Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-p...Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-proteins-for-multi-tasks"NE in terms of screening throughput and efficiency,thus often failing to meet the fast-growing demands for biocatalysts with desired properties.In this study,we design a novel"multi-enzymes-for-multi-substrates"(MEMS)DE model and establish the proof-ofconcept by running a NE-mimicking and higher-throughput screening on the basis of"two-P450 s-against-seven-substrates"(2P×7S)in one pot.With the multiplied throughput and improved hit rate,we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes(P450 BM3 and P450 cam)in laboratory.It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future.Furthermore,structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.展开更多
The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A(CsA)primarily at the 4th N-methyl leucine(MeLeu4),giving r...The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A(CsA)primarily at the 4th N-methyl leucine(MeLeu4),giving rise toγ-hydroxy-N-methyl-L-Leu4-CsA(CsA-4-OH).This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect,thus demonstrating great application potential in both pharmaceutical and cosmetic industries.However,this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine(MeLeu9),indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator.Herein,we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85Å.Together with sequence and 3D structure comparisons,Autodock-based substrate docking,molecular dynamics(MD)simulation,and site-directed mutagenesis,we identified a number of key residues including R294,E264,and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA,setting the stage for better enzymatic preparation of CsA-4-OH.This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.展开更多
Aims:Portal venous pressure frequently increases after a major hepatectomy.We aimed to assess the outcomes of patients with hepatocellular carcinoma(HCC)with different post-hepatectomy portal pressure statuses and the...Aims:Portal venous pressure frequently increases after a major hepatectomy.We aimed to assess the outcomes of patients with hepatocellular carcinoma(HCC)with different post-hepatectomy portal pressure statuses and the predictive factors correlated with prognosis and post-hepatectomy portal hypertension(PHPH).Methods:Data from consecutive patients who underwent curative-intent hepatectomy for HCC at the Eastern Hepatobiliary Surgery Hospital and Mengchao Hepatobiliary Hospital of Fujian Medical University between January 2008 and December 2016 were retrospectively analyzed.Patients were followed up until March 1,2022.PHPH was defined as new-onset portal hypertension(PH)in patients without preoperative PH within 1 month of hepatectomy.Patients were classified into two groups according to the presence or absence of PHPH:non-PH and PHPH.The clinicopathological characteristics were compared between the two groups.Univariate and multivariate analyses were used to identify independent risk factors for early and late recurrence of HCC,overall survival(OS),and PHPH.Results:Disease-free survival rates were significantly higher in the non-PH group(n=1068)than those in the PHPH group(n=423)(62.0%,39.0%,and 31.0%vs.46.2%,24.5%,and 19.3%at 1,3,and 5 years,respectively;all p<0.001).The 1-,3-,and 5-year OS rates were 91.0%,66.4%,and 51.4%in the non-PH group and 80.0%,48.9%,and 32.6%in the PHPH group,respectively(all p<0.001).Multivariate analysis revealed that PHPH was independently associated with early recurrence of HCC and poor OS(hazard ratio[HR]1.476,95%confidence interval[CI]1.279–1.704,p<0.001 and HR 1.601,95%CI 1.383–1.853,p<0.001,respectively).Furthermore,hepatitis B virus infection,cirrhosis,preoperative alanine aminotransferase>40 U/L,and major hepatectomy were identified as independent risk factors for PHPH.Conclusions:PHPH was associated with early recurrence of HCC and poor OS but not with late recurrence.Therefore,PHPH may be an attractive target for therapeutic interventions and follow-up surveillance to improve prognosis.展开更多
基金首都临床特色应用研究(No.Z181100001718055)2015年北京航天总医院创新基金项目(No.2015-045)资助Innovation Fund Project of China Aerospace Science and Technology Corporation's Technology(No.JH.2017.0579)~~
文摘背景与目的肺内多发小结节微创手术的成功与否有赖于术前定位,然而目前缺乏针对肺内多发小结节术前定位的临床研究。本研究旨在与同期肺内单发小结节定位相比,探讨行电视胸腔镜手术(video-assisted thoracoscopic surgery, VATS)术前电子计算机断层扫描(computed tomography, CT)引导下留置微弹簧圈定位肺内多发小结节的临床价值。方法回顾性分析术前行肺内小结节微弹簧圈定位者235例的临床资料。根据结节是否为单发分为:单发结节组184例(single nodule group),多发结节组51例(multiple nodules group)。单发结节组常规方式CT引导下定位,多发结节组在CT引导下分级、分批次留置微弹簧圈定位,统计分析两组定位成功率、并发症、病理结果及定位操作相关数据等。结果多发结节组定位成功率达90.2%,与同期单发结节组成功率相比无统计学差异(90.2%vs 94.6%,P=0.205),多发结节组气胸发生率与单发结节组亦无统计学差异(21.6%vs 14.1%, P=0.179),然而多发结节组的操作时间明显长于单发结节组的操作时间[(30.6±6.6) min vs (19.9±7.4) min, P=0.000]。两组均无大咯血、空气栓塞及血胸发生等严重并发症。两组均无因术中无法定位结节而中转开胸者;手术方式以亚肺叶切除为主;术后病理以非浸润性病变为主。结论对于需行胸腔镜手术的肺部多发小结节,按照一定策略,术前CT引导下分级、分批次留置微弹簧圈的定位方法安全、有效,值得推广。
