Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease(AD).In this study,a novel high-throughput screening(HTS)method aims at screening the small-molecules with amyloid-β(Aβ)bi...Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease(AD).In this study,a novel high-throughput screening(HTS)method aims at screening the small-molecules with amyloid-β(Aβ)binding affinity from natural medicines,based on the combinational use of biolayer interferometry(BLI)and ultra-high-performance liquid chromatography coupled with diode-array detector and quadrupole/time-of-flight tandem mass spectrometry(UHPLC−DAD-Q/TOF-MS/MS)has been firstly developed.Briefly,the components in natural medicines disassociated from biotinylated Aβwere collected to analyze their potential Aβbinding affinity by UHPLC−DAD-Q/TOF-MS/MS.Here,baicalein was confirmed to exhibit the highest binding affinity with Aβin Scutellaria baicalensis.Moreover,polyporenic acid C(PPAC),dehydrotumulosic acid(DTA),and tumulosic acid(TA)in Kai-Xin-San(KXS)were also identified as potent Aβinhibitors.Further bioactivity validations indicated that these compounds could inhibit Aβfibrillation,improve the viability in Aβ-induced PC-12 cells,and decrease the Aβcontent and improve the behavioral ability in Caenorhabditis elegans.The molecular docking results confirmed that PPAC,DTA,and TA possessed good binding properties with Aβ.Collectively,the present study has provided a novel and effective HTS method for the identification of natural inhibitors on Aβfibrillation,which may accelerate the process on anti-AD drugs discovery and development.展开更多
Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analys...Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.展开更多
The taste presentation and receptor perception mechanism of the salty peptide of Stropharia rugosoannulata were predicted and verified using peptide omics and molecular interaction techniques.The combination of aspart...The taste presentation and receptor perception mechanism of the salty peptide of Stropharia rugosoannulata were predicted and verified using peptide omics and molecular interaction techniques.The combination of aspartic acid(D)and glutamic acid(E),or peptide fragments composed of arginine(R),constitute the characteristic taste structural basis of salty peptides of S.rugosoannulata.The taste intensity of the salty peptide positively correlates with its concentration within a specific concentration range(0.25–1.0 mg/mL).The receptor more easily recognizes the first amino acid residue at the N-terminal of salty peptides and the aspartic acid residue in the peptides.GLU513,ASP707,and VAL508 are the critical amino acid residues for the receptor to recognize salty peptides.TRPV1 is specifically the receptor for recognizing salty peptides.Hydrogen bonds and electrostatic interactions are the main driving forces for the interactions between salty peptides and TRPV1 receptors.KSWDDFFTR has the most potent binding capacity with the receptor and has tremendous potential for application in sodium salt substitution.This study confirmed the taste receptor that specifically recognizes salty peptides,analyzed the receptor-peptide binding interaction,and provided a new idea for understanding the taste receptor perception of salty peptides.展开更多
节球藻毒素-R(nodularin-R,NOD-R)是具有强烈肝毒性的蓝藻毒素。然而,现有的NOD-R检测技术均存在一定的局限性,亟待开发一种新的检测方法。本文利用指数富集的配基系统进化(systematic evolution of ligands by exponential enrichment,...节球藻毒素-R(nodularin-R,NOD-R)是具有强烈肝毒性的蓝藻毒素。然而,现有的NOD-R检测技术均存在一定的局限性,亟待开发一种新的检测方法。本文利用指数富集的配基系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术结合高通量测序(HTSELEX),筛选出NOD-R特异的高亲和力适配体。生物膜干涉(biolayer interferometry,BLI)技术对适配体的鉴定结果表明,适配体H62与NOD-R之间的亲和力最高,达到了168 n M。而且,该适配体不与其他毒素结合,具有较高的特异性。以上结果表明,适配体H62有望作为NOD-R检测新方法——适配体生物传感器的分子识别元件。展开更多
文摘目的以革兰阴性菌外膜蛋白折叠辅助因子关键蛋白BamA为靶蛋白,基于生物膜干涉(Biolayer interferometry,BLI)技术建立化合物与BamA蛋白β折叠结构域(BamA_(β-barrel))结合活性的评价方法,为建立靶向BamA蛋白的抗革兰阴性菌先导物奠定基础。方法应用BLI方法检测BamA_(β-barrel)与已知的阳性化合物darobactin的结合活性。原核表达并纯化带有His标签的大肠埃希菌BamA_(β-barrel)蛋白,使用表面活性剂LDAO对其进行复性和折叠;使用生物素标记折叠和未折叠蛋白,并结合到超级链霉亲和素(super streptavidin,SSA)生物传感器,然后检测蛋白与不同浓度的darobactin结合信号的变化,同时做无蛋白或darobactin稀释液对照;空白对照采用未结合生物素化的BamA_(β-barrel)蛋白的传感器,检测上述系列稀释样品。相应信号采用Steady state analysis方式拟合分析,计算平衡常数(KD)值。结果成功获得高纯度的折叠状态BamA_(β-barrel)蛋白,通过BLI技术检测到折叠状态的BamA_(β-barrel)与阳性化合物darobactin具有良好结合活性且呈现浓度依赖性,R^(2)为0.9998,KD值为(2.2E-06±8.0E-08)M。结论基于BLI技术成功建立了折叠状态的BamA_(β-barrel)-化合物结合活性的评价方法,为后续BamA蛋白靶向性抗革兰阴性菌抗生素的发现建立基础。
基金This work was supported by grants from the National Natural Science Foundation of China(81903829,81801398)Department of Science and Technology of Sichuan Province,China(2018JY0474,2019JDPT0010,2021YJ0180,21RCYJ0021,202086,2019YFSY0014)+1 种基金The Joint project of Luzhou Municipal People’s Government and Southwest Medical University,China(2019LZXNYDJ02,2019LZXNYDJ05,2020LZXNYDJ37)The Science and Technology Planning Programs of Luzhou(2018-JYJ-34,China).
