This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore t...This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.展开更多
BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the ...BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the role of the NLRP3 inflammasome in DKD.METHODS Using datasets from the Gene Expression Omnibus database,30 NLRP3 inflammasome-related genes were identified.Differentially expressed genes were selected using differential expression analysis,whereas intersecting genes were selected based on overlapping differentially expressed genes and NLRP3 inflammasome-related genes.Subsequently,three machine learning algorithms were used to screen genes,and biomarkers were identified by overlapping the genes from the three algorithms.Potential biomarkers were validated by western blotting in a db/db mouse model of diabetes.RESULTS Two biomarkers,sirtuin 2(SIRT2)and caspase 1(CASP1),involved in the Leishmania infection pathway were identified.Both biomarkers were expressed in endothelial cells.Pseudo-temporal analysis based on endothelial cells showed that DKD mostly occurs during the mid-differentiation stage.Western blotting results showed that CASP1 expression was higher in the DKD group than in the control group(P<0.05),and SIRT2 content decreased(P<0.05).CONCLUSION SIRT2 and CASP1 provide a potential theoretical basis for DKD treatment.展开更多
BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untran...BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.展开更多
Mitochondrial dysfunction is a significant pathological alte ration that occurs in Parkinson's disease(PD),and the Thr61lle(T61I)mutation in coiled-coil helix coiled-coil helix domain containing 2(CHCHD2),a crucia...Mitochondrial dysfunction is a significant pathological alte ration that occurs in Parkinson's disease(PD),and the Thr61lle(T61I)mutation in coiled-coil helix coiled-coil helix domain containing 2(CHCHD2),a crucial mitochondrial protein,has been reported to cause Parkinson's disease.FIFO-ATPase participates in the synthesis of cellular adenosine triphosphate(ATP)and plays a central role in mitochondrial energy metabolism.However,the specific roles of wild-type(WT)CHCHD2 and T611-mutant CHCHD2 in regulating F1FO-ATPase activity in Parkinson's disease,as well as whether CHCHD2 or CHCHD2 T61I affects mitochondrial function through regulating F1FO-ATPase activity,remain unclea r.Therefore,in this study,we expressed WT CHCHD2 and T61l-mutant CHCHD2 in an MPP^(+)-induced SH-SY5Y cell model of PD.We found that CHCHD2 protected mitochondria from developing MPP^(+)-induced dysfunction.Under normal conditions,ove rexpression of WT CHCHD2 promoted F1FO-ATPase assembly,while T61I-mutant CHCHD2 appeared to have lost the ability to regulate F1FO-ATPase assembly.In addition,mass spectrometry and immunoprecipitation showed that there was an interaction between CHCHD2 and F1FO-ATPase.Three weeks after transfection with AAV-CHCHD2 T61I,we intraperitoneally injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into mice to establish an animal model of chronic Parkinson's disease and found that exogenous expression of the mutant protein worsened the behavioral deficits and dopaminergic neurodegeneration seen in this model.These findings suggest that WT CHCHD2 can alleviate mitochondrial dysfunction in PD by maintaining F1F0-ATPase structure and function.展开更多
AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined...AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.展开更多
Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
