Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods P...Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.展开更多
AIM:To evaluate the influence of MUC1 mucin variable number of tandem repeats (VNTR) variability on H pylori adhesion to gastric cells. METHODS: Enzyme linked immunosorbent assay (ELISA)-based adhesion assays were per...AIM:To evaluate the influence of MUC1 mucin variable number of tandem repeats (VNTR) variability on H pylori adhesion to gastric cells. METHODS: Enzyme linked immunosorbent assay (ELISA)-based adhesion assays were performed to measure the adhesion of different H pylori strains (HP26695 and HPTx30a) to gastric carcinoma cell lines (GP202 and MKN45) and GP202 clones expressing recombinant MUC1 with different VNTR lengths. RESULTS: Evaluation of adhesion results shows that H pylori pathogenic strain HP26695 has a significantly higher (P < 0.05) adhesion to all the cell lines and clones tested, when compared to the non-pathogenic strain HPTx30a. Bacteria showed a significantly higher (P < 0.05) adhesion to the GP202 cell line, when compared to the MKN45 cell line. Furthermore, both strains showed a significantly higher (P < 0.05) adhesion to GP202 clones with larger MUC1 VNTR domains. CONCLUSION: This work shows that MUC1 mucin variability conditions H pylori binding to gastric cells. The extent of bacterial adhesion depends on the size of theMUC1 VNTR domain. The adhesion is further dependent on bacterial pathogenicity and the gastric cell line. MUC1 mucin variability may contribute to determine H pylori colonization of the gastric mucosa.展开更多
Objective:Interleukin-1(IL-1)is a pro-inflammatory cytokine which may be related to urolithiasis.Genetic polymorphisms of the interleukin-1beta(IL-1β)have been proposed as markers for urolithiasis in some areas.Due t...Objective:Interleukin-1(IL-1)is a pro-inflammatory cytokine which may be related to urolithiasis.Genetic polymorphisms of the interleukin-1beta(IL-1β)have been proposed as markers for urolithiasis in some areas.Due to the high incidence of urolithiasis in Uighur children(Xinjiang,China)and existence of ethnic difference,our aim is to explore the potential of IL-1 gene polymorphisms and urolithiasis among these children.Methods:Genomic DNA extracted from peripheral blood of 115 patients and 98 controls were used for genotype polymorphisms analyses.IL-1 receptor antagonist(IL-1RN)gene variable number of tandem repeat(VNTR)gene polymorphisms were analyzed by PCR method.PCR-based restriction analysis was done for the IL-1β(-511)and IL-1β(+3954)gene polymorphisms by endonucleases Ava I and Taq I,respectively.The genotype distribution,allele frequencies,carriage rate,and haplotype frequencies were statistically analyzed.Results:No significant differences were observed in genotypic frequencies between pediatric urolithiasis patients and control group for IL-1RN gene(χ^(2)=1.906,p=0.605),IL-1β(-511)gene(χ^(2)=0.105,p=0.949),or IL-1β(+3954)gene(χ^(2)=3.635,p=0.169).There were yet no significant differences of the allele frequencies of IL-1RN VNTR gene(p=0.779),IL-1β(-511)gene(p=0.941),and IL-1β(+3954)gene(p=0.418)in the case and control groups,as well as the carriage rate and haplotype of them(all p>0.05).Conclusions:The associations between IL-1RN VNTR,IL-1β(-511)and IL-1β(+3954)genes polymorphisms and urolithiasis were not significant in Uighur children.The results need to be confirmed in studies with larger population sample size,as well as in other ethnic groups.展开更多
