Objective:To determine the mechanism of the laxative effect of rhubarb(Rheum tanguticum Maxim.ex Balf.)in rats using a combined bioinformatic and in vivo approach.Methods:Substances derived from rhubarb that are prese...Objective:To determine the mechanism of the laxative effect of rhubarb(Rheum tanguticum Maxim.ex Balf.)in rats using a combined bioinformatic and in vivo approach.Methods:Substances derived from rhubarb that are present in the colorectum were identified using high-performance liquid chromatography,coupled with linear ion-trap quadrupole Orbitrap highresolution mass spectrometry.The targets with a potential laxative effect were identified from databases and the literature,then used with rhubarb-derived substances in molecular docking modeling.The expression of candidate endogenous target molecules that bound specific components of rhubarb was then measured in constipated rats that had or had not been administered rhubarb by western blotting.Finally,potentially bioactive compounds were traced back to their prototype components.Results:We identified 17 anthraquinones and 21 anthrones derived from rhubarb in the colorectum of rats.G-scoring identified three potential mediators of the laxative effect:c-kit,5-hydroxytryptamine receptor 4(5-HT4),and aquaporin-3(AQP3).In addition,10 rhubarb-derived components(aloeemodin,emodin,rhein,chrysophanol,physcion,sennoside A,sennoside C,physcionanthrone,aloeemodinanthrone,and rheinanthrone),which have strong binding affinities for more than one of the three potential targets,were selected as likely active compounds.Rhubarb extract increased the expression of c-kit and 5-HT4,and reduced the expression of AQP3 in the colon of constipated rats,which might mediate its laxative effect.We also found that a single prototype component may be metabolized into several active metabolites,and a single active ingredient can also be generated from various prototype compounds.Conclusion:The present study demonstrates that various anthraquinones and anthrones present in rhubarb may be metabolized to form bioactive compounds that have additive or synergistic effects to promote defecation via c-kit,5-HT4 and/or AQP3.展开更多
Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic...Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.展开更多
Nanogels hold promise as soft and functional carriers and nanoreactors,but whether they will ever reach the stage of large-scale application depends crucially on efficient production methods,and on what interesting pr...Nanogels hold promise as soft and functional carriers and nanoreactors,but whether they will ever reach the stage of large-scale application depends crucially on efficient production methods,and on what interesting properties and functionalities they can have.In particular,nanogels consisting of highly charged polyelectrolyte have not yet been very much explored.Here,the authors present a novel and generic strategy for controlled and efficient synthesis of a large diversity of polyelectrolyte nanogels.The method is based on polymerizing an ionic monomer in the presence of an oppositely charged polyion-neutral diblock copolymer as template,while adding a cross-linker.The growing polymer chains assemble with the template,forming polyion complex micelles,which dissociate upon increasing salt concentration.Subsequent separation yields nanogels with well-controlled size and properties,and free template polymers that can be used again.Our design can be applied generally to a wide range of both cationic and anionic monomers,as well as various cross-linkers.Scaled-up production presents no problems as increasing monomer concentration(hundreds of mM)and reaction volume(up to 1 L)hardly compromise product quality.Moreover,the obtained nanogels with their well-controlled size,morphology,chemistry,and cross-linking degree perform well as soft nanocarriers and catalytic nanoreactors.展开更多
基金the National Natural Science Foundation of China(81673562 and 81973467)the Fundamental Research Funds for the Central Universities of China(2020-JYB-ZDGG-033).
文摘Objective:To determine the mechanism of the laxative effect of rhubarb(Rheum tanguticum Maxim.ex Balf.)in rats using a combined bioinformatic and in vivo approach.Methods:Substances derived from rhubarb that are present in the colorectum were identified using high-performance liquid chromatography,coupled with linear ion-trap quadrupole Orbitrap highresolution mass spectrometry.The targets with a potential laxative effect were identified from databases and the literature,then used with rhubarb-derived substances in molecular docking modeling.The expression of candidate endogenous target molecules that bound specific components of rhubarb was then measured in constipated rats that had or had not been administered rhubarb by western blotting.Finally,potentially bioactive compounds were traced back to their prototype components.Results:We identified 17 anthraquinones and 21 anthrones derived from rhubarb in the colorectum of rats.G-scoring identified three potential mediators of the laxative effect:c-kit,5-hydroxytryptamine receptor 4(5-HT4),and aquaporin-3(AQP3).In addition,10 rhubarb-derived components(aloeemodin,emodin,rhein,chrysophanol,physcion,sennoside A,sennoside C,physcionanthrone,aloeemodinanthrone,and rheinanthrone),which have strong binding affinities for more than one of the three potential targets,were selected as likely active compounds.Rhubarb extract increased the expression of c-kit and 5-HT4,and reduced the expression of AQP3 in the colon of constipated rats,which might mediate its laxative effect.We also found that a single prototype component may be metabolized into several active metabolites,and a single active ingredient can also be generated from various prototype compounds.Conclusion:The present study demonstrates that various anthraquinones and anthrones present in rhubarb may be metabolized to form bioactive compounds that have additive or synergistic effects to promote defecation via c-kit,5-HT4 and/or AQP3.
基金the National Key Research and Development Program of China(2018YFA0901500)the National Natural Science Foundation of China(32070083 and 32222004)+2 种基金the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008)the Innovation Fund of Haihe Laboratory of Synthetic Biology.
文摘Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.
基金supported by the 1000 Foreign Experts Program(no.WQ20163100341)the Shanghai Municipal Natural Science Foundation(nos.17ZR1440500 and 18490740100)d the Fundamental Research Funds for the Central Universities(no.222201714003).
文摘Nanogels hold promise as soft and functional carriers and nanoreactors,but whether they will ever reach the stage of large-scale application depends crucially on efficient production methods,and on what interesting properties and functionalities they can have.In particular,nanogels consisting of highly charged polyelectrolyte have not yet been very much explored.Here,the authors present a novel and generic strategy for controlled and efficient synthesis of a large diversity of polyelectrolyte nanogels.The method is based on polymerizing an ionic monomer in the presence of an oppositely charged polyion-neutral diblock copolymer as template,while adding a cross-linker.The growing polymer chains assemble with the template,forming polyion complex micelles,which dissociate upon increasing salt concentration.Subsequent separation yields nanogels with well-controlled size and properties,and free template polymers that can be used again.Our design can be applied generally to a wide range of both cationic and anionic monomers,as well as various cross-linkers.Scaled-up production presents no problems as increasing monomer concentration(hundreds of mM)and reaction volume(up to 1 L)hardly compromise product quality.Moreover,the obtained nanogels with their well-controlled size,morphology,chemistry,and cross-linking degree perform well as soft nanocarriers and catalytic nanoreactors.