Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TC...Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TCE cytotoxicity have been made in cultured liver cells. However, the molecular mechanisms by which TCE induces hepatotoxicity are not well understood. SET （also known as protein phosphatase 2A inhibitor, 12PP2A, or template-activating factor-I, TAF-D is a nuclear protein that regulates histone modification, gene transcription, DNA replication, nucleosome assembly,展开更多
The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative...The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.展开更多
Advanced age impairs bone fracture healing;the underlying mechanism of this phenomenon remains unknown.We determined that apolipoprotein E(ApoE)increases with age and causes poor fracture healing.After deletion of hep...Advanced age impairs bone fracture healing;the underlying mechanism of this phenomenon remains unknown.We determined that apolipoprotein E(ApoE)increases with age and causes poor fracture healing.After deletion of hepatic ApoE expression(ΔApoE),24-month-oldΔApoE mice displayed a 95%reduction in circulating ApoE levels and significantly improved fracture healing.ApoE treatment of aged BMSCs inhibited osteoblast differentiation in tissue culture models;RNA-seq,Western blot,immunofluorescence,and RT-PCR analyses indicated that the Wnt/β-catenin pathway is the target of this inhibition.Indeed,we showed that ApoE had no effect on cultures with stabilizedβ-catenin levels.Next,we determined that Lrp4 serves as the osteoblast cell surface receptor to ApoE,as expression of Lrp4 is required in ApoE-based inhibition of Wnt/β-catenin signaling and osteoblast differentiation.Importantly,we validated this ApoE-Lrp4-Wnt/β-catenin molecular mechanism in human osteoblast differentiation.Finally,we identified an ApoE-neutralizing antibody(NAb)and used it to treat aged,wildtype mice 3 days after fracture surgery resulting in fracture calluses with 35%more bone deposition.Our work here identifies novel liver-to-bone cross-talk and a noninvasive,translatable therapeutic intervention for aged bone regeneration.展开更多
Objective:To explore the mechanisms of fulminant hepatitis(FH) in the early stages,and to determine the critical pathways in its initiation and progression.Methods:Twelve BALB/c mice were divided into four groups:one ...Objective:To explore the mechanisms of fulminant hepatitis(FH) in the early stages,and to determine the critical pathways in its initiation and progression.Methods:Twelve BALB/c mice were divided into four groups:one group left as negative control and sacrificed immediately after injection of phosphate-buffered saline(PBS),and another three groups with concanavalin A(Con A) administration sacrificed at 1,3,and 6 h after injection.Affymetrix GeneChip Mouse 430 2.0 Array was employed to evaluate the expression profile of each of the 12 samples.Further analysis was done on the microarray data to extract the genes that were differentially expressed.Enrichment analysis was carried out to determine relevant pathways within which regulated genes were significantly enriched.Results:A total of 393,8354 and 11 344 differentially expressed genes were found,respectively,at three time points.During 0-1 h and 1-3 h,most of the pathways enriched with regulated genes were related to immune response and inflammation,among which Toll-like receptor(TLR) signaling and mitogen-activated protein kinase(MAPK) signaling appeared during both phases,while cytokine-cytokine receptor interaction,apoptosis,T cell receptor signaling,and natural killer(NK) cell-mediated cytotoxicity pathways emerged during the second phase.Pathways found to be significant during 3-6 h were mostly related to metabolic processes.Conclusion:The TLR signaling pathway dominates the early responses of Con A-induced FH in mice.It stimulates the production of type I cytokines,therefore recruiting and activating T/NK cells.Activated T/NK cells exert their cytotoxicity on hepatocytes through inducing death receptorintermediated apoptosis,resulting in liver injury.展开更多
Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at Eco...Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E.coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter,SD sequence and identical N-terminus.The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants.The findings were compared to results obtained by other workers in studies of HBcAg expression in procaryotes and their significance in the expression of eucaryotic genes in procaryotic cells were discussed.展开更多
Objective Impaired hepatic expression of protein tyrosine phosphatase delta(PTPRD)is associated with increased STAT3 transcriptional activity and reduced survival from hepatocellular carcinoma in patients with chronic...Objective Impaired hepatic expression of protein tyrosine phosphatase delta(PTPRD)is associated with increased STAT3 transcriptional activity and reduced survival from hepatocellular carcinoma in patients with chronic hepatitis C virus infection.However,the PTPRD-expressing hepatic cell types,signalling pathways responsive to PTPRD and their role in non-viral liver disease are largely unknown.Methods We studied PTPRD expression in single-cell and bulk liver transcriptomic data from mice and humans,and established a Ptprd-deficient mouse model for metabolic dysfunction-associated steatohepatitis(MASH).Identified pathways were validated by perturbation studies in human hepatocytes and PTPRD substrates by pull-down assays.The clinical relevance