Guidelines for application of high-content screening in traditional Chinese medicine:concept,equipment,and troubleshooting

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.

Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity,damaging the neurons.However,how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear.Herein,we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury.We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice.Lipid droplet accumulation could be induced by myelin debris in HT22 cells.Myelin debris degradation by phospholipase led to massive free fatty acid production,which increased lipid droplet synthesis,β-oxidation,and oxidative phosphorylation.Excessive oxidative phosphorylation increased reactive oxygen species generation,which led to increased lipid peroxidation and HT22 cell apoptosis.Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway,thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells.Motor function,lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury.The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening

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.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Development of a high-content screening method to evaluate the cellular population-based biological activity of new siRNA delivering reagent

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.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer’s disease by the high-content fluorescence imaging system

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.

Study on sulfur deposition damage model of fractured gas reservoirs with high-content H_(2)S

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.

Method Based on Time Randomization to Resist Fault Sensitivity Analysis

A fault sensitivity analysis（FSA）-resistance model based on time randomization is proposed.The randomization unit is composed of two parts,namely the configurable register array（R-A）and the decoder（chiefly random number generator,RNG）.In this way,registers chosen can be either valid or invalid depending on the configuration information generated by the decoder.Thus,the fault sensitivity information can be confusing.Meanwhile,based on this model,a defensive scheme is designed to resist both fault sensitivity analysis（FSA）and differential power analysis（DPA）.This scheme is verified with our experiments.

Modeling drug-induced liver injury and screening for anti-hepatofibrotic compounds using human PSC-derived organoids

Preclinical models that can accurately predict the toxicity and efficacy of candidate drugs to human liver tissue are in urgent need.Human liver organoid(HLO)derived from human pluripotent stem cells offers a possible solution.Herein,we generated HLOs,and demonstrated the utility of these HLOs in modeling a diversity of phenotypes associated with drug-induced liver injury(DILI),including steatosis,fibrosis,and immune responses.Phenotypic changes in HLOs after treatment with tool compounds such as acetaminophen,fialuridine,methotrexate,or TAK-875 showed high concordance with human clinical data in drug safety testings.Moreover,HLOs were able to model liver fibrogenesis induced by TGFβ or LPS treatment.We further devised a high-content analysis system,and established a high-throughput anti-fibrosis drug screening system using HLOs.SD208 and Imatinib were identified that can significantly suppress fibrogenesis induced by TGFβ,LPS,or methotrexate.Taken together,our studies demonstrated the potential applications of HLOs in drug safety testing and anti-fibrotic drug screening.

In vitro 3D phenotypic drug screen identifies celastrol as an effective in vivo inhibitor of polycystic kidney disease

Polycystic kidney disease(PKD)is a prevalent genetic disorder,characterized by the formation of kidney cysts that progressively lead to kidney failure.The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments.The signalling rewiring in PKD that drives cyst formation is highly complex and not fully understood.As a consequence,the effects of drugs are sometimes difficult to predict.We previously established a high-throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth.Here,we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules,including approved drugs.We identified 81 active molecules that inhibit cyst growth.Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties.Celastrol,a triterpenoid from Tripterygium wilfordii,was identified as a potent inhibitor of cyst growth in vitro.In an in vivo iKspCre-Pkd1^lox,lox mouse model for PKD,celastrol inhibited the growth of renal cysts and maintained kidney function.

Motion Characteristic Analysis of a Parallel Mechanism with 2-DOF for Industrial Fan Blades' Scribing

Abstract：With the application background of 16 inch industrial fan blades＇ scribing process, a simple and prac tical 2DOF parallel plane scribing device is proposed to replace the traditional manual scribing. The direct and inverse kinematics solutions, workspace and singular configuration of the mechanism are analyzed and solved. Some contour curve equations are fitted based on the data points which are measured by a three coordinate meas uring machine. According to the blade＇s contour curve, each input link＇s motion characteristic is obtained by u sing MATLAB, which provides a new way to realize blades＇ automatic scribing process technology.

Drupacine as a ootentSARS-CoV-2replication inhibitor in vitro

Despite the availability of vaccines and antiviral treatments,the continued emergence of severe acute respira-tory syndrome coronavirus 2(SARS-CoV-2)variants and breakthrough infections underscores the need for new,potent antiviral therapies.In a previous study,we established a transcription and replication-competent SARS-CoV-2 virus-like particle(trVLP)system that recapitulates the complete viral life cycle.In this study,we combined high-content screening(HCS)with the SARS-CoV-2 trVLP cell culture system,providing a powerful phenotype-oriented approach to assess the antiviral potential of compounds on a large scale.We screened a library of 3,200 natural compounds and identified drupacine as a potential candidate against SARS-CoV-2 infection.Furthermore,we utilized a SARS-CoV-2 replicon system to demonstrate that drupacine could inhibit viral genome transcription and replication.However,in vitro,enzymatic assays revealed that the inhibition could not be attributed to conventional antiviral targets,such as the viral non-structural proteins nsp5(MPro)or nsp12(RdRp).In conclusion,our findings position drupacine as a promising antiviral candidate against SARS-CoV-2,providing a novel scaffold for developing anti-coronavirus disease 2019 therapeutics.Further investigation is required to pinpoint its precise target and mechanism of action.