基金supported by the National Key Research and Development Program of China(2019YFA0706900)the National Natural Science Foundation of China(32025001,31872729,31600045,32071266,31800664,82022066,and 31800041)+5 种基金the Natural Science Foundation of Shandong Province,China(ZR2019ZD20,ZR2016CQ05,and ZR2019QC009)the Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology(Qingdao)(LMDBKF-2019-01)the Tianjin Synthetic Biotechnology Innovation Capability Improvement Project(TSBICIP-KJGG-001)the State Key Laboratory of Bio-organic and Natural Products Chemistry(SKLBNPC18242)the Fundamental Research Funds of Shandong University(2019GN030 and 2019GN033)the Foundation of Qilu University of Technology of Cultivating Subject for Biology and Biochemistry(No.202014)。
文摘Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-proteins-for-multi-tasks"NE in terms of screening throughput and efficiency,thus often failing to meet the fast-growing demands for biocatalysts with desired properties.In this study,we design a novel"multi-enzymes-for-multi-substrates"(MEMS)DE model and establish the proof-ofconcept by running a NE-mimicking and higher-throughput screening on the basis of"two-P450 s-against-seven-substrates"(2P×7S)in one pot.With the multiplied throughput and improved hit rate,we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes(P450 BM3 and P450 cam)in laboratory.It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future.Furthermore,structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.
基金This work was supported by the National Key Research and Development Program of China(2019YFA0905100 to S.L.)the National Natural Science Foundation of China(31800664 to F.L.,31872729 to S.L.and 31600045 to L.M.)+3 种基金General Support from China Postdoctoral Science Foundation(Grant No.2017M622293 to F.L.)Shandong Provincial Natural Science Foundation,China(ZR2019ZD22 to S.L.and ZR2016CQ05 to L.M.)the Applied Basic Research Programs of Science and Technology of Qingdao(17-1-1-60-jch to L.M.)National Research Foundation of Korea(No.NRF-2017R1A2A2A05069859 to E.-S.K.).
文摘The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A(CsA)primarily at the 4th N-methyl leucine(MeLeu4),giving rise toγ-hydroxy-N-methyl-L-Leu4-CsA(CsA-4-OH).This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect,thus demonstrating great application potential in both pharmaceutical and cosmetic industries.However,this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine(MeLeu9),indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator.Herein,we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85Å.Together with sequence and 3D structure comparisons,Autodock-based substrate docking,molecular dynamics(MD)simulation,and site-directed mutagenesis,we identified a number of key residues including R294,E264,and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA,setting the stage for better enzymatic preparation of CsA-4-OH.This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.
基金Shen Kang Hospital Development Center Foundation,Grant/Award Number:SHDC2020CR5007。
文摘Aims:Portal venous pressure frequently increases after a major hepatectomy.We aimed to assess the outcomes of patients with hepatocellular carcinoma(HCC)with different post-hepatectomy portal pressure statuses and the predictive factors correlated with prognosis and post-hepatectomy portal hypertension(PHPH).Methods:Data from consecutive patients who underwent curative-intent hepatectomy for HCC at the Eastern Hepatobiliary Surgery Hospital and Mengchao Hepatobiliary Hospital of Fujian Medical University between January 2008 and December 2016 were retrospectively analyzed.Patients were followed up until March 1,2022.PHPH was defined as new-onset portal hypertension(PH)in patients without preoperative PH within 1 month of hepatectomy.Patients were classified into two groups according to the presence or absence of PHPH:non-PH and PHPH.The clinicopathological characteristics were compared between the two groups.Univariate and multivariate analyses were used to identify independent risk factors for early and late recurrence of HCC,overall survival(OS),and PHPH.Results:Disease-free survival rates were significantly higher in the non-PH group(n=1068)than those in the PHPH group(n=423)(62.0%,39.0%,and 31.0%vs.46.2%,24.5%,and 19.3%at 1,3,and 5 years,respectively;all p<0.001).The 1-,3-,and 5-year OS rates were 91.0%,66.4%,and 51.4%in the non-PH group and 80.0%,48.9%,and 32.6%in the PHPH group,respectively(all p<0.001).Multivariate analysis revealed that PHPH was independently associated with early recurrence of HCC and poor OS(hazard ratio[HR]1.476,95%confidence interval[CI]1.279–1.704,p<0.001 and HR 1.601,95%CI 1.383–1.853,p<0.001,respectively).Furthermore,hepatitis B virus infection,cirrhosis,preoperative alanine aminotransferase>40 U/L,and major hepatectomy were identified as independent risk factors for PHPH.Conclusions:PHPH was associated with early recurrence of HCC and poor OS but not with late recurrence.Therefore,PHPH may be an attractive target for therapeutic interventions and follow-up surveillance to improve prognosis.
