Abstract: Objective　This study aims to explore the roles of interferon-stimulated gene cholesterol-25-hydroxylase (CH25H) on the replication of Japanese encephalitis virus (JEV) and its underlying mechanisms, providing potential targets for developing anti-JEV drugs and theoretical support for the research. Methods　First, we investigated whether CH25H could be induced by type Ⅰinterferons or JEV infection in human kidney HEK293T cells through real-time fluorescence quantitative PCR. The cells were stimulated with different concentrations of interferon α (IFN-α), and 12 hours later, CH25H expression was measured via real-time fluorescence quantitative PCR. HEK293T cells were infected with JEV at different multiplicities of infection (MOI), and 24 hours later, changes in CH25H mRNA expression levels were assessed. Eukaryotic expression plasmid pcDNA3.1-CH25H-3×HA was constructed for overexpression of CH25H in HEK293T cells, and its effect on JEV replication was analyzed using real-time fluorescence quantitative PCR and plaque formation assay. The reasonable use concentration of 25-hydroxycholesterol (25HC), the enzyme active product of CH25H, in HEK293T, BHK21, and Vero cells was obtained by CCK8 assay, and the roles of 25-hydroxycholesterol (25HC), the enzyme active product of CH25H, in JEV replication and its impact on JEV life cycle, were further studied by qPCR, viral plaque assay, immunofluorescence assay and adsorption assay. Point mutation was employed to construct an enzymatically inactive mutant, CH25HM, which was transfected into HEK293T cells. After 24 hours of JEV infection, the dependence of CH25H-mediated impact on JEV replication on its enzymatic activity was revealed through qPCR and plaque formation assay. Results　CH25H could be induced by IFN-α in HEK293T cells, but after JEV infection, CH25H mRNA expression was downregulated. Overexpression of CH25H in HEK293T cells significantly inhibited JEV replication. 25HC, the enzyme product of CH25H, can inhibit JEV replication in HEK293T, BHK21, and Vero cells. In terms of mechanism, 25HC can inhibit JEV replication by affecting the virus adsorption process. Expressing the enzymatically inactive mutant CH25HM in HEK293T cells, also significantly inhibited JEV replication. Conclusions　JEV infection down-regulates the expression of interferon-stimulated gene CH25H, while CH25H inhibits JEV replication in a manner dependent on enzyme activity and non-enzyme activity.

Objective To investigate the application value of transumbilical single-port total laparoscopic hysterectomy by conventional instrument without uterine-lifting in the treatment of cervical lesions.Methods We selected 60 cases of total laparoscopic hysterectomy due to cervical high-grade squamous intraepithelial lesion(HSIL)or cervical cancer stage ⅠA1 from December 2021 to June 2023.According to the patients'preference,30 cases of single-port laparoscopy through the umbilicus and 30 cases of multi-port laparoscopy were performed,both using conventional instruments without uterine-lifting.The surgical indicators of the two groups were compared.Results No conversion to open surgery occurred in both groups,and no intraoperative injuries to the urinary system,bowel,or major blood vessels occurred.As compared with the multi-port group,the single-port group had significantly reduced amount of bleeding during surgery[(54.6±20.5)ml vs.(67.5±27.0)ml,P = 0.041],earlier anal exhaust time[(27.6±8.0)h vs.(32.2±9.0)h,P =0.040],and shorter total hospitalization time[(4.4±1.5)d vs.(5.1±1.2)d,P = 0.044].There were no significant differences in uterine weight,surgical time,and postoperative complications between the two group(P>0.05).The healing of the abdominal wall puncture wounds in both groups of patients were satisfied.There were no short-term complications related to the puncture device(such as puncture wound infection and bleeding)or long-term complications(such as umbilical hernia and incisional hernia).Conclusion Transumbilical single-port total laparoscopic hysterectomy without uterine-lifting presents advantages of less intraoperative bleeding,faster postoperative recovery,and almost no scarring,with complications similar to traditional laparoscopic surgery.

Objective:To investigate the molecular mechanisms by which SARS-CoV-2 accessory protein ORF7a mediating NF-κB activation and thus inducing inflammatory cytokines production.Methods:Effects of ORF7a on NF-κB promoter activity was analyzed by luciferase reporter assay.qRT-PCR was used to analyze expressions of inflammatory cytokines.Effects of ORF7a on phos-phorylation and nuclear-translocation of p65 were determined by Western blot and immunofluorescence.Co-immunoprecipitation and immunofluorescence assay were performed to investigate potential target s of ORF7a.Results:Luciferase reporter assay showed that ORF7a activated NF-κB promoter activity in a dose-dependent manner(P<0.001),while has no effect on activation of AP-1 reporter gene.ORF7a significantly upregulates expressions of TNF-α,IL-1β and IL-8 mRNA levels(P<0.05).Western blot showed that ORF7a markedly increased phosphorylation of p65 protein(P<0.05)and p65 nuclear localization(P<0.01).The interaction between ORF7a and IKKβ protein of NF-κB signaling pathway was found by immunoprecipitation assay,and the co-localization of ORF7a and IKKβ was also confirmed by immunofluorescence assay.Conclusion:SARS-CoV-2 ORF7a targets IKKβ to promote active NF-κB pathway to the release of inflammatory cytokines.