was conducted with the General Information Questionnaire, the Uncertainty Stress Scale and the Occupational Well-being Scale. Pearson method was used to analyze the correlation between uncertain psychological stress and occupational well-being of medical staff, and multiple linear regression was used to analyze the influencing factors of occupational well-being of medical staff.Results:The total score of uncertainty stress of 1028 medical staff was (25.62±8.92), and the total score of occupational well-being was (75.03±14.68) at a moderate level, including 48 (4.7%) participants with a high level of occupational well-being. Uncertainty stress of medical staff was negatively correlated with occupational well-being ( r=-0.46, P<0.01). Multiple regression analysis showed that working years, hospital class, interests, leisure time, personality traits, status of self-reported health and levels of uncertainty stress had statistically significant influence on occupational well-being ( t=-2.30-10.28, all P<0.05). Conclusions:The occupational well-being of medical staff in Zhejiang Province needs to be improved. Health administrative departments should formulate targeted intervention measures to enhance the ability of medical staff to cope with uncertainty stress, so as to improve the occupational well-being of medical staff.

Objective:To screen the differentially expressed exosomal miRNAs derived from liver cancer stem cells (LCSCs) and its effect on the malignant biological characteristics of liver cancer cells.Methods:miRNA expression profile chip was used to analyze the differentially expressed exosomal miRNA derived from LCSCs. The effects of miRNA on malignant phenotypes of LCSCs were identified. The cells were further treated with doxorubicin at different concentrations (0, 150, 300 μmol/L), and the expression level of miR-196a was detected by quantitative real-time PCR (qRT-PCR). The apoptosis of liver cancer cells cultured by exosomes derived from LCSCs (Exo-NC group) and exosomes derived from miR-196a inhibited LCSCs (Exo-Inhibitor group) and the activity of caspase3/7 under the action of exosomes from LCSCs were detected. Nude mice were randomly divided into Do-PBS group, Do-Exo-Inhibitor group and Do-Exo-NC group using random number table method, with 5 mice in each group, and the effect of miR-196a on nude mice xenograft tumor model with liver cancer cells was analyzed.Results:In this study, exosomes were isolated and purified from CD133 + Huh7 stem cell culture supernatant. miR-7162-3p, miR-1910-5, miR-3613-3p, miR-196a and miR-155-5p were up-regulated, while miR-1246 and miR-3613-5p were down-regulated. miR-7162-3p, miR-196a and miR-155-5p in exosomes had important effects on the self-renewal ability of LCSCs. miR-1910-5p, miR-196a and miR-155-5p had important effects on the invasion ability of liver cancer stem cells, among which miR-196a had the most significant inhibitory effect. Treatment for 24 h, the miR-196a expression level of the 0, 150 and 300 μmol/L doxorubicin was 0.96±0.05, 1.23±0.05 and 2.33±0.03 respectively, with a statistically significant difference ( F=996.90, P<0.001). Treatment for 48 h, the miR-196a expression level of the 0, 150 and 300 μmol/L doxorubicin were 1.02±0.07, 2.35±0.05 and 2.89±0.55 respectively, with a statistically significant difference ( F=303.00, P<0.001). When the concentration of doxorubicin was 0 and 300 μmol/L, the apoptosis rates of the Exo-NC group were 9.37%±0.19% and 11.64%±0.27%, and those of the Exo-Inhibitor group were were 