Nipah virus (NiV) and related viruses form a distinct henipavirus genus within the Paramyxoviridae family. NiV continues to spillover into the humans causing deadly outbreaks with increasing human-bat interaction. NiV encodes the large protein (L) and phosphoprotein (P) to form the viral RNA polymerase machinery. Their sequences show limited homologies to those of non-henipavirus paramyxoviruses. We report two cryo-electron microscopy (cryo-EM) structures of the Nipah virus (NiV) polymerase L-P complex, expressed and purified in either its full-length or truncated form. The structures resolve the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L protein, as well as a tetrameric P protein bundle bound to the L-RdRp domain. L-protein C-terminal regions are unresolved, indicating flexibility. Two PRNTase domain zinc-binding sites, conserved in most Mononegavirales, are confirmed essential for NiV polymerase activity. The structures further reveal anchoring of the P protein bundle and P protein X domain (XD) linkers on L, via an interaction pattern distinct among Paramyxoviridae. These interactions facilitate binding of a P protein XD linker in the nucleotide entry channel and distinct positioning of other XD linkers. We show that the disruption of the L-P interactions reduces NiV polymerase activity. The reported structures should facilitate rational antiviral-drug discovery and provide a guide for the functional study of NiV polymerase.
Nipah Virus/chemistry* ; Cryoelectron Microscopy ; Viral Proteins/genetics* ; RNA-Dependent RNA Polymerase/genetics* ; Phosphoproteins/genetics* ; Humans ; Models, Molecular ; Protein Binding

ObjectiveTo analyze the epidemiological characteristics and spatial clustering patterns of brucellosis in humans in Zhangjiakou City, Heibei Province from 2018 to 2023, so as to provide a basis for the prevention and control of brucellosis. MethodsIncidence data of brucellosis in Zhangjiakou City from 2018 to 2023 were collected. Descriptive epidemiological analysis, Joinpoint regression modelling, and spatial autocorrelation analysis were used to analyze the temporal trends and spatial clustering patterns of the epidemic. ResultsA total of 3 812 cases of brucellosis were reported in Zhangjiakou City from 2018 to 2023, with no death case, yielding an average annual incidence rate of 15.43/100 000 (incidence range: 12.82/100 000‒17.76/100 000). Cases of brucellosis occurred year-round, with a distinct seasonal pattern, predominantly concentrated between March and September, peaking in May and June. The male-to-female ratio was 2.58∶1, with a higher incidence in males than that in females. The highest incidence rates were observed in the 40‒<50 years (74.98/100 000) and 50‒<60 years age group (87.14/100 000). The majority of cases were farmers and herdsmen (3 557 cases, 93.31%). Joinpoint regression analyses indicated that from 2018 to 2023, the incidence rate of human brucellosis in pastoral areas of Zhangjiakou City showed a declining trend (APC=-9.70%, 95%CI: -15.31%‒ -4.63%), while the incidence rate in mixed-use areas exhibited an increasing trend (APC=6.90%, 95%CI: 0.17%‒14.30%). Spatial clustering analyses showed that the incidence of brucellosis in Zhangjiakou from 2018 to 2023 was non-randomly distributed across the whole city, with a positive spatial correlation and significant clustering (Moran’s I>0, all P<0.001). Local spatial autocorrelation analyses showed that the high-high clusters were concentrated in the pastoral areas during 2018‒2020. From 2021 onward, the number of high-high clusters in mixed and non-pastoral regions exceeded those in traditional pastoral areas. ConclusionFrom 2018 to 2023, the incidence of brucellosis in Zhangjiakou City showed a declining trend, with significant spatial clustering observed across the city. It is recommended to intensify health education among males aged 40‒<60 years. Scientific livestock management practices should be promoted in non-pastoral and mixed areas, and cross-sectoral quarantine and joint prevention and control efforts should be strengthened as well.