Background： Medullary cystic kidney disease （MCKD） is clinically indistinguishable from several other autosomal-dominant renal diseases： thus, molecular genetic testing is needed to establish a definitive diagnosi...Background： Medullary cystic kidney disease （MCKD） is clinically indistinguishable from several other autosomal-dominant renal diseases： thus, molecular genetic testing is needed to establish a definitive diagnosis. A specific type of single cytosine insertion in the variable number tandem repeat （VNTR） of the mucin 1 （MUC1） gene is the only known cause of MCKD1; however, genetic analysis of this mutation is difficult and not yet offered routinely. To identify the causative mutation/s and establish a definitive diagnosis in a Chinese family with chronic kidney disease, clinical assessments and genetic analysis were performed, including using a modified genotyping method to identify the MUC1-VNTR single cytosine insertion. Methods： Clinical data from three patients in a Chinese family with chronic kidney disease were collected and evaluated. Linkage analysis was used to map the causative locus. Mutation analysis of uromodulin （UMOD） gene was performed using polymerase chain reaction （PCR） and direct sequencing. For MUC1 genotyping, the mutant repeat units were enriched by Mwol restriction, and then were amplified and introduced into pMD-18T vectors. The 192 clones per transformant were picked up and tested by colony PCR and second round of Mwol digestion. Finally, Sanger sequencing was used to confirm the MUC1 mutation. Results： Clinical findings and laboratory results were consistent with a tubulointerstitial lesion. Linkage analysis indicated that the family was compatible with the MCKDI locus. No mutations were found in UMOD gene. Using the modified MUC1 genotyping method, we detected the MUC1-VNTR single cytosine insertion events in three patients of the family; and mutation-containing clones were 12/192, 14/192, and 5/96, respectively, in the three patients. Conclusions： Clinical and genetic findings could support the MCKDI diagnosis. The modified strategy has been demonstrated to be a practical way to detect MUCI mutation.展开更多
目的通过对236例流产物进行低深度全基因组拷贝数变异检测(copy number variation sequencing,CNV-seq)及短串联重复序列(short tandem repeats,STR)连锁分析检测,探讨其在流产物染色体异常检测中的临床应用价值。方法采用CNV-seq和STR...目的通过对236例流产物进行低深度全基因组拷贝数变异检测(copy number variation sequencing,CNV-seq)及短串联重复序列(short tandem repeats,STR)连锁分析检测,探讨其在流产物染色体异常检测中的临床应用价值。方法采用CNV-seq和STR连锁分析对2021年3月-2024年8月于郑州大学第二附属医院产前诊断中心就诊的236例自然流产患者的流产物样本进行检测,确定具有临床意义的染色体异常并对其进行分析。结果6例流产物因严重母源污染退出研究,剩余230例流产物染色体异常的阳性率为62.61%(144/230),其中染色体非整倍体110例(69.44%),多倍体7例(4.86%),拷贝数变异27例(18.75%)。染色体非整倍体中以常染色体三体最为常见,其中16-三体最常见,其次是21-三体。高龄产妇染色体异常率显著高于低龄产妇,早期流产组染色体异常发生率显著高于晚期流产组,均以染色体非整倍体异常为主。结论染色体非整倍体是导致自然流产最常见的原因,孕妇的妊娠年龄与胎儿常染色体三体的发生密切相关。CNV-seq联合STR连锁分析对于流产物的染色体分析高效可靠,可作为流产物遗传学分析的常规方法。展开更多
基金supported by the project (grant 2005CB522904 and 2005CB522905) from the Ministry of Scientific Technologythe project (grant 2008ZX10004-001, 2008ZX10004-008, and 2009ZX10004-101) from the Ministry of Scientific Technology and the Ministry of Health of the People’s Republic of China
文摘Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.
基金Portuguese Foundation for Science and Technology(FCT)Project POCTI/CBO/44812/2002+1 种基金Project POCTI/SAU-IMI/56895/2004 National Institutes of Health, R01-CA57362
文摘AIM:To evaluate the influence of MUC1 mucin variable number of tandem repeats (VNTR) variability on H pylori adhesion to gastric cells. METHODS: Enzyme linked immunosorbent assay (ELISA)-based adhesion assays were performed to measure the adhesion of different H pylori strains (HP26695 and HPTx30a) to gastric carcinoma cell lines (GP202 and MKN45) and GP202 clones expressing recombinant MUC1 with different VNTR lengths. RESULTS: Evaluation of adhesion results shows that H pylori pathogenic strain HP26695 has a significantly higher (P < 0.05) adhesion to all the cell lines and clones tested, when compared to the non-pathogenic strain HPTx30a. Bacteria showed a significantly higher (P < 0.05) adhesion to the GP202 cell line, when compared to the MKN45 cell line. Furthermore, both strains showed a significantly higher (P < 0.05) adhesion to GP202 clones with larger MUC1 VNTR domains. CONCLUSION: This work shows that MUC1 mucin variability conditions H pylori binding to gastric cells. The extent of bacterial adhesion depends on the size of theMUC1 VNTR domain. The adhesion is further dependent on bacterial pathogenicity and the gastric cell line. MUC1 mucin variability may contribute to determine H pylori colonization of the gastric mucosa.
基金This work was supported by the Natural Science Foundation of Guangdong Province,China(No.2019A1515010891).