18.80%±1.91% and 22.79%±1.57%, with statistically significant differences ( t=4.41, P=0.048; t=4.96, P=0.038). When doxorubicin was not used, the ratios of caspase3/7 in the Exo-NC group at 24 h and 48 h were 0.94±0.08 and 0.97±0.09, and those in the Exo-Inhibitor group were 1.56±0.01 and 1.58±0.01, with statistically significant differences ( t=11.41, P=0.008; t=6.07, P=0.026). Under 300 μmol/L doxorubicin, the ratios of caspase3/7 in the Exo-NC group at 24 h and 48 h were 0.95±0.07 and 1.36±0.08, and those in the Exo-Inhibitor group were 2.84±0.08 and 3.20±0.14, with statistically significant differences ( t=24.20, P=0.002; t=15.78, P=0.004). The results of xenograft tumor in nude mice showed that the tumor volumes of Do-PBS, Do-Exo-Inhibitor and Do-Exo-NC groups increased successively, which were (1 051.86±89.90) mm 3, (1 310.91±86.66) mm 3 and (2 185.14± 352.34) mm 3 respectively, with a statistically significant difference ( F=30.28, P<0.001). The weights of the transplanted tumors in the 3 groups increased successively, which were (0.36±0.10) g, (0.39±0.12) g and (0.76±0.16) g respectively, with a statistically significant difference ( F=11.81, P=0.002). The expression of miR-196a in tumors was significantly decreased after miR-196a inhibitor transfection. The expression levels of the 3 groups were 1.05±0.16, 0.38±0.08 and 2.17±0.26, with a statistically significant difference ( F=48.93, P<0.001). Conclusion:The exosomal secreted by LCSCs can enhance the resistance of liver cancer cells to doxorubicin by miR-196a.

Objective: To understand the molecular evolution characteristics of the nucleoprotein (N) genes and epidemiological feature of 118 rabies virus (RABV) strains isolated in Yunnan province, China from 2006 to 2015. Methods: The brain tissue samples from mad dogs, suspicious sick dogs, sick cow, and human brain tissue, saliva and CSF samples from rabies patients were collected in Yunnan province to detect the viral antigen by direct immunofluorescence assay (DFA). The viral RNA from positive samples was extracted. Coding region of N gene was amplified by RT-PCR and sequenced. The phylogenetic tree was constructed by Neighbor-Joining method of MEGA5.0 software. Results: The sequences of N genes of 91 RABV strains in Yunnan from 2012 to 2015 were obtained. With the sequences of N genes of 27 RABV strains in Yunnan from 2006 to 2011 and 29 RABV strains from Southeast Asian Countries, the phylogenetic analysis was performed. RABV strains in Yunnan were divided into clades YN-A (105 strains), YN-B (6 strains), YN-C (7 strains), which belonged to clades China-I, China-VI, China-II respectively. Clade YN-A was epidemic every year from 2006 to 2015, of them, 14 strains from 2006 to 2011 and 91 strains from 2012 to 2015 were distributed in 13 prefectures (cities) of Yunnan. Clades YN-B and YN-C were epidemic only from 2006 to 2010 and from 2008 to 2011 respectively. The regional distribution of clades YN-B and YN-C was limited. The strains of YN-A and YN-C were closely related to the strains of clades China-I and China-II from neighboring Sichuan, Guizhou, Guangxi and Hunan provinces. The strains of YN-B were closely related to the strains from Myanmar, Laos, Vietnam and Cambodia. Conclusions Three RABV clades with multiple transmission sources were identified in Yunnan. Clade YN-A was widely distributed in rabies endemic area in Yunnan from 2006 to 2015, and it has strong ability to spread as principal clade in Yunnan. Since 2012, clades YN-B and YN-C were not found again in Yunnan.