Objective To investigate the therapeutic efficacy of artesunate(AS)on polycystic ovary syndrome(PCOS)in mice and explore the potential mechanism primarily.Methods Twenty-five female C57BL/6J mice were randomly divided into Control group,model group(PCOS group),low-and high-dose AS groups(AS15 and AS30 groups)and metformin group(Met group).In addition to the Control group,the mouse model of PCOS was established by subcutaneous injection of dehydroepiandrosterone(DHEA,60 mg/kg)following by a high-fat diet for 21 d.After modeling,AS of 15 and 30 mg/kg was intraperitoneally injected into the mice of the AS 15 and AS30 groups,respectively,and 200 mg/kg Met was given to those of the Met group by gavage,once per day,for 6 weeks.ELISA was used to detect serum testosterone(T),fasting insulin(FINS),luteinizing hormone(LH)and follicle-stimulating hormone(FSH),and the LH/FSH ratio was calculated.The levels of fasting blood glucose(FBG),triglyceride(TG)and total cholesterol(TC)were detected by automatic biochemical analyzer,and the homeostasis model assessment of insulin resistance(HOMA-IR)was calculated.The estrous cycle was observed,and HE staining was performed for pathological changes in the ovary and uterus.Immunofluorescence assay was employed to measure the expression of p-eIF2α,ATF4 and CHOP in the ovarian tissue.After steroidogenic human granulosa-like tumor cell line KGN were exposed to 100 μmol/L DHEA to simulate the hyperandrogen environment of PCOS,and then treated with 5 and 10 μg/mL AS for 24 h,the protein levels of endoplasmic reticulum stress signaling pathway was detected by Western blotting.Results Compared with the Control group,the PCOS mice had disturbed estrous cycle,polycystic changes in the ovaries,and significantly increased serum T level and LH/FSH ratio(P＜0.05),and obviously elevated HOMA-IR,TC and TG levels in terms of metabolism(P＜0.01).The expression levels of p-eIF2α,ATF4 and CHOP were notably up-regulated in the ovarian granulosa cells of PCOS mice and KGN cells after DHEA exposure(P＜0.05).Additionally,AS treatment attenuated the pathological changes of ovary and uterine expression,decreased the serum T level and the LH/FSH ratio(P＜0.05),and reduced HOMA-IR,TC and TG levels(P＜0.05)when compared with the PCOS mice.Moreover,the expression levels of p-eIF2α,ATF4 and CHOP were significantly down-regulated after AS treatment in both ovarian granulosa cells of PCOS mice and KGN cells(P＜0.05).Conclusion AS significantly improves glycolipid metabolic disorder and reproductive dysfunction in PCOS mice,which may be associated with its suppressing endoplasmic reticulum stress by inhibiting the PERK/eIF2α/ATF4/CHOP pathway.

Objective To investigate the role of transcription factor EB(TFEB)in endotoxin-tolerant macrophages.Methods The RAW264.7 cells were divided into blank group(DMEM medium),LPS 5 group(5 ng/mL LPS treatment for 4 h),LPS 100 group(100 ng/mL LPS treatment for 4 h),and tolerance group(5 ng/mL LPS for 12 h followed by 100 ng/mL LPS for 4 h).The releases of inflammatory factors TNF-α and IL-6 were measured using ELISA.Western blotting and immunofluorescence assay were used to evaluate the distribution of autophagy-related proteins LC3 and P62,as well as TFEB in the cytoplasm and nucleus.Lentiviral overexpression of TFEB or siRNA-mediated knockdown of TFEB were performed to observe the changes in autophagy levels and bacterial clearance ability in the tolerant cells.Results The cells in the tolerance group had significantly lower contents of TNF-α and IL-6,as well as reduced bacterial clearance ability(P<0.01),down-regulated LC3 expression while up-regulated P62 level,and decreased expression of TFEB in both the cytoplasm and nucleus(P<0.01)when compared with the cells of the LPS 100 group.Overexpression of TFEB significantly increased LC3 level,reduced P62 level,and enhanced bacterial clearance ability in the endotoxin-tolerant cells(P<0.01).In contrast,siRNA-mediated knockdown of TFEB had no significant impacts on LC3 and P62 expression levels or bacterial clearance ability.Conclusion Overexpression of TFEB can restore the autophagy of endotoxin-tolerant cells and enhance their bacterial clearance capacity,thereby alleviating the immunosuppressive state of sepsis.These findings suggest that TFEB holds promise as a potential therapeutic target for the prevention and treatment of sepsis.