文摘Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease(AD).In this study,a novel high-throughput screening(HTS)method aims at screening the small-molecules with amyloid-β(Aβ)binding affinity from natural medicines,based on the combinational use of biolayer interferometry(BLI)and ultra-high-performance liquid chromatography coupled with diode-array detector and quadrupole/time-of-flight tandem mass spectrometry(UHPLC−DAD-Q/TOF-MS/MS)has been firstly developed.Briefly,the components in natural medicines disassociated from biotinylated Aβwere collected to analyze their potential Aβbinding affinity by UHPLC−DAD-Q/TOF-MS/MS.Here,baicalein was confirmed to exhibit the highest binding affinity with Aβin Scutellaria baicalensis.Moreover,polyporenic acid C(PPAC),dehydrotumulosic acid(DTA),and tumulosic acid(TA)in Kai-Xin-San(KXS)were also identified as potent Aβinhibitors.Further bioactivity validations indicated that these compounds could inhibit Aβfibrillation,improve the viability in Aβ-induced PC-12 cells,and decrease the Aβcontent and improve the behavioral ability in Caenorhabditis elegans.The molecular docking results confirmed that PPAC,DTA,and TA possessed good binding properties with Aβ.Collectively,the present study has provided a novel and effective HTS method for the identification of natural inhibitors on Aβfibrillation,which may accelerate the process on anti-AD drugs discovery and development.
基金funded by the National Natural Sci-ence Foundation of China(No.31801620).
文摘Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.
基金funded by Natural Science Foundation of Shanghai,China(No.23ZR1426100)SAAS program for Excellent Research Team(No.G202203)。
文摘The taste presentation and receptor perception mechanism of the salty peptide of Stropharia rugosoannulata were predicted and verified using peptide omics and molecular interaction techniques.The combination of aspartic acid(D)and glutamic acid(E),or peptide fragments composed of arginine(R),constitute the characteristic taste structural basis of salty peptides of S.rugosoannulata.The taste intensity of the salty peptide positively correlates with its concentration within a specific concentration range(0.25–1.0 mg/mL).The receptor more easily recognizes the first amino acid residue at the N-terminal of salty peptides and the aspartic acid residue in the peptides.GLU513,ASP707,and VAL508 are the critical amino acid residues for the receptor to recognize salty peptides.TRPV1 is specifically the receptor for recognizing salty peptides.Hydrogen bonds and electrostatic interactions are the main driving forces for the interactions between salty peptides and TRPV1 receptors.KSWDDFFTR has the most potent binding capacity with the receptor and has tremendous potential for application in sodium salt substitution.This study confirmed the taste receptor that specifically recognizes salty peptides,analyzed the receptor-peptide binding interaction,and provided a new idea for understanding the taste receptor perception of salty peptides.
文摘节球藻毒素-R(nodularin-R,NOD-R)是具有强烈肝毒性的蓝藻毒素。然而,现有的NOD-R检测技术均存在一定的局限性,亟待开发一种新的检测方法。本文利用指数富集的配基系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术结合高通量测序(HTSELEX),筛选出NOD-R特异的高亲和力适配体。生物膜干涉(biolayer interferometry,BLI)技术对适配体的鉴定结果表明,适配体H62与NOD-R之间的亲和力最高,达到了168 n M。而且,该适配体不与其他毒素结合,具有较高的特异性。以上结果表明,适配体H62有望作为NOD-R检测新方法——适配体生物传感器的分子识别元件。