BACKGROUND： As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 （CRP2） has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previo...BACKGROUND： As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 （CRP2） has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previous study has demonstrated that CRP2 can be detected in the embryonic rat inner ear but not in the adult rat inner ear. However, at present, the expression of LIM protein family H members in stem or precursor cells has not been described. OBJECTIVE： To determine the expression and sub-cellular localization of CRP2 in olfactory stem cells. DESIGN, TIME AND SETTING： An experiment with repeated measures was performed in the Laboratory of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, the Fourth Military Medical University from February 2008 to April 2008. MATERIALS： Olfactory stem cells, and rabbit-anti-CRP2 polyclonal antibody were prepared and kept in our laboratory. METHODS： Reverse transcription polymerase chain reaction and Western blot analysis were used to detect expression of CRP2 in olfactory stem cells. Immunocytochemistry was also used to localize CRP2 in olfactory stem cells. MAIN OUTCOME MEASURES： The expression and sub-cellular localization of CRP2 in rat olfactory stem cells. RESULTS： CRP2 expression was found in olfactory stem cells, and CRP2 was distributed in both the nucleus and the cytoplasm. CONCLUSION： Confirmation of the expression and distribution of CRP2 in olfactory stem cells.展开更多
目的 分析X-pert联合核苷酸结合寡聚化结构域蛋白2(NOD2)、自噬相关蛋白16样蛋白1(ATG16L1)在活动性肺结核患者疾病转归评估中的应用价值。方法 前瞻性选取2023年4月至2024年4月池州市人民医院收治的110例活动性肺结核患者为研究对象。...目的 分析X-pert联合核苷酸结合寡聚化结构域蛋白2(NOD2)、自噬相关蛋白16样蛋白1(ATG16L1)在活动性肺结核患者疾病转归评估中的应用价值。方法 前瞻性选取2023年4月至2024年4月池州市人民医院收治的110例活动性肺结核患者为研究对象。所有患者均行抗结核治疗、疾病转归评估,将转归患者60例作为转归组,未转归患者50例作为未转归组。收集两组患者的临床资料(年龄、体重指数、性别、吸烟史、贫血、累及肺野数、肺部空洞病变、利福平耐药)。治疗前行X-pert、NOD2、ATG16L1检测,比较两组X-pert阳性率及NOD2、ATG16L1表达水平。采用多因素Logistics回归分析分析活动性肺结核患者疾病转归的影响因素,采用受试者操作特征(ROC)曲线分析X-pert、NOD2、ATG16L1对活动性肺结核患者疾病转归的预测价值。结果 两组体重指数、吸烟史、贫血、累及肺野数、利福平耐药比较,差异均无统计学意义(P>0.05);与转归组相比,未转归组患者年龄较大[(56.15±19.34)vs.(63.18±12.84)岁],男性(71.67 vs. 90.00)%、肺部空洞病变(11.67 vs. 32.00)%比例较高,差异均有统计学意义(P<0.05)。与转归组相比,未转归组患者X-pert阳性率(75.00 vs. 90.00)%、NOD2[(164.31±15.55)vs.(199.29±24.63)ng/L]、ATG16L1[(8.95±1.1.74)vs.(12.15±2.26)ng/L]表达水平均较高,差异均有统计学意义(P<0.05)。多因素Logistics回归分析结果显示,年龄、性别、肺部空洞病变、X-pert、NOD2、ATG16L1为活动性肺结核患者疾病转归的危险因素(P<0.05)。与X-pert、NOD2、ATG16L1单项诊断相比,X-pert、NOD2、ATG16L1联合检测对活动性肺结核患者疾病转归的预测价值较高(P<0.05)。结论 疾病未转归活动性肺结核患者X-pert阳性率、NOD2、ATG16L1表达水平均高于转归患者,X-pert、NOD2、ATG16L1为活动性肺结核患者疾病转归的危险因素,X-pert联合NOD2、ATG16L1对活动性肺结核患者疾病转归的预测价值较高,为活动性肺结核患者疾病转归评估提供了有效依据。展开更多
Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- Ap...Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- ApoE^-/-） mice （n=12, 24 eyes, experimental group） and MBD2 （wt） ApoE^-/- mice （n=12, 24 eyes, control group） were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch＇s membrane （BM） of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 （ICAM-1） immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group （P〉0.05）. Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium （RPE） were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group （P〈0.05）. Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.展开更多
文摘This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.
基金Joint Funds for the Innovation of Science and Technology,Fujian Province,No.2021Y9106Fujian Provincial Health Technology Project,No.2021GGA033the Natural Science Foundation of Fujian Province,No.2024J011234.