Objective To investigate the role and underlying mechanism of activating transcription factor 3(ATF3)in suppressing lipopolysaccharide(LPS)-induced autophagy and inflammatory responses in macrophages.Methods Firstly,the gene expression omnibus(GEO)database was used to analyze ATF3 expression in peripheral blood mononuclear cells(PBMCs)from sepsis patients,and gene set enrichment analysis(GSEA)was performed to identify enriched signaling pathways.Secondly,RAW264.7 macrophages were divided into a blank control group and an LPS-stimulated group(100 ng/mL LPS).Western blotting and immunofluorescence assay were used to detect ATF3 protein expression and observe its subcellular localization,respectively.Lentiviral transduction was used to generate ATF3 knockdown and overexpression cell lines to evaluate their effects on cytokine release and bacterial clearance.Cleavage Under Targets and Tagmentation(CUT&Tag)sequencing was employed to identify downstream target genes transcriptionally regulated by ATF3.Furthermore,the impact of ATF3 knockdown or overexpression on autophagy-related gene 5(ATG5),autophagy-related gene 16-like 1(ATG16L1),and autophagy levels was evaluated.Results GEO analysis revealed that ATF3 expression was significantly elevated in PBMCs from sepsis patients(P<0.01),and GSEA showed significant enrichment of autophagy-related and inflammation-related pathways(P<0.01).In RAW264.7 cells,100 ng/mL LPS stimulation significantly increased ATF3 expression in the nucleus than the blank control group(P<0.01).ATF3 knockdown led to increased secretions of TNF-α and IL-6 and enhanced bacterial clearance of macrophages(P<0.01),whereas ATF3 overexpression significantly suppressed TNF-α and IL-6 releases,and remained bacterial clearance at a low level when compared with the conditions in the negative control(NC)group(P<0.01).CUT&Tag results demonstrated that ATF3 was enriched at the promoter regions of key autophagy genes Atg5 and Atg16l1.Compared with the NC group,ATF3 knockdown significantly up-regulated the protein levels of LC3-II/I,ATG5,and ATG16L1 while decreased p62 expression(P<0.01).Conversely,ATF3 overexpression inhibited the expression of LC3-II/I,ATG5,and ATG16L1(P<0.01),but had no significant effect on p62 level.Conclusion Sepsis induces elevated ATF3 expression in macrophages,and suppresses autophagic activity and down-regulates pro-inflammatory cytokines TNF-α and IL-6,which probably mediated by ATF3 regulating transcription of ATG5 and ATG16L1,suggesting ATF3 as a potential therapeutic target for autophagy-inflammation imbalance.

Objective To investigate the role of p300 in lipid metabolism disorders.Methods Bioinformatics analysis was performed to analyze the expression patterns of p300 in lipid metabolism disorder-related diseases and its correlation with SREBP-1c and downstream lipid metabolic enzymes.Immunofluorescence assay was used to detect the expression of p300 in the liver tissues of the patients with varying disease severity of non-alcoholic fatty liver disease(NAFLD).A mouse model of lipid metabolism disorder was established in male C57BL/6J mice by feeding high-fat diet(HFD)for 12 weeks.Western blotting was employed to assess p300 expression level in the liver tissues of HFD-fed mice.A cell model of lipid metabolism disorder was established in HepG2/AML-12 cells induced with free fatty acid(FFA).The effects of siRNA-mediated knockdown of p300 was observed to measure the levels of intracellular total cholesterol(TC)and triglyceride(TG),lipid deposition,and production of reactive oxygen species(ROS).Results Clinically,p300 was highly expressed in lipid metabolism disorders,and its level was positively correlated with NAFLD severity(P<0.05).Gene Set Enrichment Analysis(GSEA)revealed that p300 expression was significantly associated with fatty acid metabolism,cholesterol homeostasis,lipogenesis,PPAR signaling pathway,and peroxisome pathway.In vivo,p300 was significantly up-regulated in the livers of HFD-fed mice(P<0.01).In vitro,FFA stimulation markedly increased p300 expression in both HepG2 and AML-12 cells(P<0.01),whereas p300 knockdown significantly reduced intracellular TG and TC levels(P<0.01),attenuated lipid droplet accumulation,and reversed FFA-induced ROS elevation(P<0.01).Furthermore,p300 expression was positively correlated with the expression of SREBP-1c and its downstream key lipid synthesis enzymes.Conclusion p300 may promote hepatic lipid accumulation by acetylating and activating SREBP-1c and regulating downstream lipid metabolic enzymes,thereby affecting lipid synthesis and oxidative stress.These findings suggest that p300 may be a potential therapeutic target for lipid metabolism disorder-related diseases.

