当归具有良好的活血作用,但目前对其发挥“活血”功效的现代科学原理缺乏系统研究,限制了其现代化开发。该研究采用基于结构相似性的靶点反向预测、复杂网络分析、分子对接、结合自由能计算、聚类分析、ADMET计算等计算机辅助药物设计...当归具有良好的活血作用,但目前对其发挥“活血”功效的现代科学原理缺乏系统研究,限制了其现代化开发。该研究采用基于结构相似性的靶点反向预测、复杂网络分析、分子对接、结合自由能计算、聚类分析、ADMET计算等计算机辅助药物设计方法结合体外酶活性测定实验系统探讨了当归发挥活血作用的成分及作用机制。靶点反向预测以及复杂网络分析结果显示当归抗凝血的潜在作用靶点有40个。GO(Gene Ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析表明预测靶点可能通过作用于补体与凝血级联信号通路发挥抗凝血作用。其中,凝血级联反应和血栓形成中的关键酶THR(thrombin)和FXa(coagulation factor Xa)是治疗血栓栓塞性疾病的药物靶点。全成分分子对接以及聚类分析显示当归针对FXa具有更好的选择性。结合自由能打分值筛选出8个潜在活性成分进行体外酶活实验,结果显示8个化合物分别对THR和FXa具有不同程度的抑制作用,且对FXa活性的抑制作用比对THR更强。对8个活性化合物进行药效团模型构建,得到药效特征AAHH,其中A为氢键受体,H为疏水中心。ADMET计算表明8个活性化合物都具有良好的药动学性质,且安全性较高。该研究以靶点反向预测、复杂网络分析、分子对接及结合自由能计算、体外抗凝活性、分子与靶点的空间结合构象、药效团模型构建及ADMET参数计算为依据,从大数据层面初步阐明了当归活血作用的物质基础及分子机制,并对活性成分进行了药理、毒理计算,首次从全成分计算结合酶活实验揭示了当归对不同凝血蛋白具有明显的选择性和针对性,反映出不同中药的独特功效具有其生物学基础。该研究可为当归的精准用药及其活血成分的现代化开发提供线索。展开更多
Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine...Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 （DE3）. Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside （IPTG）, and the interest protein was identified by SDS-PAGE and purified using one-step nickel （Ni） affinity chromatography. Under the optimal expres- sion condition （0.05 mM IPTG for 6 h at 37 ℃）, the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.展开更多
从定性和定量两方面分析了目标医院现有手术室的流程。首先,通过现场调研获得医院手术流程数据,并采用Minitab进行处理;其次,利用故障树分析法(Fault Tree Analysis,FTA),在定性方面分析现有流程的问题;最后,对现有流程进行Pro-model仿...从定性和定量两方面分析了目标医院现有手术室的流程。首先,通过现场调研获得医院手术流程数据,并采用Minitab进行处理;其次,利用故障树分析法(Fault Tree Analysis,FTA),在定性方面分析现有流程的问题;最后,对现有流程进行Pro-model仿真建模,得到流程各节点运行效果,又从定量方面得到了流程的相关优化指标。展开更多
Coagulation Factor Xa(FXa)is the crucial enzyme at the convergent point of the intrinsic and extrinsic coagulation pathways.The inhibition of FXa is an effective approach against thrombotic diseases.In the present stu...Coagulation Factor Xa(FXa)is the crucial enzyme at the convergent point of the intrinsic and extrinsic coagulation pathways.The inhibition of FXa is an effective approach against thrombotic diseases.In the present study,a specific strategy is reported to discover 10 novel FXa inhibitors based on ligand-based(pharmacophore)virtual screening and molecular docking analysis from a dataset of specs(containing 220000 molecules).The binding modes analysis provide insights into the contribution of particular structural moieties of the compounds towards their activity against FXa,and 10 novel structural compounds were discovered as potent candidate molecules.This work could be helpful in further design and development of FXa inhibitors.展开更多
PCR method was used to introduce the code sequence of Factor Xa cleavage site to the 5′ end of cecropin CMIV mutant gene X,then the gene was cloned into the expression vector pGEX KG,and was highly expressed in E.col...PCR method was used to introduce the code sequence of Factor Xa cleavage site to the 5′ end of cecropin CMIV mutant gene X,then the gene was cloned into the expression vector pGEX KG,and was highly expressed in E.coli BL21 by IPTG induction.The fusion protein was purified by affinity chromatography and was cleaved by Factor Xa.Cecropin X with antibacterial activity was obtained after purified by ion exchange chromatography.展开更多
