Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is ...Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.展开更多
目的探讨胸腰段椎体(包括T11~L2、L1~L2及单个椎体)基于MRI平扫多序列优化的椎体骨质量评分(vertebral bone quality score based on MRI,VBQ)与定量计算机体层成像(quantitative computed tomography,QCT)测得的体积骨密度(volume bone...目的探讨胸腰段椎体(包括T11~L2、L1~L2及单个椎体)基于MRI平扫多序列优化的椎体骨质量评分(vertebral bone quality score based on MRI,VBQ)与定量计算机体层成像(quantitative computed tomography,QCT)测得的体积骨密度(volume bone density,vBMD)的相关性及对骨质疏松症(osteoporosis,OP)的诊断价值,明确筛查及诊断分组的最佳“单个椎体窗口”及序列。材料与方法收集在开封市中心医院医学影像科同时(扫描时间间隔小于3天)行胸腰段椎体1.5 T MRI及CT平扫且年龄大于18岁的健检者的临床及影像资料,共69例。分别测量胸腰段椎体及L3后方脑脊液(cerebrospinal fluid,CSF)感兴趣区(region of interest,ROI)的MRI平均信号强度(signal intensity,SI),进一步计算多序列(包括T11、T12、L1、L2、T11~L2及L1~L2)及优化的VBQ,其中T11~L2、L1~L2椎体的VBQ取平均值(多序列及优化的VBQ,包括VBQT1、VBQT2、VBQT2-STIR、VBQFLAIR、VBQ_(压脂优化)、VBQ_(压水优化)、VBQ_(共同优化))。测量T11、T12、L1、L2的vBMD,进一步取平均值计算T11~L2、L1~L2椎体vBMD。依次分别进行两次分组:(1)筛查分组,以vBMD=120 mg/cm^(3)为界限分为骨量正常组及非骨量正常组(OP+骨量减少);(2)诊断分组,以vBMD=80 mg/cm^(3)为界限分为OP组及非OP组(骨量正常+骨量减少)。探讨胸腰椎段椎体多序列及优化的VBQ与QCT-vBMD的相关性;使用受试者工作特征(receiver operating characteristic,ROC)曲线探究多序列及优化的VBQ对OP的诊断价值。结果(1)相关性分析显示:在单个椎体VBQ中,VBQ_((L2)压脂优化)与vBMD负相关性最强(r=-0.531,P<0.001);在L1~L2椎体平均值的VBQ中,VBQ_((L1~L2)压脂优化)与vBMD负相关性最强(r=-0.561,P<0.001);在T11~L2椎体平均值的VBQ中,VBQ_((总体)压脂优化)与vBMD负相关性最强(r=-0.562,P<0.001)。值得注意的是,在L1椎体的VBQ中,VBQ_((L1)压水优化)与vBMD负相关性最强(r=-0.463,P<0.001)。(2)从T11到L2,单椎体vBMD的均值依次递减,压脂序列优化的VBQ值依次递增。(3)ROC曲线分析显示:单个椎体中L2椎体诊断VBQ_((L2)压脂优化)的曲线下面积(area under the curve,AUC)=0.928,最佳截断值(Cut-off)为8.765,敏感度为88.9%,特异度为80.8%。(4)筛查分组:在T11~L2水平的VBQ中,VBQ_((总体)压脂优化)的AUC最高(0.791),Cut-off值为7.829;在L1~L2的VBQ中,VBQ_((L1~L2)压脂优化)的AUC最高(0.756),最大约登指数为0.492,敏感度为63.2%,特异度为86.0%,Cut-off值为8.406。(5)诊断分组:在T11~L2的VBQ中,VBQ_((总体)压脂优化)的AUC最高(0.791),Cut-off值为7.829;在L1~L2的VBQ中,VBQ_((L1~L2)压脂优化)的AUC最高(0.912),最大约登指数为0.680,敏感度为86.0%,特异度为82.0%,Cut-off值为8.406。结论(1)多序列优化的VBQ与vBMD具有负相关性。(2)从T11到L2,单椎体的骨密度逐渐降低,脂肪含量逐渐增加;单个椎体可以预测OP的发生,在体检人群中比T11~L2或L1~L2的平均值预测价值高,单个椎体VBQ可以无辐射精准评价椎体骨质量,可以提示临床。(3)无论采用筛查标准还是诊断标准分组、无论是T11~L2、L1~L2,压脂优化的VBQ诊断效能均最高。展开更多
基金Project(31000350)supported by the National Natural Science Foundation of China
文摘Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.