文摘BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the role of the NLRP3 inflammasome in DKD.METHODS Using datasets from the Gene Expression Omnibus database,30 NLRP3 inflammasome-related genes were identified.Differentially expressed genes were selected using differential expression analysis,whereas intersecting genes were selected based on overlapping differentially expressed genes and NLRP3 inflammasome-related genes.Subsequently,three machine learning algorithms were used to screen genes,and biomarkers were identified by overlapping the genes from the three algorithms.Potential biomarkers were validated by western blotting in a db/db mouse model of diabetes.RESULTS Two biomarkers,sirtuin 2(SIRT2)and caspase 1(CASP1),involved in the Leishmania infection pathway were identified.Both biomarkers were expressed in endothelial cells.Pseudo-temporal analysis based on endothelial cells showed that DKD mostly occurs during the mid-differentiation stage.Western blotting results showed that CASP1 expression was higher in the DKD group than in the control group(P<0.05),and SIRT2 content decreased(P<0.05).CONCLUSION SIRT2 and CASP1 provide a potential theoretical basis for DKD treatment.
基金Supported by General Program of National Natural Science Foundation of China,No.81770197Scientific and Technological Research Major Program of Chongqing Municipal Education Commission,No.KJZD-M202312802+1 种基金Chongqing Natural Science Foundation of China,No.CSTB2022NSCQ-MSX0190,No.CSTB2022NSCQ-MSX0176,and No.cstc2020jcyj-msxmX0051Xinqiao Young Postdoc Talent Incubation Program,No.2022YQB098.
文摘BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.
基金supported by the National Natural Science Foundation of China(Youth Program),No.81901282(to XC)the National Natural Science Foundation of China,Nos.81401416(to PX),81870992(to PX),81870856(to XC and MZ)+3 种基金Guangdong Basic and Applied Basic Research Foundation the Science Foundation,No.2019A1515011189(to XC)Central Government Guiding Local Science and Technology Development Projects,No.ZYYD2022C17(to PX)Key Project of Guangzhou Health Commission,No.2019-ZD-09(to PX)Science and Technology Planning Project of Guangzhou,Nos.202102020029(to XC),202102010010(to PX)。
文摘Mitochondrial dysfunction is a significant pathological alte ration that occurs in Parkinson's disease(PD),and the Thr61lle(T61I)mutation in coiled-coil helix coiled-coil helix domain containing 2(CHCHD2),a crucial mitochondrial protein,has been reported to cause Parkinson's disease.FIFO-ATPase participates in the synthesis of cellular adenosine triphosphate(ATP)and plays a central role in mitochondrial energy metabolism.However,the specific roles of wild-type(WT)CHCHD2 and T611-mutant CHCHD2 in regulating F1FO-ATPase activity in Parkinson's disease,as well as whether CHCHD2 or CHCHD2 T61I affects mitochondrial function through regulating F1FO-ATPase activity,remain unclea r.Therefore,in this study,we expressed WT CHCHD2 and T61l-mutant CHCHD2 in an MPP^(+)-induced SH-SY5Y cell model of PD.We found that CHCHD2 protected mitochondria from developing MPP^(+)-induced dysfunction.Under normal conditions,ove rexpression of WT CHCHD2 promoted F1FO-ATPase assembly,while T61I-mutant CHCHD2 appeared to have lost the ability to regulate F1FO-ATPase assembly.In addition,mass spectrometry and immunoprecipitation showed that there was an interaction between CHCHD2 and F1FO-ATPase.Three weeks after transfection with AAV-CHCHD2 T61I,we intraperitoneally injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into mice to establish an animal model of chronic Parkinson's disease and found that exogenous expression of the mutant protein worsened the behavioral deficits and dopaminergic neurodegeneration seen in this model.These findings suggest that WT CHCHD2 can alleviate mitochondrial dysfunction in PD by maintaining F1F0-ATPase structure and function.
