High-content screening(HCS)technology combines automated high-speed imaging hardware and single-cell quantitative analysis.It can greatly accelerate data acquisition in cellular fluorescence imaging and is a powerful ...High-content screening(HCS)technology combines automated high-speed imaging hardware and single-cell quantitative analysis.It can greatly accelerate data acquisition in cellular fluorescence imaging and is a powerful research technique in traditional Chinese medicine(TCM).An increasing number of laboratories and platforms,including TCM laboratories,have begun utilizing HCS systems.However,this technology is still in its infancy in TCM research and there is a lack of sufficient experience with the associated concepts,instrument configurations,and analysis methods.To improve the understanding of HCS among researchers in the field of TCM,this paper summarizes the concept of HCS,software and hardware configuration,the overall research process,as well as common problems and related solutions of HCS in TCM research based on our team’s previous research experience,providing several research examples and an outlook on future perspectives,aiming to provide a technical guide for HCS in TCM research.展开更多
Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-conte...Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening （HCS） strategy to evaluate a novel delivery system （named CLD） of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics （siBraf） better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified.展开更多
Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-ne...Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.展开更多
Hand,foot,and mouth disease(HFMD)is a common pediatric illness mainly caused by enteroviruses,which are important human pathogens.Currently,there are no available antiviral agents for the therapy of enterovirus infect...Hand,foot,and mouth disease(HFMD)is a common pediatric illness mainly caused by enteroviruses,which are important human pathogens.Currently,there are no available antiviral agents for the therapy of enterovirus infection.In this study,an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed.Using this screening system,we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors.Fangchinoline(FAN),a bis-benzylisoquinoline alkaloid,exhibits potential inhibitory effects against various enteroviruses that cause HFMD,such as EV-A71,CV-A10,CV-B3 and CV-A16.Further investigations revealed that FAN targets the early stage of the enterovirus life cycle.Through the selection of FAN-resistant EV-A71 viruses,we demonstrated that the VP1 protein could be a potential target of FAN,as two mutations in VP1(E145G and V258I)resulted in viral resistance to FAN.Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.展开更多
Due to extreme poison,strong corrosion,and complex precipitation and deposition of H_(2)S in the reservoir,it is very difficult and risky to investigate and explore drilling,completion,production and gas transportatio...Due to extreme poison,strong corrosion,and complex precipitation and deposition of H_(2)S in the reservoir,it is very difficult and risky to investigate and explore drilling,completion,production and gas transportation.In the course of production of fractured gas reservoir with high H_(2)S content,formation pressure falls continually,which lead to decline of solubility of sulfur particles in gas phase.Sulfur particles which dissolve in gas phase originally in the formation should precipitate from gas phase after running up to saturation state and deposit at pore space and throat,sequentially resulting in formation porosity and permeability reduction.At present,the researches of sulfur deposition are mainly focused on the conventional gas reservoirs and sulfur deposition in the near wellbore region is generally estimated using Roberts'model.However,most of the gas reservoirs with high-content H2S are fractured gas reservoirs,classical damage model is no longer applicable to fractured gas reservoirs with high H2S content.In the present study,a sulfur deposition damage model is established.The refined model,based on non-Darcy flow,takes into consideration the effects of sulfur deposition,variation in gas properties and fracture.In addition,the effect of gas well production rate on formation permeability is also studied.The results show that formation permeability decreases with fracture aperture and gas well production rate increasing.The bigger gas well production rate is,the quicker sulfur precipitates.The sulfur deposition of fractured gas reservoir with high-content H_(2)S is mainly in the near wellbore zone,and the fracture aperture has a significant impact on the formation permeability in the near wellbore zone.展开更多
基金supported by grants from the Construction Fund of Key Medical Disciplines of Hangzhou(OO20200121,China)“Pioneer”and“Leading Goose”R&D Program of Zhejiang(2023C03004,2024C03143).