was further explored in a cohort with metabolic disease by ranking patients according to PTPRD expression and analysing its association with metabolic disease markers.Results The analysis of individuals ranked according to PTPRD expression and Ptprd-deficient mice,showed that PTPRD levels were associated with hepatic glucose/lipid signalling and peroxisome function.Hepatic PTPRD expression is impaired in aetiologies of chronic liver diseases that are associated with metabolic disease.We further validated PTPRD as a STAT3 phosphatase in the liver,acting as a regulator of peroxisomal fatty acid metabolism.During MASH,low PTPRD led to increased liver steatosis in Ptprd+/−mice and a pronounced unfolded protein response,which impacts insulin signalling.Accordingly,silencing of PTPRD blunted insulin-induced AKT phosphorylation.Patients with obesity and low hepatic PTPRD expression exhibit increased levels of metabolic risk factors.Conclusion Our data revealed an important regulatory role of the hepatic PTPRD-STAT3 axis in maintaining glucose/lipid homeostasis,which is recapitulated in clinical manifestations of metabolic liver disease.展开更多
基金supported by NSFC (the National Natural Science Foundation of China) [81273126, 30972454]the Key Project of Guangdong Natural Science Foundation [S2012020010903]+2 种基金the Project of Shenzhen Basic Research Plan [JCYJ20120616 154222545]the Upgrade Scheme of Shenzhen Municipal Key Laboratory [CXB201005260068A]Medical Scientific Research Foundation of Guangdong Province (A2012577)
文摘Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TCE cytotoxicity have been made in cultured liver cells. However, the molecular mechanisms by which TCE induces hepatotoxicity are not well understood. SET （also known as protein phosphatase 2A inhibitor, 12PP2A, or template-activating factor-I, TAF-D is a nuclear protein that regulates histone modification, gene transcription, DNA replication, nucleosome assembly,
基金grants from China National 973 Project （Grant G 1999054105） National Natural Science Foundation of China （No. 30530040）.
文摘The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.
基金supported by a Borden Scholars awardDuke Claude D.Pepper Older Americans Independence Center Pilot Award(P30AG028716)by the NIH/NIA(R01AG081393)。
文摘Advanced age impairs bone fracture healing;the underlying mechanism of this phenomenon remains unknown.We determined that apolipoprotein E(ApoE)increases with age and causes poor fracture healing.After deletion of hepatic ApoE expression(ΔApoE),24-month-oldΔApoE mice displayed a 95%reduction in circulating ApoE levels and significantly improved fracture healing.ApoE treatment of aged BMSCs inhibited osteoblast differentiation in tissue culture models;RNA-seq,Western blot,immunofluorescence,and RT-PCR analyses indicated that the Wnt/β-catenin pathway is the target of this inhibition.Indeed,we showed that ApoE had no effect on cultures with stabilizedβ-catenin levels.Next,we determined that Lrp4 serves as the osteoblast cell surface receptor to ApoE,as expression of Lrp4 is required in ApoE-based inhibition of Wnt/β-catenin signaling and osteoblast differentiation.Importantly,we validated this ApoE-Lrp4-Wnt/β-catenin molecular mechanism in human osteoblast differentiation.Finally,we identified an ApoE-neutralizing antibody(NAb)and used it to treat aged,wildtype mice 3 days after fracture surgery resulting in fracture calluses with 35%more bone deposition.Our work here identifies novel liver-to-bone cross-talk and a noninvasive,translatable therapeutic intervention for aged bone regeneration.
基金Project supported by the National Natural Science Foundation of China (No 30771918)the National Basic Research Program (973) of China (No 2007CB512905)the State S & T Projects (11th Five Year) (No 2008ZX10002-007) of China
文摘Objective:To explore the mechanisms of fulminant hepatitis(FH) in the early stages,and to determine the critical pathways in its initiation and progression.Methods:Twelve BALB/c mice were divided into four groups:one group left as negative control and sacrificed immediately after injection of phosphate-buffered saline(PBS),and another three groups with concanavalin A(Con A) administration sacrificed at 1,3,and 6 h after injection.Affymetrix GeneChip Mouse 430 2.0 Array was employed to evaluate the expression profile of each of the 12 samples.Further analysis was done on the microarray data to extract the genes that were differentially expressed.Enrichment analysis was carried out to determine relevant pathways within which regulated genes were significantly enriched.Results:A total of 393,8354 and 11 344 differentially expressed genes were found,respectively,at three time points.During 0-1 h and 1-3 h,most of the pathways enriched with regulated genes were related to immune response and inflammation,among which Toll-like receptor(TLR) signaling and mitogen-activated protein kinase(MAPK) signaling appeared during both phases,while cytokine-cytokine receptor interaction,apoptosis,T cell receptor signaling,and natural killer(NK) cell-mediated cytotoxicity pathways emerged during the second phase.Pathways found to be significant during 3-6 h were mostly related to metabolic processes.Conclusion:The TLR signaling pathway dominates the early responses of Con A-induced FH in mice.It stimulates the production of type I cytokines,therefore recruiting and activating T/NK cells.Activated T/NK cells exert their cytotoxicity on hepatocytes through inducing death receptorintermediated apoptosis,resulting in liver injury.
