Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Objective To explore the role and possible molecular mechanism of Isocitrate dehydrogenase 1(IDH1)gene in proliferation and migration of intrahepatic cholangiocarcinoma(iCCA)cell HuCCT1.Methods HuCCT1 cells with IDH1 gene knockout(HuCCT1IDH1-/-)were constructed by CRISPR/Cas9 gene editing technology.To investigate the capacities of proliferation,migration and invasion of HuCCT1WT(HuCCT1 cells with wild-type IDH1 gene)and HuCCT1IDH1-/-cells,assays of CCK-8,clone formation,scratch and transwell were performed.Western blotting was used to detect the expression levels of epithelial-mesenchymal transition(EMT)associated proteins E-cadherin,N-cadherin,Vimentin,MMP-9,Wnt3a and β-catenin in two groups of cells.The transcriptome sequencing data of HuCCT1WT and HuCCT1IDH1-/-cells were analyzed by bioinformatics methods,Western blotting was used to verify the expression of signaling pathway-related proteins.Results Compared with HuCCT1WT cells,HuCCT1IDH1-/-cells showed the number of proliferation and clone formation significantly reduced(P<0.05),the proportion of cells blocked in G2/M phase was significantly increased(P<0.01),the rate of scratch healing was significantly decreased(P<0.01),and the number of migrated cells(P<0.001)and invaded cells(P<0.05)was significantly reduced.qRT-PCR assay showed that the expression levels of IDH1,Vimentin,MMP-9 and genes related to the regulation of G2/M cycle proliferation,Cyclin A2,Cyclin B1 and CDK1 mRNA were down-regulated in HuCCT1IDH1-/-cells(P<0.05),and the expression of CDH1 mRNA encoding E-cadherin was up-regulated(P<0.01);Western blotting assay showed that the expression level of E-cadherin in HuCCT1IDH1-/-cells was significantly increased(P<0.05),and the expression level of N-cadherin,Vimentin and MMP-9 protein was significantly decreased(P<0.05)than that in HuCCT1WT cells.Data of transcriptome sequencing revealed 1476 differentially expressed genes(DEGs)between two groups of HuCCT1 cells.Go enrichment analysis showed the DEGs were significantly enriched in cell biological processes associated with inflammatory response,cell signaling and cell metabolism.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis suggested that the DEGs may be involved in some signaling pathways such as Wnt,MAPK,Rap1,Hippo and TNF,which are closely related to the regulation of proliferation and invasion of tumor cells.Western blotting verification results showed that compared with HuCCT1WT cells,the relative expression of Wnt3a and β-catenin proteins of HuCCT1IDH1-/-cells was significantly decreased(P<0.05).Conclusions IDH1 gene may participate in the control of biological functions of HuCCT1 cells,including cell proliferation,migration,invasion and epithelial mesenchymal transition.The mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.

ObjectiveTePixD (Tll0078) is a blue light-using flavin (BLUF) photoreceptor protein from Thermosynechococcus elongatus BP-1. TePixD protein has a conserved Tyr8-Gln50-Met93 triad around the FAD pocket to mediate the proton-coupled electron transfer (PCET) process. But the detailed light response mechanism needs further study. We aimed to elucidate the structure and biochemical properties of TePixD mutants at key light response sites to analyze the light response process of TePixD. MethodsWe employed X-ray crystallography to resolve the crystal structure of the TePixD Y8F mutant. The side chain of Tyr8 is involved in PCET while Phe8 in mutation loses the function due to the loss of its hydroxyl group. We compared the structure of TePixD Y8F mutation to TePixD wild type (WT) and its homology protein SyPixD Y8F. Using multi-angle light scattering (MALS), we analyzed the oligomerization of multiple TePixD mutations (Y8F, Q50L, W91F, Y8F/W91F, and Q50L/W91F), focusing specifically on mutational sites that are critical residues for the protein’s photo response to dark and light conditions. ResultsWe resolved the crystal structure of TePixD Y8F mutant at a resolution of 2.54 Å and found that it shares a similar overall structure with the TePixD WT but exhibits significant differences from the SyPixD Y8F structure. Biochemical analysis revealed differences in molecular mass and elution profiles between the TePixD mutants and the WT under dark and light conditions, indicating the perturbation on the light-induced conformational change by the mutants. ConclusionOur structure determination and biochemical analyses will add information to reveal the light response mechanism of BLUF proteins.

