AIM： TO determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis. METHODS： The Ku70/80 DNA-binding activity w...AIM： TO determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis. METHODS： The Ku70/80 DNA-binding activity was determined by electrophoretic mobility shift assays in 20 colon adenoma and 15 colorectal cancer samples as well as matched normal colonic tissues. Nuclear and cytoplasmic protein expression was determined by immunohistochemistry and Western blot analysis. RESULTS： A statistical found in both adenomas y significant difference was and carcinomas as compared to matched normal colonic mucosa （P〈0.00）. However, changes in binding activity were not homogenous with approximately 50% of the tumors showing a clear increase in the binding activity, 30% displaying a modest increase and 15% showing a decrease of the activity.Tumors, with increased DNA-binding activity, also showed a statistically significant increase in Ku70 and Ku86 nuclear expression, as determined by Western blot and immunohistochemical analyses （P〈0.001）. Cytoplasmic protein expression was found in pathological samples, but not in normal tissues either from tumor patients or from healthy subjects. CONCLUSION： Overall, our DNA-binding activity and protein level are consistent with a substantial activation of the NHEJ pathway in colorectal tumors. Since the NHEJ is an error prone mechanism, its abnormal activation can result in chromosomal instability and ultimately lead to tumorigenesis.展开更多
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different ch...Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.展开更多
CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells（Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014）.The most widely us...CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells（Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014）.The most widely used customized CRISPR/Cas9（Sp Cas9）is derived from Streptococcus pyogenes（Cong et al.,2013）.展开更多
DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end ...DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.展开更多
Using one step solid-phase extraction for urine pretreatment and a new sample focusing mode-dynamic pH junction injection for increasing sensitivity, a simple and sensitive method for the analysis of urinary 8OHdG by ...Using one step solid-phase extraction for urine pretreatment and a new sample focusing mode-dynamic pH junction injection for increasing sensitivity, a simple and sensitive method for the analysis of urinary 8OHdG by capillary electrophoresis with end-column amprometric detection has been developed. The limit of detection was 20 nmol/L(signal to noise ratio S/N=3). The urinary concentration of 8OHdG in nine healthy persons and 28 cancer patients was determined. It was found that urinary concentration of 8OHdG in cancer patients was significantly higher than that in healthy persons[(35.26±27.96) nmol/L) vs. (13.51±5.08) nmol/L, P<0.05], the result demonstrated that 8OHdG may be a good biomarker for measurement of oxidative DNA damage in cancer patients. Furthermore, the excretion levels of urinary 8OHdG from cancer patients receiving surgical therapy are investigated, and the results demonstrated that the smoking has a strong effect on 8OHdG content.展开更多