文摘Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E.coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter,SD sequence and identical N-terminus.The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants.The findings were compared to results obtained by other workers in studies of HBcAg expression in procaryotes and their significance in the expression of eucaryotic genes in procaryotic cells were discussed.
基金supported by the European Union(ERC-AdG-2014 HEPCIR ERC POC PRELICAN 755460,ERC POC HEPCAN 862551 to TFB,EU H2020 HEPCAR 667273 to TFB and JL,HORIZON-HLTH-2021-DISEASE-04-07 D-SOLVE#101057917 to TFB and JL,IP-cure-B project#847939 to FZ,ERC-AdG-2020-101021417 to YH)the French Cancer Agency(TheraHCC2.0 IHU201901299 to TFB)+9 种基金the Agence Nationale de la Recherche(ANR-21-RHUS-001 to TFB)the ANRS Maladies infectieusesémergentes(ANRS-MIE)(ECTZ103701,ECTZ131760,ECTZ160436,ECTZ171594 to JL,ECTZ104017 and ECTZ75178 to TFB,ECTZ4446 and ECTZ206376 to AARS)the Fondation de l’Universitéde Strasbourg(HEPKIN)(TBA-DON-0002),SATT Conectus,University of Strasbourg(CANCLAU)(TFB)the Inserm Plan Cancer 2019-2023the US National Institutes of Health(CA233794,CA255621,CA282178,CA288375 and CA283935 to YH)the Cancer Prevention and Research Institute of Texas(RR180016,RP200554 to YH)The AEPIC animal facility platform is financially supported by the CoRTecS network of the University of StrasbourgThis work of the Interdisciplinary Thematic Institute IMCBio,as part of the ITI 2021-2028 programme of the University of Strasbourg,CNRS and Inserm,was supported by IdEx Unistra(ANR-10-IDEX-0002)SFRI-STRAT’US project(ANR 20-SFRI-0012)EUR IMCBio(ANR-17-EURE-0023)under the framework of the French Investments for the Future Programme,as well as state funds managed within the France 2030 programme(reference ANR-21-RHUS-0001).
文摘Objective Impaired hepatic expression of protein tyrosine phosphatase delta(PTPRD)is associated with increased STAT3 transcriptional activity and reduced survival from hepatocellular carcinoma in patients with chronic hepatitis C virus infection.However,the PTPRD-expressing hepatic cell types,signalling pathways responsive to PTPRD and their role in non-viral liver disease are largely unknown.Methods We studied PTPRD expression in single-cell and bulk liver transcriptomic data from mice and humans,and established a Ptprd-deficient mouse model for metabolic dysfunction-associated steatohepatitis(MASH).Identified pathways were validated by perturbation studies in human hepatocytes and PTPRD substrates by pull-down assays.The clinical relevance was further explored in a cohort with metabolic disease by ranking patients according to PTPRD expression and analysing its association with metabolic disease markers.Results The analysis of individuals ranked according to PTPRD expression and Ptprd-deficient mice,showed that PTPRD levels were associated with hepatic glucose/lipid signalling and peroxisome function.Hepatic PTPRD expression is impaired in aetiologies of chronic liver diseases that are associated with metabolic disease.We further validated PTPRD as a STAT3 phosphatase in the liver,acting as a regulator of peroxisomal fatty acid metabolism.During MASH,low PTPRD led to increased liver steatosis in Ptprd+/−mice and a pronounced unfolded protein response,which impacts insulin signalling.Accordingly,silencing of PTPRD blunted insulin-induced AKT phosphorylation.Patients with obesity and low hepatic PTPRD expression exhibit increased levels of metabolic risk factors.Conclusion Our data revealed an important regulatory role of the hepatic PTPRD-STAT3 axis in maintaining glucose/lipid homeostasis,which is recapitulated in clinical manifestations of metabolic liver disease.
