Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorio...Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.展开更多
Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunopreci...Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.展开更多
Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms ...Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.展开更多
[Objective] The aim was to investigate the possible interaction between SET and eEF1A1 in human liver cells. [Method] Firstly the total proteins of human L-02 liver cells were extracted under non-denaturing conditions...[Objective] The aim was to investigate the possible interaction between SET and eEF1A1 in human liver cells. [Method] Firstly the total proteins of human L-02 liver cells were extracted under non-denaturing conditions; then,mouse anti-human SET and rabbit anti-human eEF1A1 antibodies were used to perform the co-immunoprecipitation respectively; subsequently,the immunoprecipitations was correspondingly detected with rabbit anti-human eEF1A1 and mouse anti-human SET antibodies by Western Blot. [Result] EEF1A1 was detected in protein complex from the immunoprecipitations by using anti-SET antibody,and SET also was detected in immunoprecipitations by using anti-eEF1A1 antibody. [Conclusion] The interaction between SET and eEF1A1 in human liver cells was confirmed.展开更多
Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whethe...Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whether smaller volume could save testing time.Result:Time consumption of elution in Group T was significantly shorter than that in Group C,while the efficiency of eluted m6A-containing fragments and the performance of m^(6)A-Seq as indicated by m6A peak distributions showed no difference between the two groups.Conclusion:A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.展开更多
目的探讨CMTM6、PD-L1在子宫内膜异位症(endometriosis,EM)中的表达及意义。方法收集50例EM患者腹腔镜下手术切除的卵巢异位子宫内膜组织、刮除的在位子宫内膜组织(实验组)以及30例非EM患者刮除的在位子宫内膜组织(对照组),采用免疫组...目的探讨CMTM6、PD-L1在子宫内膜异位症(endometriosis,EM)中的表达及意义。方法收集50例EM患者腹腔镜下手术切除的卵巢异位子宫内膜组织、刮除的在位子宫内膜组织(实验组)以及30例非EM患者刮除的在位子宫内膜组织(对照组),采用免疫组化法检测CMTM6、PD-L1的表达水平。收集实验组相关临床病理资料,包括患者年龄、体重指数(body mass index,BMI)、病灶大小、血清CA-125浓度及rAFS评分等,采用Spearman系数法分析CMTM6、PD-L1的表达与上述病理参数的相关性。培养人EM上皮细胞系12Z,采用Western blot及免疫蛋白共沉淀法检测CMTM6、PD-L1的表达水平及相互作用。结果CMTM6、PD-L1在EM患者中高表达,EM患者异位、在位子宫内膜中的表达水平高于非EM患者在位子宫内膜组织(P<0.05),CMTM6、PD-L1表达共同定位于异位、在位子宫内膜组织上皮细胞质、细胞膜中,且两种蛋白表达呈正相关(P<0.05)。患者年龄、BMI、病灶大小、血清CA-125浓度及rAFS评分与CMTM6、PD-L1的表达呈正相关(P<0.01)。CMTM6、PD-L1蛋白在12Z中表达,且CMTM6和PD-L能够相互结合。结论CMTM6、PD-L1在EM患者中高表达,CMTM6、PDL1的表达水平与EM病变严重程度相关,CMTM6、PD-L1在EM中能相互结合共同参与EM的发生、发展,CMTM6、PD-L1或可成为EM的潜在生物学标志物,为EM的临床诊治提供新靶点。展开更多
BACKGROUND The Warburg effect is common in cancers.Lactate and its receptor GPR81 play an important role in cancer progression.It is widely accepted that membrane receptor nuclear translocation plays some novel role i...BACKGROUND The Warburg effect is common in cancers.Lactate and its receptor GPR81 play an important role in cancer progression.It is widely accepted that membrane receptor nuclear translocation plays some novel role in cancer pathology.The mechanism by which the lactate/GPR81 axis regulates cancer malignancy remains unclear.AIM To elucidate the mechanism of GPR81 nuclear transportation promoted by exogenous lactate.METHODS Lung cancer cells were stimulated with exogenous lactate and GPR81 levels were measured by immunofluoresence and western blot analysis in membrane,cytoplasmic,and nuclear fractions.Lung cancer cells were transduced with a mutant GPR81 nuclear localization signal(NLS)construct,wild type GPR81 or empty vector and used to examine how GPR81 nuclear transportation affects lung cancer cells malignancy in vitro and in vivo.Immunoprecipitation Proteomics analysis and Chromatin immunoprecipitation(ChIP)sequencing were used to determine GPR81 interacting proteins and genes.RESULTS In response to hypoxia/Lactate stimulation,GPR81 translocates and accumulates in the nucleus of lung cancer cells.Functionally,GPR81 nuclear translocation promotes cancer cell proliferation and motility.Depletion of the GPR81 NLS depletes GPR81 nuclear levels and decreases cancer cell growth and invasion in vitro,as well as cancer cell malignancy in vivo.Proteomics analysis revealed a set of proteins including SFPQ,that interact with GPR81 in the cancer cell nucleus.Notably,the interaction of GPR81 with SFPQ promotes cancer cell growth and motility.ChIP sequencing analysis discovered that there is a set of genes targeted by GPR81.CONCLUSION The interaction of GPR81 with SFPQ promotes cancer cell malignancy.GPR81 nuclear translocation is critical in conferring cancer progression and may be a potential therapeutic target for limiting cancer progression.展开更多