目的 观察雷公藤多甙 (TWP)对终板膜乙酰胆碱受体 (n ACh R)的反应性的作用。方法 在不均匀牵张法制动的蟾蜍缝匠肌标本上 ,利用微电泳氯化乙酰胆碱 (ACh Cl)技术 ,在终板区引起的乙酰胆碱电位 (ACh P)来反映终板膜 n ACh R的反应性...目的 观察雷公藤多甙 (TWP)对终板膜乙酰胆碱受体 (n ACh R)的反应性的作用。方法 在不均匀牵张法制动的蟾蜍缝匠肌标本上 ,利用微电泳氯化乙酰胆碱 (ACh Cl)技术 ,在终板区引起的乙酰胆碱电位 (ACh P)来反映终板膜 n ACh R的反应性。结果 12 0 mg/ L TWP组 ,用药 2 0 m in后 ACh P振幅明显升高 ,4 0 mg/ L 和 6 0mg/ L TWP组 ,用药 10 m in后 ACh P振幅明显升高 ;2 2 0 mg/ L 和 4 0 m g/ L TWP组内 ,ACh P振幅除 80 m in组外 ,其它各时间组间有显著性差异 (P<0 .0 5 ) ,6 0 min达最大值 ;36 0 mg/ L TWP组 ,在用药后 4 0 m in、 6 0 m in和80 m in均记录到动作电位 (AP) ;4药物各浓度组引起的 ACh P振幅变化率之间有显著差异 (P<0 .0 5 ) ,其中 6 0mg/ L TWP组引起的变化率最大。结论 TWP能提高终板膜 n ACh R的反应性 。展开更多
Congenital abnormalities of the kidney and urinary tract(CAKUT) include a wide range of abnormalities ranging from asymptomatic ectopic kidneys to life threatening renal agenesis(bilateral). Many of them are detected ...Congenital abnormalities of the kidney and urinary tract(CAKUT) include a wide range of abnormalities ranging from asymptomatic ectopic kidneys to life threatening renal agenesis(bilateral). Many of them are detected in the antenatal or immediate postnatal with a significant proportion identified in the adult population with varying degree of severity. CAKUT can be classified on embryological basis in to abnormalities in the renal parenchymal development, aberrant embryonic migration and abnormalities of the collecting system. Renal parenchymal abnormalities include multi cystic dysplastic kidneys, renal hypoplasia, number(agenesis or supernumerary), shape and cystic renal diseases. Aberrant embryonic migration encompasses abnormal location and fusion anomalies. Collecting system abnormalities include duplex kidneys and Pelvi ureteric junction obstruction. Ultrasonography(US) is typically the first imaging performed as it is easily available, noninvasive and radiation free used both antenatally and postnatally. Computed tomography(CT) and magnetic resonance imaging(MRI) are useful to confirm the ultrasound detected abnormality, detection of complex malformations, demonstration of collecting system and vascular anatomy and more importantly for early detection of complications like renal calculi, infection and malignancies. As CAKUT are one of the leading causes of end stage renal disease, it is important for the radiologists to be familiar with the varying imaging appearances of CAKUT on US, CT and MRI, thereby helping in prompt diagnosis and optimal management.展开更多
目的分析初诊Ⅳ期胃癌/食管胃结合部癌肝转移患者的临床病理特征,探索此类患者的预后因素。方法通过美国国立癌症研究所监测、流行病学和最终结果(surveillance,epidemiology,and end results,SEER)*Stat软件收集SEER数据库中2010—2015...目的分析初诊Ⅳ期胃癌/食管胃结合部癌肝转移患者的临床病理特征,探索此类患者的预后因素。方法通过美国国立癌症研究所监测、流行病学和最终结果(surveillance,epidemiology,and end results,SEER)*Stat软件收集SEER数据库中2010—2015年1614例Ⅳ期胃癌/食管胃结合部癌患者的临床资料。采用χ2检验比较肝转移(641例)和非肝转移(973例)患者临床病理特征的差异,采用二项分类Logistic回归分析探讨发生肝转移的影响因素;采用Kaplan-Meier法计算总生存(overall survival,OS),通过Log-Rank检验计算各因素OS的差异。通过多因素COX回归模型分析Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立危险因素。结果肝转移和非肝转移患者临床病理特征中差异有统计学意义的因素包括:诊断年龄、种族、性别、原发灶部位、T分期、N分期、组织学分型、分化程度(均P<0.05)。二项分类Logistic回归分析结果提示,诊断年龄、种族、性别、原发灶部位、组织学分型、分化程度、骨转移、肺转移均为Ⅳ期胃癌/食管胃结合部癌患者发生肝转移的危险因素(均P<0.05)。641例胃癌/食管胃结合部癌肝转移患者的中位生存时间为3个月。单因素生存分析结果显示,诊断年龄、N分期、组织学分型、分化程度、原发灶手术、肺转移均与Ⅳ期胃癌/食管胃结合部癌肝转移患者生存情况有显著相关性(均P<0.05)。多因素COX回归模型分析显示,诊断年龄、N分期、组织学分型、分化程度、原发灶手术、肺转移均为Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立影响因素(均P<0.05)。结论Ⅳ期胃癌/食管胃结合部癌具有独特的临床病理特征。发生肝转移的患者整体预后较差,诊断年龄、N分期、组织学分型、分化程度、原发灶手术和肺转移均为Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立影响因素。展开更多
基金Supported by Italian Ministero della Salute, IRCCS, RC0302TG13 by Ministero dell'Istruzíone, Università e Ricerca scientifica e tecnologica (MIUR), COFIN2002, to the Universita Campus Bio-Medico
文摘AIM： TO determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis. METHODS： The Ku70/80 DNA-binding activity was determined by electrophoretic mobility shift assays in 20 colon adenoma and 15 colorectal cancer samples as well as matched normal colonic tissues. Nuclear and cytoplasmic protein expression was determined by immunohistochemistry and Western blot analysis. RESULTS： A statistical found in both adenomas y significant difference was and carcinomas as compared to matched normal colonic mucosa （P〈0.00）. However, changes in binding activity were not homogenous with approximately 50% of the tumors showing a clear increase in the binding activity, 30% displaying a modest increase and 15% showing a decrease of the activity.Tumors, with increased DNA-binding activity, also showed a statistically significant increase in Ku70 and Ku86 nuclear expression, as determined by Western blot and immunohistochemical analyses （P〈0.001）. Cytoplasmic protein expression was found in pathological samples, but not in normal tissues either from tumor patients or from healthy subjects. CONCLUSION： Overall, our DNA-binding activity and protein level are consistent with a substantial activation of the NHEJ pathway in colorectal tumors. Since the NHEJ is an error prone mechanism, its abnormal activation can result in chromosomal instability and ultimately lead to tumorigenesis.
基金supported by Major Program of the National Natural Science Foundation of China(21621004)the Natural Science Foundation of Tianjin City(19JCQNJC09200)the Young Elite Scientists Sponsorship Program by Tianjin(TJSQNTJ-2018-16).
文摘Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.
基金supported by the grants from the Natural Science Foundation of China (No.81201181 to F.G.81473295 and 81670882 to Z.M.S and 81700885 to X.L.G.)+5 种基金Zhejiang Provincial & Ministry of Health research fund for medical sciences (WKJ2013-2-023 to F.G.WKJ-ZJ-1828 to J.Z.Z.and 2016KYA145 to X.L.G.)Science Technology Project of Zhejiang Province (2014C33260 to Z.M.S.and 2017C37176 to F.G.)Eye Hospital at Wenzhou Medical University (YNZD201602 to F.G.)Wenzhou City (Y20160008 to J.Z.Z.)Research Fund for Lin He's Academician Workstation of New Medicine and Clinical Translation (17331209 to C.B.L.)
文摘CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells（Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014）.The most widely used customized CRISPR/Cas9（Sp Cas9）is derived from Streptococcus pyogenes（Cong et al.,2013）.
基金This work was supported by grants from the National Natural Science Foundation of China[31730003,31670077]Natural Science Foundation of Shandong Province[ZR2017ZB0210].
文摘DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.
文摘Using one step solid-phase extraction for urine pretreatment and a new sample focusing mode-dynamic pH junction injection for increasing sensitivity, a simple and sensitive method for the analysis of urinary 8OHdG by capillary electrophoresis with end-column amprometric detection has been developed. The limit of detection was 20 nmol/L(signal to noise ratio S/N=3). The urinary concentration of 8OHdG in nine healthy persons and 28 cancer patients was determined. It was found that urinary concentration of 8OHdG in cancer patients was significantly higher than that in healthy persons[(35.26±27.96) nmol/L) vs. (13.51±5.08) nmol/L, P<0.05], the result demonstrated that 8OHdG may be a good biomarker for measurement of oxidative DNA damage in cancer patients. Furthermore, the excretion levels of urinary 8OHdG from cancer patients receiving surgical therapy are investigated, and the results demonstrated that the smoking has a strong effect on 8OHdG content.