Objective:To investigate the effect of myogenic differentiation protein 1(Myod1)on the proliferation inhibition and apoptosis of the SH-SY5Y cells induced by oxygen-glucose deprivation(OGD),and to elucidate its mechanism.Methods:Real-time quantitative fluorescence PCR(RT-qPCR)method was used to detect the mRNA levels of Myod1 and long non-coding RNA(lncRNA)small nucleolar RNA host gene 15(SNHG15)in peripheral blood of the subjects in normal group and the patients in ischemic cerebral infarction group as well as the normal cultured SH-SY5Y cells(control group)and the cells in OGD model(OGD group).After transfecting SH-SY5Y cells with si-Myod1,pcDNA3.0-Myod1,si-SNHG15,pcDNA3.0-SNHG15、si-NC,Vector,miR-NC,and miR-24-3p mimics,the cells were treated with OGD,and then the SH-SY5Y cells were divided into control group,OGD group,OGD+Vector group,OGD+Myod1 group,OGD+si-NC group,OGD+si-Myod1 group,OGD+si-SNHG15 group,OGD+si-SNHG15+Vector group,OGD+si-SNHG15+Myod1 group,OGD+miR-NC group,OGD+miR-mimics group,OGD+miR-mimics+Vector group,and OGD+miR-mimics+SNHG15 group.CCK-8 method was used to detect the cell activities in various groups;5-ethynyl-2'-deoxyuridine(EdU)staining was used to detect the rates of EDU positive cells in various groups;the rates of TdT-mediated dUTP nick end labeling(TUNEL)positive cells in various groups were detected by TUNEL staining;Western blotting method was used to detect the expression levels of cleaved caspase-3,cleaved caspase-9,B-cell lymphoma 2(Bcl-2)and Bcl-2 associated X protein(Bax)proteins in the cells in various groups;the association between Myod1 and SNHG15 was evaluated by chromatin immunoprecipitate(CHIP);dual luciferase reporter gene experiment was used to evaluate the targeting relationships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p.Results:Compared with normal control group,the expression levels of Myod1 and SNHG15 mRNA in peripheral blood of the patients in ischemic cerebral infarction group were significantly increased(P＜0.05).Compared with control group,the expression levels of Myod1 and SNHG15 mRNA in the SH-SY5Y cells in OGD group were significantly increased(P＜0.05).Compared with OGD group,the cell activities and rates of EdU positive cells in OGD+Myod1 group at 48 and 72 h were decreased(P＜0.01),and the rates of TUNEL positive cells were increased(P＜0.05);the cell activities and rates of EdU positive cells in OGD+si-Myod1 group were increased(P＜0.05),while the rates of TUNEL positive cells were decreased(P＜0.01).Myod1 binded to the promoter sequence of SNHG15.SNHG15 could absorb miR-24-3p,and there were target relatronships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p.After SNHG15 knockdown,compared with OGD group,the cell activities and rates of EdU positive cells in OGD+si-SNHG15 group at 48 and 72 h were increased(P＜0.01),and the rates of TUNEL positive cells were decreased(P＜0.01),the expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 proteins were decreased(P＜0.01),and the expression levels of Bcl-2 protein were increased(P＜0.01).Compared with OGD+si-SNHG15 group,the cell activities and rates of EdU positive cells in OGD+si-SNHG15+Myod1 group at 48 and 72 h were decreased(P＜0.05),the rates of TUNEL positive cells were(P＜0.05),the expression levels of Bax,cleaved caspase-3,and cleaved caspase-9 proteins were increased(P＜0.05),and the expression levels of Bcl-2 were decreased(P＜0.05).After over-expression of miR-24-3p and SNHG15,compared with OGD group,the cell activities and rates of EdU positive cells in OGD+miR-mimics group at 48 and 72 h were increased(P＜0.01),the rates of TUNEL positive cells were significantly decreased(P＜0.01),the protein expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 were decreased(P＜0.05),and the expression levels of Bcl-2 were increased(P＜0.01).Compared with OGD+miR-mimics group,the cell activities and rates of EdU positive cells in OGD+miR-mimics+SNHG15 group at 48 and 72 h were decreased(P＜0.05),and the rates of TUNEL positive cells were increased(P＜0.05),the expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 proteins were increased(P＜0.05),and the expression levels of Bcl-2 protein were decreased(P＜0.05).Conclusion:Myod1 can promote the proliferation inhibition and apoptosis of OGD-induced SH-SY5Y cells by binding to the SNHG15 promoter region and then absorbing miRNA-24.