BACKGROUND The exact mechanisms underlying diabetic nephropathy(DN)remain incompletely elucidated,prompting researchers to explore new perspectives and identify novel intervention targets in this field.AIM To explore ...BACKGROUND The exact mechanisms underlying diabetic nephropathy(DN)remain incompletely elucidated,prompting researchers to explore new perspectives and identify novel intervention targets in this field.AIM To explore the role and underlying mechanisms of farnesoid X receptor(FXR)in the development of DN by regulating endoplasmic reticulum stress(ERS)molecular chaperone binding immunoglobulin protein(BiP)expression.METHODS Bioinformatics analyses identified potential FXR-binding elements in the BiP promoter.Dual-luciferase and chromatin immunoprecipitation(ChIP)assays confirmed FXR-BiP binding sites.In vitro studies used SV40 MES 13 cells under varying glucose conditions and treatments with FXR modulators[obeticholic acid(INT-747)and guggulsterones]or BiP small interfering RNA.The expression of BiP and ERS-related proteins[protein kinase R-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme 1(IRE1),activating transcription factor 6(ATF6)]was assessed alongside cell proliferation and extracellular matrix(ECM)synthesis.In vivo studies in DN mice(db/db)examined the effects of FXR activation on renal function and morphology.RESULTS FXR bound to the target sequence in the BiP promoter region,enhancing transcriptional activity,as confirmed by ChIP experiments.FXR expression decreased in SV40 MES 13 cells stimulated with high glucose and in renal tissues of DN mice compared with control.Treatment of SV40 MES 13 cells with the FXR agonist INT-747 significantly increased intracellular BiP expression,whereas silencing the FXR gene led to the downregulation of BiP levels.In vivo administration of INT-747 significantly elevated BiP levels in renal tissues,improved renal function and fibrosis in DN mice,while inhibiting the expression of ERS-related signaling proteins PERK,IRE1,and ATF6.CONCLUSION FXR promotes BiP expression by binding to its promoter,suppressing ERS pathways,and reducing mesangial cell proliferation and ECM synthesis.These findings highlight FXR as a potential therapeutic target for diabetic glomerulosclerosis.展开更多
文摘Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.
文摘Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
基金supported by the National Program on Key Research Project of China(2018YFE0200400,2019YFC1200700)the National Natural Science Foundation of China(U20A20135)+1 种基金the Strategic Biological Resources Capacity Building Project of Chinese Academy of Sciences(KFJ-BRP-017-06)the Key deployment projects of Chinese Academy of Sciences(KJZD-SW-L11)
文摘Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.
基金Supported by National Natural Science Foundation (30972454)Shenzhen Science and Technology Plan Major Project (200801010)~~
文摘[Objective] The aim was to investigate the possible interaction between SET and eEF1A1 in human liver cells. [Method] Firstly the total proteins of human L-02 liver cells were extracted under non-denaturing conditions; then,mouse anti-human SET and rabbit anti-human eEF1A1 antibodies were used to perform the co-immunoprecipitation respectively; subsequently,the immunoprecipitations was correspondingly detected with rabbit anti-human eEF1A1 and mouse anti-human SET antibodies by Western Blot. [Result] EEF1A1 was detected in protein complex from the immunoprecipitations by using anti-SET antibody,and SET also was detected in immunoprecipitations by using anti-eEF1A1 antibody. [Conclusion] The interaction between SET and eEF1A1 in human liver cells was confirmed.
文摘Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whether smaller volume could save testing time.Result:Time consumption of elution in Group T was significantly shorter than that in Group C,while the efficiency of eluted m6A-containing fragments and the performance of m^(6)A-Seq as indicated by m6A peak distributions showed no difference between the two groups.Conclusion:A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.
