The dried mature peel of Citrus reticulata, a plant in the Rutaceae family and its cultivated varieties, is a commonly used Chinese medicinal material known as Chenpi(Citri Reticulatae Pericarpium). It is rich in nutritional components and medicinal value, with pharmacological effects including relieving cough and eliminating phlegm, strengthening the spleen and drying dampness, protecting the liver and benefiting the stomach, tonifying Qi, and calming the mind. Huanglongbing(HLB), also known as Citrus Huanglongbing, is a destructive disease in citrus production that seriously threatens the development of the citrus industry. HLB causes symptoms such as the inability of Rutaceae plants to produce mature fruit, gradual weakening of the tree, and eventual death, posing a significant threat to the yield and quality of Chenpi. Due to the uneven distribution of the HLB pathogen in infected plants, accurate detection of the pathogen requires the collection of a large number of plant samples. Current sample pretreatment methods, such as traditional extraction methods and commercial extraction kits, are time-consuming and involve multiple steps, which significantly increase the difficulty and workload of HLB diagnosis and have become a bottleneck in HLB detection. In this study, a rapid high-throughput detection method combining alkali lysis and TaqMan qPCR was developed. This method allows the pretreatment of multiple samples within 5 min, and the entire detection process can be completed within 45 min, with a detection limit of 6.67 fg·μL~(-1). The alkali lysis method and commercial kits were used for parallel detection of field-collected citrus samples, and the results showed no significant difference. The sample pretreatment method established in this study is characterized by low cost, simplicity, and high efficiency. Combined with TaqMan qPCR, it can provide technical support for early and on-site diagnosis of HLB. This method is of great significance for disease prevention and control in the citrus industry and is expected to help improve the yield and quality of citrus medicinal materials.
Citrus/microbiology* ; Plant Diseases/microbiology* ; Rhizobiaceae/physiology* ; High-Throughput Screening Assays/methods* ; Liberibacter/physiology*

Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase from Actinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.
Transaminases/metabolism* ; Fluorescent Dyes/chemistry* ; High-Throughput Screening Assays/methods* ; Ketones/metabolism* ; Actinobacteria/enzymology*

The 5-hydroxytryptamine 2A receptor (5-HT_2AR) is one of the key targets in the development of novel antidepressants. To develop new antidepressants targeting the 5-HT_2A receptor, this study established a high-throughput screening method for drugs targeting the 5-HT_2A receptor based on the principle of detecting calcium flux signals. The immunofluorescence assay and western blotting were employed to evaluate receptor expression levels in the 5-HT_2AR-CHO cell line. The reaction system parameters, including cell seeding density, DMSO concentration, and dye incubation time, were optimized with Z'-factor and signal window values as evaluation indicators. The specificity, precision, stability, and applicability of the method were assessed. Results indicated that the 5-HT_2AR-CHO cell line stably expressed high levels of the 5-HT_2A receptor. The optimized screening method involved a reaction system with 10 000 cells/well, 0.2% DMSO, and 2 h incubation with Calcium 6 dye. The method demonstrated excellent specificity, with inter-batch precision below 10% for the detection of 5-hydroxytryptamine (5-HT) at low, medium, and high concentrations. Testing four compounds that target the 5-HT_2A receptor- agonists 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and lysergic acid diethylamide (LSD), along with the antagonist MDL100907-yielded Z'-factors (at EC₈₀) greater than 0.85 and signal window values over 0.91. The EC₅₀ values of these compounds were in the nanomolar range, and their potency rank order aligned with previously reported data, confirming the reliability of the established method. When being applied to the detection of 38 known active compounds, the method efficiently identified 5-HT_2A receptor agonists and antagonists while showing no response to non-target compounds. In conclusion, this study successfully constructs a high-throughput screening approach for 5-HT_2A receptor-targeting drugs based on calcium flux signals. The method possesses strong specificity, high sensitivity, and robust stability, being suitable for screening antidepressants targeting the 5-HT_2A receptor.
High-Throughput Screening Assays/methods* ; Receptor, Serotonin, 5-HT2A/metabolism* ; Animals ; CHO Cells ; Cricetulus ; Calcium Signaling/drug effects* ; Antidepressive Agents/pharmacology* ; Humans ; Serotonin 5-HT2 Receptor Antagonists/pharmacology* ; Calcium/metabolism*