Manganese superoxide dismutase catalyzes the dismutation of two molecules of superoxide radicals to one molecule of oxygen and one molecule of hydrogen peroxide. The oxidation of superoxide anion to oxygen by Mn³⁺SOD proceeds at a rate close to diffusion. The reduction of superoxide anion to hydrogen peroxide by Mn²⁺SOD can be progressed parallelly in either a fast or a slow cycle pathway. In the slow cycle pathway, Mn²⁺SOD forms a product inhibitory complex with superoxide anion, which is protonated and then slowly releases hydrogen peroxide out. In the fast cycle pathway, superoxide anion is directly converted into product hydrogen peroxide by Mn²⁺SOD, which facilitates the revival and turnover of the enzyme. We proposed for the first time that temperature is a key factor that regulates MnSOD into the slow- or fast-cycle catalytic pathway. Normally, the Mn²⁺ rest in the pent-coordinated state with four amino acid residues (His26, His74, His163 and Asp159) and one water (WAT1) in the active center of MnSOD. The sixth coordinate position on Mn (orange arrow) is open for water (WAT2, green) or O₂^• to coordinate. With the cold contraction in the active site as temperature decreases, WAT2 is closer to Mn, which may spatially interfere with the entrance of O₂^• into the inner sphere, and avoid O₂^•/Mn²⁺ coordination to reduce product inhibition. Low temperature compels the reaction into the faster outer sphere pathway, resulting in a higher gating ratio for the fast-cycle pathway. As the temperature increases in the physiological temperature range, the slow cycle becomes the mainstream of the whole catalytic reaction, so the increasing temperature in the physiological range inhibits the activity of the enzyme. The biphasic enzymatic kinetic properties of manganese superoxide dismutase can be rationalized by a temperature-dependent coordination model of the conserved active center of the enzyme. When the temperature decreases, a water molecule (or OH^-) is close to or even coordinates Mn, which can interfere with the formation of product inhibition. So, the enzymatic reaction occurs mainly in the fast cycle pathway at a lower temperature. Finally, we describe the several chemical modifications of the enzyme, indicating that manganese superoxide dismutase can be rapidly regulated in many patterns (allosteric regulation and chemical modification). These regulatory modulations can rapidly and directly change the activation of the enzyme, and then regulate the balance and fluxes of superoxide anion and hydrogen peroxide in cells. We try to provide a new theory to reveal the physiological role of manganese superoxide dismutase and reactive oxygen species.

Osteoporosis(OP)is a systemic bone disease characterized by low bone mass and damage to the microstructure of bone tissue,leading to increased bone fragility and susceptibility to fracture.Owing to its high prevalence,disability and mortality rates,as well as more sequelae,it brings heavy burden to patients and society.In recent years,as the research on the chemical and pharmacological effects of Psoralea corylifolia has made great progress,scholars have isolated compounds such as coumarins,flavonoids and monoterpene phenols,which have anti-OP,anti-oxidation,and anti-inflammatory properties,Psoralea corylifolia has been widely used in the treatment of OP.This article reviews the regulatory mechanism of active ingredients of Psoralea corylifolia in OP lipid metabolism,bone metabolism and oxidative stress,as well as related therapeutic strategies targeting Wnt/β-catenin,PI3K/Akt,PPAR-γ/Wnt,RANKL/RANK/MAPK and NF-κB signaling pathways,in order to provide further reference for the prevention and treatment of OP.

