Development of a high-throughput cell based 384-well influenza A quantification assay for interpandemic and highly pathogenic avian strains

Influenza remains a world wide health threat, thus the need for a high-throughput and robust assay to quantify both seasonal and avian in-fluenza A strains. Therefore, a 384-well plate format was developed for the median tissue culture infectious dose assay (TCID50) utilizing the detection of nucleoprotein by an in situ en-zyme linked immunosorbent assay (ELISA) which was optimized for sensitivity in this assay. Highly pathogenic avian influenza, A/Vietnam/ 1203/04 (H5N1), and interpandemic strains, A/ New Caledonia/20/99 (H1N1) and A/Brisbane/ 10/07 (H3N2), were quantified using this high- throughput assay. Each 384-well plate can be used to analyze ten viral samples in quadrupli-cate, eight dilutions per sample, including all necessary assay controls. The results obtained from 384-well plates were comparable to tradi-tional 96-well plates and also demonstrate re-peatability, intermediate precision, and assay linearity. Further, the use of 384-well plates in-creased the throughput of sample analysis and the precision and accuracy of the resulting titer.

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction.IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration.Baicalein,a known IN inhibitor,was found to be an IN-CCD inhibitor.The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.

Development of a Colorimetric Loop-mediated Isothermal Amplification Assay for the Visual Detection of Fusarium oxysporum f.sp. melonis

Fusarium wilt of melon, Fusarium oxysporum f.sp. melonis is one of the most important diseases, causing tremendous losses in melon growing areas in Iran. There is a real need for a rapid and inexpensive assay to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. One of the procedures designed for detection of this disease is loop-mediated isothermal amplification(LAMP)assay; its efficiency has been contrasted with polymerase chain reaction(PCR). The translation elongation factor 1-alpha gene is basically used for designing the LAMP(i.e. F3, B3, FIP, and BIP) together with PCR(F and B). Using hydroxynaphthol blue(HNB) dye, LAMP was placed in a water bath after the optimization was done. The results show LAMP is an advantageous method because it is highly sensitive(100-fold), quite cheap,user-friendly, and safe; in addition, it is performed quickly by visual detection and does not require DNA extraction(in direct-LAMP). The LAMP is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually.

Development and Characterization of West Nile Virus Replicon Expressing Secreted Gaussia Luciferase

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

Autoantigen Microarray for High-throughput Autoantibody Profiling in Systemic Lupus Erythematosus

Systemic lupus erythematosus （SLE） is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components （nucleic acids and associated proteins）, cytoplas- mic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epi- topes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection.

A cell-based high-throughput approach to identify inhibitors of influenza A virus

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors.In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA,which expresses Gaussia luciferase upon influenza A virus infection.Using this cell line,an assay was developed and optimized to search for inhibitors of influenza virus replication.Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format(Z 0 factor value40.8).A pilot screening provides further evidence for validation of the assay.Taken together,this work provides a simple,convenient,and reliable HTS assay to identify compounds with anti-influenza activity.

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2(SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2(SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76 A. After initial screening of our in-house compound library(w2300 compounds), we identified 4 new SHP2-PTP inhibitors(0.17%hit rate) and 28 novel allosteric SHP2 inhibitors(1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP(SYK-85: IC_(50) Z 0.32 mmol/L;WS-635: IC_(50) Z 4.13 mmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently(IC_(50) Z 0.25 mmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.

Quality control of cell-based high-throughput drug screening

The pharmaceutical industry is presently suffering difficult times due to low productivity of new molecular entities.As a major source of drug leads,high-throughput screening(HTS)has been often criticized for its‘dead end’lead compounds.However,the fruitful achievements resulting from HTS technology indicate that it remains a feasible way for drug innovation.Because of increasing considerations of earlier stage ADMET(absorption,distribution,metabolism,excretion and toxicity)in drug development,cell-based HTS is highly recommended in modern drug discovery for its ability to detect more biologically relevant characteristics of compounds in living systems.This review provides a systematic and practical description of vital points for conducting high quality cell-based HTS,from assay development to optimization,compound management,data analyses,hit validation as well as lead identification.Potential problems and solutions are also covered.

Visual Detection of COVID‑19 from Materials Aspect

In the recent COVID-19 pandemic,World Health Organization emphasized that early detection is an effective strategy to reduce the spread of SARS-CoV-2 viruses.Several diagnostic methods,such as reverse transcription-polymerase chain reaction(RT-PCR)and lateral flow immunoassay(LFIA),have been applied based on the mechanism of specific recognition and binding of the probes to viruses or viral antigens.Although the remarkable progress,these methods still suffer from inadequate cellular materials or errors in the detection and sampling procedure of nasopharyngeal/oropharyngeal swab collection.Therefore,developing accurate,ultrafast,and visualized detection calls for more advanced materials and technology urgently to fight against the epidemic.In this review,we first summarize the current methodologies for SARS-CoV-2 diagnosis.Then,recent representative examples are introduced based on various output signals(e.g.,colorimetric,fluorometric,electronic,acoustic).Finally,we discuss the limitations of the methods and provide our perspectives on priorities for future test development.

Enhanced lateral flow assay with double conjugates for the detection of exosomes

Exosomes are promising biological biomarkers for monitoring a number of diseases, especially cancers. Here, we developed a double gold nanoparticles （GNPs） conjugates based lateral flow assay （D-LFA） for rapidly and sensitively detecting and molecular profiling of exosomes. Based on these two GNPs conjugates, the signal amplification can be achieved without any additional operation. The antibody on the 1st GNPs conjugate could recognize exosomes and form a sandwich format on the test zone. The 2nd GNPs conjugate was designed to bind to the 1st GNPs conjugate to realize signal amplification. This biosensor enabled visual and quantitative detection of exosomes by the accumulation of GNPs on the test zone and showed a low detection limit of 1.3x10^3 particles/laL, which has been improved 13-fold compared with the normal lateral flow assay. The D-LFA exhibited good sensitivity and reproducibility and has been successfully used for the detection ofexosomes in fetal bovine serum, which proved its potential application in practical diagnostics.

Optical substrates for drug-metabolizing enzymes: Recent advances and future perspectives

Drug-metabolizing enzymes(DMEs),a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics,have been recognized as the critical determinants to drug safety and efficacy.Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity,as well as characterizing the interactions of central DMEs with xenobiotics require reliable,practical and highly specific tools for sensing the activities of these enzymes in biological systems.In the last few decades,the scientists have developed a variety of optical substrates for sensing human DMEs,parts of them have been successfully used for studying target enzyme(s)in tissue preparations and living systems.Herein,molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically.Furthermore,the challenges and future perspectives in this field are also highlighted.The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems,which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb-drug interactions,as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes.