Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitiv...Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.展开更多
Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease dia...Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.展开更多
目的:探讨全髋关节置换术的CroweⅢ-Ⅳ型发育性髋关节发育不良(developmental dysplasia of the hip,DDH)患者的满意度及造成不满意的相关因素。方法:回顾性分析2013年3月至2018年3月行全髋关节置换术的169例CroweⅢ-Ⅳ型DDH患者,通过...目的:探讨全髋关节置换术的CroweⅢ-Ⅳ型发育性髋关节发育不良(developmental dysplasia of the hip,DDH)患者的满意度及造成不满意的相关因素。方法:回顾性分析2013年3月至2018年3月行全髋关节置换术的169例CroweⅢ-Ⅳ型DDH患者,通过微信进行调查问卷,调查患者对手术总体满意度、10项日常功能满意度和患者认为对自己日常生活影响比较大的前5个问题。手术前后采用髋关节Harris评分进行功能评价。结果:收到完整调查问卷145份,所有患者获随访,时间1~5(3.23±1.22)年。145例患者分成两组,其中对手术疗效满意的118例,不满意的27例,手术总体满意率81.38%(118/145)。患者认为对生活影响比较大的前5个问题分别是术后髋部疼痛,肢体明显不等长、行走、上下楼梯、蹲起。两组术前Harris评分比较,差异无统计学意义(P>0.05),不满意组术后Harris评分较低。术后髋关节疼痛、肢体不等长是影响手术不满意的直接因素。结论:采用全髋关节置换术治疗CroweⅢ-Ⅳ型DDH患者手术难度大;术后髋关节疼痛(轻度以上),肢体不等长(>2 cm)是术后不满意的独立危险因素。展开更多
Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutat...Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.展开更多
文摘Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.
基金financially supported by the National Natural Science Foundation of China(Grant No.:21904045)the Fundamental Research Funds for the Central Universities(HUST:Grant No.:2019kfyXJJS169)Training Program of Innovation and Entrepreneurship for Undergraduates of Hubei Province(Grant No.:S202010487225).
文摘Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.
文摘目的:探讨全髋关节置换术的CroweⅢ-Ⅳ型发育性髋关节发育不良(developmental dysplasia of the hip,DDH)患者的满意度及造成不满意的相关因素。方法:回顾性分析2013年3月至2018年3月行全髋关节置换术的169例CroweⅢ-Ⅳ型DDH患者,通过微信进行调查问卷,调查患者对手术总体满意度、10项日常功能满意度和患者认为对自己日常生活影响比较大的前5个问题。手术前后采用髋关节Harris评分进行功能评价。结果:收到完整调查问卷145份,所有患者获随访,时间1~5(3.23±1.22)年。145例患者分成两组,其中对手术疗效满意的118例,不满意的27例,手术总体满意率81.38%(118/145)。患者认为对生活影响比较大的前5个问题分别是术后髋部疼痛,肢体明显不等长、行走、上下楼梯、蹲起。两组术前Harris评分比较,差异无统计学意义(P>0.05),不满意组术后Harris评分较低。术后髋关节疼痛、肢体不等长是影响手术不满意的直接因素。结论:采用全髋关节置换术治疗CroweⅢ-Ⅳ型DDH患者手术难度大;术后髋关节疼痛(轻度以上),肢体不等长(>2 cm)是术后不满意的独立危险因素。
基金financially supported by the National Natural Science Foundation of China(No.31361140364)the National Major Project for Developing New GM Crops(No.2016ZX080009-001)the Agricultural Science and Technology Innovation Program(ASTIP)of CAAS to Chuanxiao Xie
文摘Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.