Objective To examine the changing antimicrobial resistance profile of Enterobacter spp.isolates in 53 hospitals across China from 2015 t0 2021.Methods The clinical isolates of Enterobacter spp.were collected from 53 hospitals across China during 2015-2021 and tested for antimicrobial susceptibility using Kirby-Bauer method or automated testing systems according to the CHINET unified protocol.The results were interpreted according to the breakpoints issued by the Clinical & Laboratory Standards Institute(CLSI)in 2021(M100 31st edition)and analyzed with WHONET 5.6 software.Results A total of 37 966 Enterobacter strains were isolated from 2015 to 2021.The proportion of Enterobacter isolates among all clinical isolates showed a fluctuating trend over the 7-year period,overall 2.5％in all clinical isolates amd 5.7％in Enterobacterale strains.The most frequently isolated Enterobacter species was Enterobacter cloacae,accounting for 93.7％(35 571/37 966).The strains were mainly isolated from respiratory specimens(44.4±4.6)％,followed by secretions/pus(16.4±2.3)％and urine(16.0±0.9)％.The strains from respiratory samples decreased slightly,while those from sterile body fluids increased over the 7-year period.The Enterobacter strains were mainly isolated from inpatients(92.9％),and only(7.1±0.8)％of the strains were isolated from outpatients and emergency patients.The patients in surgical wards contributed the highest number of isolates(24.4±2.9)％compared to the inpatients in any other departement.Overall,≤ 7.9％of the E.cloacae strains were resistant to amikacin,tigecycline,polymyxin B,imipenem or meropenem,while ≤5.6％of the Enterobacter asburiae strains were resistant to these antimicrobial agents.E.asburiae showed higher resistance rate to polymyxin B than E.cloacae(19.7％vs 3.9％).Overall,≤8.1％of the Enterobacter gergoviae strains were resistant to tigecycline,amikacin,meropenem,or imipenem,while 10.5％of these strains were resistant to polycolistin B.The overall prevalence of carbapenem-resistant Enterobacter was 10.0％over the 7-year period,but showing an upward trend.The resistance profiles of Enterobacter isolates varied with the department from which they were isolated and whether the patient is an adult or a child.The prevalence of carbapenem-resistant E.cloacae was the highest in the E.cloacae isolates from ICU patients.Conclusions The results of the CHINET Antimicrobial Resistance Surveillance Program indicate that the proportion of Enterobacter strains in all clinical isolates fluctuates slightly over the 7-year period from 2015 to 2021.The Enterobacter strains showed increasing resistance to multiple antimicrobial drugs,especially carbapenems over the 7-year period.

Interleukin-35(IL-35)is an important immunosuppressive cytokine that has been shown to play a role in the immune response of various diseases.In this study,we cloned the coding sequence of human IL-35 gene,constructed single subunit expression vectors pXC17.4-p35 and pcDNA3.1(+)-EBI3,and co-transfected CHO-K1 cells to express IL-35 in vitro.No binding was found between subunits of p35 and EBI3.Knobs-into-Holes(KIH)can solve the problem of heavy chain mismatch of heterolo-gous antibodies.Therefore,expression vectors pXC17.4-p35-Fch and pcDNA3.1(+)-EBI3-Fck were constructed by fusing KIH structures on the basis of the original sequences to express the recombinant fu-sion protein of KIH-IL-35.The expression vectors of two subunits were exchanged at the same time to verify the influence of different vectors on the expression level of KIH-IL-35.The analysis of various pro-tein detection methods showed that the correct expression rate of KIH-IL-35 structure was significantly im-proved.Affinity purification of KIH-IL-35 was performed after large amount of expression,and the bind-ing activity of KIH-IL-35 to glycoprotein 130(gp130)was detected by ELISA.The results showed that the binding of KIH-IL-35 to gp130 was concentration dependent.The indirect activity of KIH-IL-35 and M1 cells was detected by cell activity assay.Further results showed that the inhibition rate of M1 cells in-creased in a dose-dependent manner with the concentration of KIH-IL-35.In addition,a method for de-termining IL-35 activity by activated human peripheral blood mononuclear cells was successfully estab-lished.Activated PBMCs increased in a dose-dependent manner with KIH-IL-35 concentration.In sum-mary,this study utilized the KIH-IL-35 model to enhance the expression of recombinant human IL-35 and validated its high activity in vitro,providing new ideas for the study of IL-35 and the recombinant expres-sion of similar heterodimeric cytokines.

Objective:To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC ) from human umbilical cords and evaluate its cell biological properties.Methods:In control group,mesenchymal stem cells (MSC) were isolated by digesting human umbilical cord Wharton's Jelly tissues with 0.2％ collagenase Ⅱ,and the released cells were collected and cultured in an animal serum-free culture medium.In AO-MSC group,incompletely collagenase Ⅱ-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture. The conventional digestion and culture method was used as control.MSC colony forming ability was evaluated by fibroblast colony forming assay (CFU-F).MSC proliferative capacity was evaluated by CCK-8 assay.The MSC surface markers were detected by using flow cytometry and immunofluorescence staining.The adipogenic and osteogenic capacity of MSC was evaluated by multi-differentiation in vitro,and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR (RT-qPCR );Moreover,the mRNA expression of antioxidant substances such as SOD-1,GSH,GAT,and NQO1 in MSC was also evaluated by RT-qPCR.Results:The AO-MSC isolated by this strategy reached a confluence of 80％-90％ at around 18 days and grew in a swirling pattern.Flow cytometry and immunofluorescence staining assays showed that CD73,CD29,CD105,CD90 were highly expressed and CD31,CD45,HLA-DR were scarcely expressed in AO-MSC.AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC.However,the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC.RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.Conclusion:Human AO-MSC is successfully prepared from human umbilical cord without animal serum.