文摘High-content screening(HCS)technology combines automated high-speed imaging hardware and single-cell quantitative analysis.It can greatly accelerate data acquisition in cellular fluorescence imaging and is a powerful research technique in traditional Chinese medicine(TCM).An increasing number of laboratories and platforms,including TCM laboratories,have begun utilizing HCS systems.However,this technology is still in its infancy in TCM research and there is a lack of sufficient experience with the associated concepts,instrument configurations,and analysis methods.To improve the understanding of HCS among researchers in the field of TCM,this paper summarizes the concept of HCS,software and hardware configuration,the overall research process,as well as common problems and related solutions of HCS in TCM research based on our team’s previous research experience,providing several research examples and an outlook on future perspectives,aiming to provide a technical guide for HCS in TCM research.
基金Ministry of Science and Technology of China(Grant No.2012CB720604)NSFC(Grant No.20932001)
文摘Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening （HCS） strategy to evaluate a novel delivery system （named CLD） of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics （siBraf） better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified.
基金the Joint Project of Luzhou Municipal People’s Government and Southwest Medical University(No.2020LZXNYDJ37)the National Natural Science Foundation of China(Nos.81903829 and 81801398)+2 种基金the Science and Technology Planning Project of Sichuan Province(Nos.2019JDPT0010 and 2020YJ0494)the Project of Southwest Medical University(Nos.2021ZKZD015,2021ZKZD018,and 2021-ZKMS046)Sichuan University Student Innovation and Entrepreneurship Project(Nos.2019424 and 201816032066).
文摘Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.
基金funded by Guangzhou Municipal Science and Technology Project(202102020241)the National Natural Science Foundation of China(32100110 and 32300132)the National Key Research and Development Program of China(2021YFC2701800,2021YFC2701801).
文摘Hand,foot,and mouth disease(HFMD)is a common pediatric illness mainly caused by enteroviruses,which are important human pathogens.Currently,there are no available antiviral agents for the therapy of enterovirus infection.In this study,an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed.Using this screening system,we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors.Fangchinoline(FAN),a bis-benzylisoquinoline alkaloid,exhibits potential inhibitory effects against various enteroviruses that cause HFMD,such as EV-A71,CV-A10,CV-B3 and CV-A16.Further investigations revealed that FAN targets the early stage of the enterovirus life cycle.Through the selection of FAN-resistant EV-A71 viruses,we demonstrated that the VP1 protein could be a potential target of FAN,as two mutations in VP1(E145G and V258I)resulted in viral resistance to FAN.Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.
基金This work was supported by a Sichuan Youth Science and Technology Innovative Research Team of Safe and Efficient Development of Sour Gas Reservoir(2014TD0009).
文摘Due to extreme poison,strong corrosion,and complex precipitation and deposition of H_(2)S in the reservoir,it is very difficult and risky to investigate and explore drilling,completion,production and gas transportation.In the course of production of fractured gas reservoir with high H_(2)S content,formation pressure falls continually,which lead to decline of solubility of sulfur particles in gas phase.Sulfur particles which dissolve in gas phase originally in the formation should precipitate from gas phase after running up to saturation state and deposit at pore space and throat,sequentially resulting in formation porosity and permeability reduction.At present,the researches of sulfur deposition are mainly focused on the conventional gas reservoirs and sulfur deposition in the near wellbore region is generally estimated using Roberts'model.However,most of the gas reservoirs with high-content H2S are fractured gas reservoirs,classical damage model is no longer applicable to fractured gas reservoirs with high H2S content.In the present study,a sulfur deposition damage model is established.The refined model,based on non-Darcy flow,takes into consideration the effects of sulfur deposition,variation in gas properties and fracture.In addition,the effect of gas well production rate on formation permeability is also studied.The results show that formation permeability decreases with fracture aperture and gas well production rate increasing.The bigger gas well production rate is,the quicker sulfur precipitates.The sulfur deposition of fractured gas reservoir with high-content H_(2)S is mainly in the near wellbore zone,and the fracture aperture has a significant impact on the formation permeability in the near wellbore zone.