Objective:To investigate the current situation of the use of transjugular intrahepatic portosystemic shunt (TIPS) for portal hypertension, which should aid the development of TIPS in China.Methods:The China Portal Hypertension Alliance (CHESS) initiated this study that comprehensively investigated the basic situation of TIPS for portal hypertension in China through network research. The survey included the following: the number of surgical cases, main indications, the development of Early-TIPS, TIPS for portal vein cavernous transformation, collateral circulation embolization, intraoperative portal pressure gradient measurement, commonly used stent types, conventional anticoagulation and time, postoperative follow-up, obstacles, and the application of domestic instruments.Results:According to the survey, a total of 13 527 TIPS operations were carried out in 545 hospitals participating in the survey in 2021, and 94.1% of the hospital had the habit of routine follow-up after TIPS. Most hospitals believed that the main indications of TIPS were the control of acute bleeding (42.6%) and the prevention of rebleeding (40.7%). 48.1% of the teams carried out early or priority TIPS, 53.0% of the teams carried out TIPS for the cavernous transformation of the portal vein, and 81.0% chose routine embolization of collateral circulation during operation. Most of them used coils and biological glue as embolic materials, and 78.5% of the team routinely performed intraoperative portal pressure gradient measurements. In selecting TIPS stents, 57.1% of the hospitals woulel choose Viator-specific stents, 57.2% woulel choose conventional anticoagulation after TIPS, and the duration of anticoagulation was between 3-6 months (55.4%). The limitation of TIPS surgery was mainly due to cost (72.3%) and insufficient understanding of doctors in related departments (77.4%). Most teams accepted the domestic instruments used in TIPS (92.7%).Conclusions:This survey shows that TIPS treatment is an essential part of treating portal hypertension in China. The total number of TIPS cases is far from that of patients with portal hypertension. In the future, it is still necessary to popularize TIPS technology and further standardize surgical indications, routine operations, and instrument application.

Objective:To analyze the expression of silent information regulator 2-related enzyme 1 (SIRT1) and its relationship with chondrocyte apoptosis in patients with Kashin-Beck disease (KBD).Methods:Twenty patients with KBD were selected as the KBD group from Guide County, Qinghai Province, and 40 healthy subjects matched by age and sex were selected as the control group. Fasting elbow venous blood of the study subjects was collected, and peripheral blood mRNA levels of SIRT1 and selenoprotein genes [glutathione peroxidase (GPX) 2, GPX3, thioredoxin reductase (TXNRD) 1, TXNRD3, iodothyronine deiodinase Ⅰ (DIO1), and selenophosphate synthetase 2 (SPS2)] were detected by real-time PCR. The correlation between SIRT1 expression and selenoprotein genes in peripheral blood of KBD patients was analyzed by curve fitting method. Meanwhile, normal human chondrocytes cultured in vitro were divided into control group (without any treatment), resveratrol (RES) group (to verify the activation effect of RES on SIRT1), tert-butyl hydroperoxide (tBHP) injury group (oxidative injury model of chondrocyte), and RES protection group (tBHP injury after RES pre protection). The mRNA levels of SIRT1, selenoprotein genes, and apoptosis-related genes [B lymphoblastoma-2 gene (BCL2), BCL2-associated X protein (BAX), nuclear transcription factor κB (NF-κB) p65, and tumor suppressor gene P53] in each group of cells were detected by real-time PCR. Results:In the population study, the peripheral blood SIRT1 mRNA level in the KBD group (1.12 ± 0.38) was lower than that of control group (1.87 ± 0.97), and the difference was statistically significant ( t = 3.31, P = 0.002). According to curve fitting analysis, the mRNA levels of GPX3, TXNRD1, and TXNRD3 in peripheral blood of KBD group increased with the increase of SIRT1 mRNA level ( R2 = 0.48, 0.66, 0.95, P < 0.001). The level of DIO1 mRNA showed a trend of decreased first and then increased with the increase of SIRT1 mRNA level ( R2 = 0.51, P = 0.024). The mRNA levels of GPX2 and SPS2 showed no significant change trend with the increase of SIRT1 mRNA level ( R2 = 0.16, 0.12, P = 0.064, 0.114). In cell studies, compared with the control group (1.00 ± 0.10), the SIRT1 mRNA level in the RES group (1.79 ± 0.07) was higher ( P < 0.05). Compared with tBHP injury group, the RES protection group had higher mRNA levels of selenoprotein genes GPX3, TXNRD1, TXNRD3, and DIO1 ( P < 0.05); the mRNA levels of apoptosis-related genes BAX, P53 and the ratio of BAX/BCL2 were lower, while the mRNA levels of BCL2 and NF-κB p65 were higher ( P < 0.05). Conclusions:KBD patients have low expression of SIRT1. And RES activation of SIRT1 may enhance the antioxidant capacity of chondrocyte by up-regulating the expression of selenoprotein genes, thus inhibiting chondrocyte apoptosis.

