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

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

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

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

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.
Alphavirus ; drug effects ; genetics ; metabolism ; Animals ; Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; High-Throughput Screening Assays ; methods ; Luciferases ; genetics ; metabolism

Cytometric bead array (CBA) is a new analytical technique, which can achieve real-time and rapid detection of targeted components in a small amount of sample. With many advantages of high throughput screening, high specificity and sensitivity, low cost, easy operation and good repeatability, this CBA technique has been widely used for the detection of various components in foods, agricultural products and environmental samples. Recently, it has got significant development in rapid detection of small molecules. This review briefly introduced the theory of CBA technique, summarized the application in the analysis of small molecules, such as mycotoxins, pesticide residues, shellfish toxins, and then prospected the application of trace small molecules detection in the complex matrices of traditional Chinese medicine and the development trend of it.
Drug Contamination ; High-Throughput Screening Assays ; instrumentation ; methods ; Immunoassay ; instrumentation ; methods ; Microspheres ; Pesticides ; analysis ; Toxins, Biological ; analysis

Industrial bioprocess is one of the most important research fields supports the promoting of biological manufacturing industry in China, and guarantees the implementation of bioscience and biotechnology research results into the industrial applications. For improving the interconnection between academic researchers and industrial stuffs and pushing research achievement into industrial implementation, bioprocess modelling and control committee of Chinese Society for Microbiology organized two tandem conferences separately in 2012 and 2014 on the topic of "Industrial bioprocess technology", focusing mainly technique front of industrial bioprocess. A special session on industrial technique applications was hold to stimulate cooperation. The conference received many good submissions from academic and industrial sectors. This special issue is based on selected excellent papers from the submissions, together with free submissions. The special issue consists of reviews and original papers, mainly involving the aspects closely related to the bio-industrial sectors including, i) high yield strain constructing and high throughput screening; ii) optimization and modification of industrial enzymes, and iii) bioprocess modelling and high efficient scale-up method.
China ; Congresses as Topic ; Enzymes ; biosynthesis ; Fermentation ; High-Throughput Screening Assays ; Industrial Microbiology ; methods ; Societies, Scientific

Diagnostic Imaging ; methods ; Genes, Neoplasm ; High-Throughput Screening Assays ; Humans ; Molecular Diagnostic Techniques ; Molecular Targeted Therapy ; Neoplasms ; genetics ; pathology ; therapy ; Pathology, Molecular ; methods ; Precision Medicine ; methods