文摘当归具有良好的活血作用,但目前对其发挥“活血”功效的现代科学原理缺乏系统研究,限制了其现代化开发。该研究采用基于结构相似性的靶点反向预测、复杂网络分析、分子对接、结合自由能计算、聚类分析、ADMET计算等计算机辅助药物设计方法结合体外酶活性测定实验系统探讨了当归发挥活血作用的成分及作用机制。靶点反向预测以及复杂网络分析结果显示当归抗凝血的潜在作用靶点有40个。GO(Gene Ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析表明预测靶点可能通过作用于补体与凝血级联信号通路发挥抗凝血作用。其中,凝血级联反应和血栓形成中的关键酶THR(thrombin)和FXa(coagulation factor Xa)是治疗血栓栓塞性疾病的药物靶点。全成分分子对接以及聚类分析显示当归针对FXa具有更好的选择性。结合自由能打分值筛选出8个潜在活性成分进行体外酶活实验,结果显示8个化合物分别对THR和FXa具有不同程度的抑制作用,且对FXa活性的抑制作用比对THR更强。对8个活性化合物进行药效团模型构建,得到药效特征AAHH,其中A为氢键受体,H为疏水中心。ADMET计算表明8个活性化合物都具有良好的药动学性质,且安全性较高。该研究以靶点反向预测、复杂网络分析、分子对接及结合自由能计算、体外抗凝活性、分子与靶点的空间结合构象、药效团模型构建及ADMET参数计算为依据,从大数据层面初步阐明了当归活血作用的物质基础及分子机制,并对活性成分进行了药理、毒理计算,首次从全成分计算结合酶活实验揭示了当归对不同凝血蛋白具有明显的选择性和针对性,反映出不同中药的独特功效具有其生物学基础。该研究可为当归的精准用药及其活血成分的现代化开发提供线索。
基金National Technology Graveness Special Purpose Fund (Grant No. 2009zx09301-010)
文摘Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 （DE3）. Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside （IPTG）, and the interest protein was identified by SDS-PAGE and purified using one-step nickel （Ni） affinity chromatography. Under the optimal expres- sion condition （0.05 mM IPTG for 6 h at 37 ℃）, the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.
文摘从定性和定量两方面分析了目标医院现有手术室的流程。首先,通过现场调研获得医院手术流程数据,并采用Minitab进行处理;其次,利用故障树分析法(Fault Tree Analysis,FTA),在定性方面分析现有流程的问题;最后,对现有流程进行Pro-model仿真建模,得到流程各节点运行效果,又从定量方面得到了流程的相关优化指标。
基金the Science and Technology Innovation Talent Project of Sichuanprovince (Grant number 2016073).
文摘Coagulation Factor Xa(FXa)is the crucial enzyme at the convergent point of the intrinsic and extrinsic coagulation pathways.The inhibition of FXa is an effective approach against thrombotic diseases.In the present study,a specific strategy is reported to discover 10 novel FXa inhibitors based on ligand-based(pharmacophore)virtual screening and molecular docking analysis from a dataset of specs(containing 220000 molecules).The binding modes analysis provide insights into the contribution of particular structural moieties of the compounds towards their activity against FXa,and 10 novel structural compounds were discovered as potent candidate molecules.This work could be helpful in further design and development of FXa inhibitors.
文摘PCR method was used to introduce the code sequence of Factor Xa cleavage site to the 5′ end of cecropin CMIV mutant gene X,then the gene was cloned into the expression vector pGEX KG,and was highly expressed in E.coli BL21 by IPTG induction.The fusion protein was purified by affinity chromatography and was cleaved by Factor Xa.Cecropin X with antibacterial activity was obtained after purified by ion exchange chromatography.