Objective:To investigate the role of oxidative stress and silent information regulator 2 homolog 1 (SIRT1) in cartilage injury in Kashin-Beck disease (KBD) by evaluating the level of oxidative stress and the effect of oxidative injury on SIRT1 expression in patients with KBD.Methods:In May 2017, Twenty patients with KBD were selected from Guide County of Qinghai Province as the KBD group, and 40 healthy subjects were selected as the control group, 5 ml elbow venous blood was collected, centrifuged, and the upper plasma was retained. The glutathione peroxidase (GPX) activity and reactive oxygen species (ROS) level were determined by enzyme linked immunosorbent assay (ELISA), and SIRT1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Meanwhile, 150 μmol/L tert-butyl hydroperoxide (tBHP) was selected to damage chondrocytes; and different concentrations of sodium selenite (Na 2SeO 3) were used to intervene in chondrocytes to detect cell viability, and appropriate concentration of Na 2SeO 3 was selected for pre protection. Total RNA and DNA of chondrocytes were extracted. The mRNA levels of SIRT1, DNA methyltransferase 1 (DNMT1), and the DNA methylation level in the SIRT1 promoter region were determined by RT-qPCR. At the same time, Hoechst 33342 staining was used to detect chondrocyte apoptosis. Results:The plasma GPX activity [(35.48 ± 8.82) U/g·Hb] in KBD group was lower than that in control group [(40.43 ± 6.68) U/g·Hb, t = - 2.43, P = 0.018], and the ROS level [(577.10 ± 96.92) U/ml] was higher than that in control group [(526.44 ± 62.63) U/ml, t = 2.13, P = 0.043]. GPX activity was positively correlated with SIRT1 mRNA level ( r s = 0.44, P = 0.005), while ROS level was negatively correlated with SIRT1 mRNA level ( r s = - 0.39, P = 0.006). After 48 hours of treatment with 150 μmol/L tBHP (tBHP injury group), the survival rate of chondrocytes decreased to (55.27 ± 2.96)%; and the survival rate of chondrocytes pre-protected with 0.10 μg/ml Na 2SeO 3 (selenium protection group) was significantly higher than that of tBHP injury group ( P < 0.05). Compared with control group, the SIRT1 mRNA level of chondrocytes in tBHP injury group was significantly decreased; while the DNA methylation level in the SIRT1 promoter region, DNMT1 mRNA level and cell apoptosis rate were significantly increased ( P < 0.05). Compared with tBHP injury group, the selenium protection group had higher levels of SIRT1 mRNA in chondrocytes, lower levels of DNA methylation in the SIRT1 promoter region, DNMT1 mRNA, and cell apoptosis rate ( P < 0.05). The apoptosis rate was negatively correlated with SIRT1 mRNA level ( r s = - 0.78, P = 0.004), and positively correlated with the DNA methylation level in the SIRT1 promoter region ( r s = 0.76, P = 0.006). Conclusions:KBD patients have increased levels of oxidative stress, which may be associated with low expression of SIRT1. Oxidative injury may down-regulate SIRT1 expression and promote chondrocytes apoptosis by catalyzing DNA methylation in the SIRT1 promoter region.