Actinomycetes are important producers of high-value natural products, and the engineering of actinomycetes to enhance the biosynthesis of target natural products has long been a hot research topic in the scientific community. However, non-rational engineering methods suffer from low beneficial mutation rates, which limit the efficiency of mutant screening. The integration of high-throughput screening (HTS) technologies can effectively enhance the screening efficiency of elite mutants and significantly shorten the cycle of actinomycete strain engineering. This review comprehensively discusses various HTS technologies suitable for the engineering of actinomycete strains and compares them in terms of application scenarios, advantages, and disadvantages. HTS technologies include microplate-based screening, antimicrobial activity screening, antibiotic resistance screening, fluorescence-activated cell sorting (FACS), and fluorescence-activated droplet sorting (FADS). Additionally, this review summarizes the applications of these technologies in assisting actinomycete strain engineering and enhancing the yields of target compounds. The development and application of HTS technologies have not only facilitated the exploration of natural product resources in actinomycetes but also provided strong support for the rapid and efficient construction of high-performance engineered actinomycete strains.
Actinobacteria/metabolism* ; High-Throughput Screening Assays/methods* ; Genetic Engineering/methods* ; Biological Products/metabolism* ; Flow Cytometry ; Metabolic Engineering/methods*

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.
Emulsions ; Flow Cytometry ; High-Throughput Screening Assays ; Microfluidics ; methods

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.
Animals ; Bacteria ; enzymology ; Enzymes ; genetics ; Gene Library ; High-Throughput Screening Assays ; methods ; Metagenome ; genetics ; Metagenomics ; methods ; Plants ; enzymology

For screening the potential drugs as anti-liver fibrosis candidates, we established a high- throughput drug screening cell model based on COL1A1 promoter. The activity of COL1A1 promoter and luciferase reporter gene can be elevated by TGF-β1, and inhibited by candidate drugs. We constructed a recombined plasmid with COL1A1 promoter and luciferase reporter gene pGL4.17, the activity of COL1A1 promoter was reflected by fluorescence intensity. COL1A1 promoter activity was detected by Dual-Luciferase Reporter Assay System, it came that the relative luciferase activity of COL1A1 promoter was 15.98 times higher than that of control group induced by TGF-β1, showing the recombined plasmid could be used in cell model. The recombined plasmid was transfected into human hepatic stellate cells LX2, detected the effect of potential drugs, and obtained a stable expression system through stable transfection and monoclonal cell culture. A sample which could reduce COL1A1 promoter activity signally by our cell model, decreased collagen I mRNA and protein expression detected by real-time RT-PCR and Western blotting. It indicates this novel cell model can be used in high-throughput drug screening of potential anti-liver fibrosis drugs.
Collagen Type I ; genetics ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; Hepatic Stellate Cells ; High-Throughput Screening Assays ; Humans ; Liver Cirrhosis ; drug therapy ; Luciferases ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

High-throughput screening is a regular approach available for identitying new lead compounds for the growing validated drug targets in drug screening. However, it has also introduced a large number of peculiar molecules which interfere drug screening. Pan assay interference compounds (PAINS) interfere with the progress of drug screening in various ways, such as interfering with a biochemical assay, modifying the protein, aggregate-based inhibitors and so on. So it is of vital significance to remove them. This paper has consulted the concept, category of PAINS and reviewed the way of PAINS interfering and the countermeasures to cope with them to direct the approach of high through screening and improve the hits percent.
Drug Discovery ; High-Throughput Screening Assays ; methods

The lack of effective in vitro infection model for hepatitis E virus (HEV) has greatly hindered the quantitative analysis of neutralizing titers of anti-HEV antibodies and human sera, thus impeding further studies of HEV-stimulated antibody responses and the immunological mechanisms. In order to improve this situation, the infection of HepG2 cells that are inefficient for HEV replication was continuously monitored until the viral load reached the limit of detection on day 13, the results of which confirmed the feasibility of using this cell line to establish the infection model. Then, neutralization assays of five anti-HEV murine monoclonal antibodies and serum samples collected from four HEV vaccine recipients (collected before and after vaccination) were performed by 96 multi-channel parallel infections, nucleic acid extraction, and qPCR. The results showed that the cell model can be applied for quantitative evaluation of the neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients. In this study, we have successfully established a high-throughput in vitro HEV replication model, which will prove to be useful for the evaluation of HEV vaccines and studies of HEV epitopes.
Animals ; Antibodies, Viral ; analysis ; immunology ; Hepatitis Antibodies ; analysis ; immunology ; Hepatitis E ; immunology ; virology ; Hepatitis E virus ; chemistry ; immunology ; physiology ; High-Throughput Screening Assays ; methods ; Humans ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; methods ; Virus Replication