文摘目的探讨胸腰段椎体(包括T11~L2、L1~L2及单个椎体)基于MRI平扫多序列优化的椎体骨质量评分(vertebral bone quality score based on MRI,VBQ)与定量计算机体层成像(quantitative computed tomography,QCT)测得的体积骨密度(volume bone density,vBMD)的相关性及对骨质疏松症(osteoporosis,OP)的诊断价值,明确筛查及诊断分组的最佳“单个椎体窗口”及序列。材料与方法收集在开封市中心医院医学影像科同时(扫描时间间隔小于3天)行胸腰段椎体1.5 T MRI及CT平扫且年龄大于18岁的健检者的临床及影像资料,共69例。分别测量胸腰段椎体及L3后方脑脊液(cerebrospinal fluid,CSF)感兴趣区(region of interest,ROI)的MRI平均信号强度(signal intensity,SI),进一步计算多序列(包括T11、T12、L1、L2、T11~L2及L1~L2)及优化的VBQ,其中T11~L2、L1~L2椎体的VBQ取平均值(多序列及优化的VBQ,包括VBQT1、VBQT2、VBQT2-STIR、VBQFLAIR、VBQ_(压脂优化)、VBQ_(压水优化)、VBQ_(共同优化))。测量T11、T12、L1、L2的vBMD,进一步取平均值计算T11~L2、L1~L2椎体vBMD。依次分别进行两次分组:(1)筛查分组,以vBMD=120 mg/cm^(3)为界限分为骨量正常组及非骨量正常组(OP+骨量减少);(2)诊断分组,以vBMD=80 mg/cm^(3)为界限分为OP组及非OP组(骨量正常+骨量减少)。探讨胸腰椎段椎体多序列及优化的VBQ与QCT-vBMD的相关性;使用受试者工作特征(receiver operating characteristic,ROC)曲线探究多序列及优化的VBQ对OP的诊断价值。结果(1)相关性分析显示:在单个椎体VBQ中,VBQ_((L2)压脂优化)与vBMD负相关性最强(r=-0.531,P<0.001);在L1~L2椎体平均值的VBQ中,VBQ_((L1~L2)压脂优化)与vBMD负相关性最强(r=-0.561,P<0.001);在T11~L2椎体平均值的VBQ中,VBQ_((总体)压脂优化)与vBMD负相关性最强(r=-0.562,P<0.001)。值得注意的是,在L1椎体的VBQ中,VBQ_((L1)压水优化)与vBMD负相关性最强(r=-0.463,P<0.001)。(2)从T11到L2,单椎体vBMD的均值依次递减,压脂序列优化的VBQ值依次递增。(3)ROC曲线分析显示:单个椎体中L2椎体诊断VBQ_((L2)压脂优化)的曲线下面积(area under the curve,AUC)=0.928,最佳截断值(Cut-off)为8.765,敏感度为88.9%,特异度为80.8%。(4)筛查分组:在T11~L2水平的VBQ中,VBQ_((总体)压脂优化)的AUC最高(0.791),Cut-off值为7.829;在L1~L2的VBQ中,VBQ_((L1~L2)压脂优化)的AUC最高(0.756),最大约登指数为0.492,敏感度为63.2%,特异度为86.0%,Cut-off值为8.406。(5)诊断分组:在T11~L2的VBQ中,VBQ_((总体)压脂优化)的AUC最高(0.791),Cut-off值为7.829;在L1~L2的VBQ中,VBQ_((L1~L2)压脂优化)的AUC最高(0.912),最大约登指数为0.680,敏感度为86.0%,特异度为82.0%,Cut-off值为8.406。结论(1)多序列优化的VBQ与vBMD具有负相关性。(2)从T11到L2,单椎体的骨密度逐渐降低,脂肪含量逐渐增加;单个椎体可以预测OP的发生,在体检人群中比T11~L2或L1~L2的平均值预测价值高,单个椎体VBQ可以无辐射精准评价椎体骨质量,可以提示临床。(3)无论采用筛查标准还是诊断标准分组、无论是T11~L2、L1~L2,压脂优化的VBQ诊断效能均最高。