文摘目的 观察雷公藤多甙 (TWP)对终板膜乙酰胆碱受体 (n ACh R)的反应性的作用。方法 在不均匀牵张法制动的蟾蜍缝匠肌标本上 ,利用微电泳氯化乙酰胆碱 (ACh Cl)技术 ,在终板区引起的乙酰胆碱电位 (ACh P)来反映终板膜 n ACh R的反应性。结果 12 0 mg/ L TWP组 ,用药 2 0 m in后 ACh P振幅明显升高 ,4 0 mg/ L 和 6 0mg/ L TWP组 ,用药 10 m in后 ACh P振幅明显升高 ;2 2 0 mg/ L 和 4 0 m g/ L TWP组内 ,ACh P振幅除 80 m in组外 ,其它各时间组间有显著性差异 (P<0 .0 5 ) ,6 0 min达最大值 ;36 0 mg/ L TWP组 ,在用药后 4 0 m in、 6 0 m in和80 m in均记录到动作电位 (AP) ;4药物各浓度组引起的 ACh P振幅变化率之间有显著差异 (P<0 .0 5 ) ,其中 6 0mg/ L TWP组引起的变化率最大。结论 TWP能提高终板膜 n ACh R的反应性 。
文摘Congenital abnormalities of the kidney and urinary tract(CAKUT) include a wide range of abnormalities ranging from asymptomatic ectopic kidneys to life threatening renal agenesis(bilateral). Many of them are detected in the antenatal or immediate postnatal with a significant proportion identified in the adult population with varying degree of severity. CAKUT can be classified on embryological basis in to abnormalities in the renal parenchymal development, aberrant embryonic migration and abnormalities of the collecting system. Renal parenchymal abnormalities include multi cystic dysplastic kidneys, renal hypoplasia, number(agenesis or supernumerary), shape and cystic renal diseases. Aberrant embryonic migration encompasses abnormal location and fusion anomalies. Collecting system abnormalities include duplex kidneys and Pelvi ureteric junction obstruction. Ultrasonography(US) is typically the first imaging performed as it is easily available, noninvasive and radiation free used both antenatally and postnatally. Computed tomography(CT) and magnetic resonance imaging(MRI) are useful to confirm the ultrasound detected abnormality, detection of complex malformations, demonstration of collecting system and vascular anatomy and more importantly for early detection of complications like renal calculi, infection and malignancies. As CAKUT are one of the leading causes of end stage renal disease, it is important for the radiologists to be familiar with the varying imaging appearances of CAKUT on US, CT and MRI, thereby helping in prompt diagnosis and optimal management.
文摘目的分析初诊Ⅳ期胃癌/食管胃结合部癌肝转移患者的临床病理特征,探索此类患者的预后因素。方法通过美国国立癌症研究所监测、流行病学和最终结果(surveillance,epidemiology,and end results,SEER)*Stat软件收集SEER数据库中2010—2015年1614例Ⅳ期胃癌/食管胃结合部癌患者的临床资料。采用χ2检验比较肝转移(641例)和非肝转移(973例)患者临床病理特征的差异,采用二项分类Logistic回归分析探讨发生肝转移的影响因素;采用Kaplan-Meier法计算总生存(overall survival,OS),通过Log-Rank检验计算各因素OS的差异。通过多因素COX回归模型分析Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立危险因素。结果肝转移和非肝转移患者临床病理特征中差异有统计学意义的因素包括:诊断年龄、种族、性别、原发灶部位、T分期、N分期、组织学分型、分化程度(均P<0.05)。二项分类Logistic回归分析结果提示,诊断年龄、种族、性别、原发灶部位、组织学分型、分化程度、骨转移、肺转移均为Ⅳ期胃癌/食管胃结合部癌患者发生肝转移的危险因素(均P<0.05)。641例胃癌/食管胃结合部癌肝转移患者的中位生存时间为3个月。单因素生存分析结果显示,诊断年龄、N分期、组织学分型、分化程度、原发灶手术、肺转移均与Ⅳ期胃癌/食管胃结合部癌肝转移患者生存情况有显著相关性(均P<0.05)。多因素COX回归模型分析显示,诊断年龄、N分期、组织学分型、分化程度、原发灶手术、肺转移均为Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立影响因素(均P<0.05)。结论Ⅳ期胃癌/食管胃结合部癌具有独特的临床病理特征。发生肝转移的患者整体预后较差,诊断年龄、N分期、组织学分型、分化程度、原发灶手术和肺转移均为Ⅳ期胃癌/食管胃结合部癌肝转移患者预后的独立影响因素。