文摘Objective:Interleukin-1(IL-1)is a pro-inflammatory cytokine which may be related to urolithiasis.Genetic polymorphisms of the interleukin-1beta(IL-1β)have been proposed as markers for urolithiasis in some areas.Due to the high incidence of urolithiasis in Uighur children(Xinjiang,China)and existence of ethnic difference,our aim is to explore the potential of IL-1 gene polymorphisms and urolithiasis among these children.Methods:Genomic DNA extracted from peripheral blood of 115 patients and 98 controls were used for genotype polymorphisms analyses.IL-1 receptor antagonist(IL-1RN)gene variable number of tandem repeat(VNTR)gene polymorphisms were analyzed by PCR method.PCR-based restriction analysis was done for the IL-1β(-511)and IL-1β(+3954)gene polymorphisms by endonucleases Ava I and Taq I,respectively.The genotype distribution,allele frequencies,carriage rate,and haplotype frequencies were statistically analyzed.Results:No significant differences were observed in genotypic frequencies between pediatric urolithiasis patients and control group for IL-1RN gene(χ^(2)=1.906,p=0.605),IL-1β(-511)gene(χ^(2)=0.105,p=0.949),or IL-1β(+3954)gene(χ^(2)=3.635,p=0.169).There were yet no significant differences of the allele frequencies of IL-1RN VNTR gene(p=0.779),IL-1β(-511)gene(p=0.941),and IL-1β(+3954)gene(p=0.418)in the case and control groups,as well as the carriage rate and haplotype of them(all p>0.05).Conclusions:The associations between IL-1RN VNTR,IL-1β(-511)and IL-1β(+3954)genes polymorphisms and urolithiasis were not significant in Uighur children.The results need to be confirmed in studies with larger population sample size,as well as in other ethnic groups.
文摘Background： Medullary cystic kidney disease （MCKD） is clinically indistinguishable from several other autosomal-dominant renal diseases： thus, molecular genetic testing is needed to establish a definitive diagnosis. A specific type of single cytosine insertion in the variable number tandem repeat （VNTR） of the mucin 1 （MUC1） gene is the only known cause of MCKD1; however, genetic analysis of this mutation is difficult and not yet offered routinely. To identify the causative mutation/s and establish a definitive diagnosis in a Chinese family with chronic kidney disease, clinical assessments and genetic analysis were performed, including using a modified genotyping method to identify the MUC1-VNTR single cytosine insertion. Methods： Clinical data from three patients in a Chinese family with chronic kidney disease were collected and evaluated. Linkage analysis was used to map the causative locus. Mutation analysis of uromodulin （UMOD） gene was performed using polymerase chain reaction （PCR） and direct sequencing. For MUC1 genotyping, the mutant repeat units were enriched by Mwol restriction, and then were amplified and introduced into pMD-18T vectors. The 192 clones per transformant were picked up and tested by colony PCR and second round of Mwol digestion. Finally, Sanger sequencing was used to confirm the MUC1 mutation. Results： Clinical findings and laboratory results were consistent with a tubulointerstitial lesion. Linkage analysis indicated that the family was compatible with the MCKDI locus. No mutations were found in UMOD gene. Using the modified MUC1 genotyping method, we detected the MUC1-VNTR single cytosine insertion events in three patients of the family; and mutation-containing clones were 12/192, 14/192, and 5/96, respectively, in the three patients. Conclusions： Clinical and genetic findings could support the MCKDI diagnosis. The modified strategy has been demonstrated to be a practical way to detect MUCI mutation.
文摘目的通过对236例流产物进行低深度全基因组拷贝数变异检测(copy number variation sequencing,CNV-seq)及短串联重复序列(short tandem repeats,STR)连锁分析检测,探讨其在流产物染色体异常检测中的临床应用价值。方法采用CNV-seq和STR连锁分析对2021年3月-2024年8月于郑州大学第二附属医院产前诊断中心就诊的236例自然流产患者的流产物样本进行检测,确定具有临床意义的染色体异常并对其进行分析。结果6例流产物因严重母源污染退出研究,剩余230例流产物染色体异常的阳性率为62.61%(144/230),其中染色体非整倍体110例(69.44%),多倍体7例(4.86%),拷贝数变异27例(18.75%)。染色体非整倍体中以常染色体三体最为常见,其中16-三体最常见,其次是21-三体。高龄产妇染色体异常率显著高于低龄产妇,早期流产组染色体异常发生率显著高于晚期流产组,均以染色体非整倍体异常为主。结论染色体非整倍体是导致自然流产最常见的原因,孕妇的妊娠年龄与胎儿常染色体三体的发生密切相关。CNV-seq联合STR连锁分析对于流产物的染色体分析高效可靠,可作为流产物遗传学分析的常规方法。