基金Supported by National Natural Science Foundation of China,No. 81071998Hubei Natural Science Foundation,No.2008CDB159Specialized Research Fund for the Doctoral Program of Higher Education,No. 20070487114
文摘AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
文摘BACKGROUND： As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 （CRP2） has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previous study has demonstrated that CRP2 can be detected in the embryonic rat inner ear but not in the adult rat inner ear. However, at present, the expression of LIM protein family H members in stem or precursor cells has not been described. OBJECTIVE： To determine the expression and sub-cellular localization of CRP2 in olfactory stem cells. DESIGN, TIME AND SETTING： An experiment with repeated measures was performed in the Laboratory of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, the Fourth Military Medical University from February 2008 to April 2008. MATERIALS： Olfactory stem cells, and rabbit-anti-CRP2 polyclonal antibody were prepared and kept in our laboratory. METHODS： Reverse transcription polymerase chain reaction and Western blot analysis were used to detect expression of CRP2 in olfactory stem cells. Immunocytochemistry was also used to localize CRP2 in olfactory stem cells. MAIN OUTCOME MEASURES： The expression and sub-cellular localization of CRP2 in rat olfactory stem cells. RESULTS： CRP2 expression was found in olfactory stem cells, and CRP2 was distributed in both the nucleus and the cytoplasm. CONCLUSION： Confirmation of the expression and distribution of CRP2 in olfactory stem cells.
文摘目的 分析X-pert联合核苷酸结合寡聚化结构域蛋白2(NOD2)、自噬相关蛋白16样蛋白1(ATG16L1)在活动性肺结核患者疾病转归评估中的应用价值。方法 前瞻性选取2023年4月至2024年4月池州市人民医院收治的110例活动性肺结核患者为研究对象。所有患者均行抗结核治疗、疾病转归评估,将转归患者60例作为转归组,未转归患者50例作为未转归组。收集两组患者的临床资料(年龄、体重指数、性别、吸烟史、贫血、累及肺野数、肺部空洞病变、利福平耐药)。治疗前行X-pert、NOD2、ATG16L1检测,比较两组X-pert阳性率及NOD2、ATG16L1表达水平。采用多因素Logistics回归分析分析活动性肺结核患者疾病转归的影响因素,采用受试者操作特征(ROC)曲线分析X-pert、NOD2、ATG16L1对活动性肺结核患者疾病转归的预测价值。结果 两组体重指数、吸烟史、贫血、累及肺野数、利福平耐药比较,差异均无统计学意义(P>0.05);与转归组相比,未转归组患者年龄较大[(56.15±19.34)vs.(63.18±12.84)岁],男性(71.67 vs. 90.00)%、肺部空洞病变(11.67 vs. 32.00)%比例较高,差异均有统计学意义(P<0.05)。与转归组相比,未转归组患者X-pert阳性率(75.00 vs. 90.00)%、NOD2[(164.31±15.55)vs.(199.29±24.63)ng/L]、ATG16L1[(8.95±1.1.74)vs.(12.15±2.26)ng/L]表达水平均较高,差异均有统计学意义(P<0.05)。多因素Logistics回归分析结果显示,年龄、性别、肺部空洞病变、X-pert、NOD2、ATG16L1为活动性肺结核患者疾病转归的危险因素(P<0.05)。与X-pert、NOD2、ATG16L1单项诊断相比,X-pert、NOD2、ATG16L1联合检测对活动性肺结核患者疾病转归的预测价值较高(P<0.05)。结论 疾病未转归活动性肺结核患者X-pert阳性率、NOD2、ATG16L1表达水平均高于转归患者,X-pert、NOD2、ATG16L1为活动性肺结核患者疾病转归的危险因素,X-pert联合NOD2、ATG16L1对活动性肺结核患者疾病转归的预测价值较高,为活动性肺结核患者疾病转归评估提供了有效依据。
基金supported by grants from Natural Science Foundation of Hubei Province(No:2012FFB02304)Scientific Research Foundation of Ministry of Education(No:2013-1792),China
文摘Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- ApoE^-/-） mice （n=12, 24 eyes, experimental group） and MBD2 （wt） ApoE^-/- mice （n=12, 24 eyes, control group） were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch＇s membrane （BM） of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 （ICAM-1） immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group （P〉0.05）. Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium （RPE） were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group （P〈0.05）. Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.