文摘目的探讨CMTM6、PD-L1在子宫内膜异位症(endometriosis,EM)中的表达及意义。方法收集50例EM患者腹腔镜下手术切除的卵巢异位子宫内膜组织、刮除的在位子宫内膜组织(实验组)以及30例非EM患者刮除的在位子宫内膜组织(对照组),采用免疫组化法检测CMTM6、PD-L1的表达水平。收集实验组相关临床病理资料,包括患者年龄、体重指数(body mass index,BMI)、病灶大小、血清CA-125浓度及rAFS评分等,采用Spearman系数法分析CMTM6、PD-L1的表达与上述病理参数的相关性。培养人EM上皮细胞系12Z,采用Western blot及免疫蛋白共沉淀法检测CMTM6、PD-L1的表达水平及相互作用。结果CMTM6、PD-L1在EM患者中高表达,EM患者异位、在位子宫内膜中的表达水平高于非EM患者在位子宫内膜组织(P<0.05),CMTM6、PD-L1表达共同定位于异位、在位子宫内膜组织上皮细胞质、细胞膜中,且两种蛋白表达呈正相关(P<0.05)。患者年龄、BMI、病灶大小、血清CA-125浓度及rAFS评分与CMTM6、PD-L1的表达呈正相关(P<0.01)。CMTM6、PD-L1蛋白在12Z中表达,且CMTM6和PD-L能够相互结合。结论CMTM6、PD-L1在EM患者中高表达,CMTM6、PDL1的表达水平与EM病变严重程度相关,CMTM6、PD-L1在EM中能相互结合共同参与EM的发生、发展,CMTM6、PD-L1或可成为EM的潜在生物学标志物,为EM的临床诊治提供新靶点。
文摘BACKGROUND The Warburg effect is common in cancers.Lactate and its receptor GPR81 play an important role in cancer progression.It is widely accepted that membrane receptor nuclear translocation plays some novel role in cancer pathology.The mechanism by which the lactate/GPR81 axis regulates cancer malignancy remains unclear.AIM To elucidate the mechanism of GPR81 nuclear transportation promoted by exogenous lactate.METHODS Lung cancer cells were stimulated with exogenous lactate and GPR81 levels were measured by immunofluoresence and western blot analysis in membrane,cytoplasmic,and nuclear fractions.Lung cancer cells were transduced with a mutant GPR81 nuclear localization signal(NLS)construct,wild type GPR81 or empty vector and used to examine how GPR81 nuclear transportation affects lung cancer cells malignancy in vitro and in vivo.Immunoprecipitation Proteomics analysis and Chromatin immunoprecipitation(ChIP)sequencing were used to determine GPR81 interacting proteins and genes.RESULTS In response to hypoxia/Lactate stimulation,GPR81 translocates and accumulates in the nucleus of lung cancer cells.Functionally,GPR81 nuclear translocation promotes cancer cell proliferation and motility.Depletion of the GPR81 NLS depletes GPR81 nuclear levels and decreases cancer cell growth and invasion in vitro,as well as cancer cell malignancy in vivo.Proteomics analysis revealed a set of proteins including SFPQ,that interact with GPR81 in the cancer cell nucleus.Notably,the interaction of GPR81 with SFPQ promotes cancer cell growth and motility.ChIP sequencing analysis discovered that there is a set of genes targeted by GPR81.CONCLUSION The interaction of GPR81 with SFPQ promotes cancer cell malignancy.GPR81 nuclear translocation is critical in conferring cancer progression and may be a potential therapeutic target for limiting cancer progression.
基金Supported by the Talent Launch Fund of Chongqing Medical University Affiliated University City HospitalChongqing Medical University Affiliated University City Hospital Youth Program,No.2021ZD05.
文摘BACKGROUND The exact mechanisms underlying diabetic nephropathy(DN)remain incompletely elucidated,prompting researchers to explore new perspectives and identify novel intervention targets in this field.AIM To explore the role and underlying mechanisms of farnesoid X receptor(FXR)in the development of DN by regulating endoplasmic reticulum stress(ERS)molecular chaperone binding immunoglobulin protein(BiP)expression.METHODS Bioinformatics analyses identified potential FXR-binding elements in the BiP promoter.Dual-luciferase and chromatin immunoprecipitation(ChIP)assays confirmed FXR-BiP binding sites.In vitro studies used SV40 MES 13 cells under varying glucose conditions and treatments with FXR modulators[obeticholic acid(INT-747)and guggulsterones]or BiP small interfering RNA.The expression of BiP and ERS-related proteins[protein kinase R-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme 1(IRE1),activating transcription factor 6(ATF6)]was assessed alongside cell proliferation and extracellular matrix(ECM)synthesis.In vivo studies in DN mice(db/db)examined the effects of FXR activation on renal function and morphology.RESULTS FXR bound to the target sequence in the BiP promoter region,enhancing transcriptional activity,as confirmed by ChIP experiments.FXR expression decreased in SV40 MES 13 cells stimulated with high glucose and in renal tissues of DN mice compared with control.Treatment of SV40 MES 13 cells with the FXR agonist INT-747 significantly increased intracellular BiP expression,whereas silencing the FXR gene led to the downregulation of BiP levels.In vivo administration of INT-747 significantly elevated BiP levels in renal tissues,improved renal function and fibrosis in DN mice,while inhibiting the expression of ERS-related signaling proteins PERK,IRE1,and ATF6.CONCLUSION FXR promotes BiP expression by binding to its promoter,suppressing ERS pathways,and reducing mesangial cell proliferation and ECM synthesis.These findings highlight FXR as a potential therapeutic target for diabetic glomerulosclerosis.
