High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.

A Cell-based High-throughput Screening Assay for Farnesoid X Receptor Agonists

Objective To develop a high-throughput screening assay for Farnesoid X receptor （FXR） agonists based on mammalian one-hybrid system （a chimera receptor gene system） for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain （LBD） was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain （DBD） of yeast GAL4 of pBIND to construct a GAL4-FXR （LBD） chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD （189-472） chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z＇ factor value of 0.65, Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

Development of a New High-throughput Screening Model for Human High Density Lipoprotein Receptor (CLA-1) Agonists

To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

High-Throughput Screening of Nanoparticle-Stabilizing Ligands:Application to Preparing Antimicrobial Curcumin Nanoparticles by Antisolvent Precipitation

Water-dispersible curcumin nanoparticles were prepared by bottom-up antisolvent precipitation approach. A new high-throughput screening technique was developed for selecting appropriate ligands stabilizing the nanoparticles in aqueous medium and improving their performance. The initial set of twenty-eight potential stabilizing ligands was evaluated based on their capacity to improve curcumin dispersibility in aqueous medium. The performance of four promising ligands(amino acid proline, polyphenol tannic acid, polycation Polyquaternium 10, and neutral polymer polyvinylpyrrolidone) was tested in ultrasound-aided antisolvent precipitation trials. Using the selected stabilizing ligands diminished the average particle size from ca. 1,200 to 170–230 nm, reduced their dispersity, improved stability, and allowed reaching curcumin concentration of up to 1.4 m M in aqueous medium. Storage stability of the aqueous nanodispersions varied from 2 days to 2 weeks, depending on stabilizing ligand. Studying the effects of ionic strength and pH on size and f-potential of the particles suggested that electrostatic forces and hydrophobic interactions could be the major factors affecting their stability. The ligand-protected nanoparticles showed minimal inhibitory concentration of 400 or500 μM toward Escherichia coli. We suggest that the presented screening approach may be useful for preparing nanoparticles of various poorly water-soluble bioactive materials.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades,high-throughput screening(HTS)has made great contributions to new drug discovery.HTS technology is equipped with higher throughput,minimized platforms,more automated and computerized operating systems,more efficient and sensitive detection devices,and rapid data processing systems.At the same time,in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes.However,challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional(3D)in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions.Here,we review the applications of HTS technologies on in vitro neurogenesis,especially aiming at identifying the essential genes,chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids.We also discuss the developmental tendency of HTS technology,e.g.,so-called next-generation screening,which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.

High-Throughput Screening Using Fourier-Transform Infrared Imaging

Efficient parallel screening of combinatorial libraries is one of the most challenging aspects of the high- throughput （HT） heterogeneous catalysis workflow. Today, a number of methods have been used in HT catalyst studies, including various optical, mass-spectrometry, and gas- chromatography techniques. Of these, rapid-scanning Fourier-transform infrared （FTIR） imaging is one of the fastest and most versatile screening techniques. Here, the new design of the 16-channel HT reactor is presented and test results for its accuracy and reproducibility are shown. The performance of the system was evaluated through the oxidation of CO over commercial Pd/AI203 and cobalt oxide nanoparticles synthesized with different reducer-reductant molar ratios, surfactant types, metal and surfactant concentrations, synthesis temperatures, and ramp rates.

An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.

A high-throughput screening method for breeding Aspergillus niger with 12C6+ ion beam-improved cellulase

In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable for screening a large number of mutants and improving the breeding efficiency after heavy-ion beam irradiation.Seventeen strains showed higher cellulase activity compared with the initial strain after the screening of plate and MTP fermentation.The filter paper activity and β-glucosidase activity of Aspergillus niger H11201 had increased 38.74 and 63.23%separately compared with A.niger H11 by shaking flask fermentation,and it was genetically stable after being passaged to nine generations.The results indicate that the high-throughput screening method can be used for the quick breeding of A.niger with high cellulase activity.

Computational high-throughput screening and in vitro approaches identify CB-006-3;A novel PI3K-BRAF^(V600E) dual targeted inhibitor against melanoma

Malignant melanoma is characterized by both genetic and molecular alterations that activate phosphoinositide 3-kinase(PI3K),and RAS/BRAF pathways.In this work,through diversity-based high-throughput virtual screening we identified a lead molecule that selectively targets PI3K and BRAF^(V600E) kinases.Computational screening,Molecular dynamics simulation and MMPBSA calculations were performed.PI3K and BRAF^(V600E) kinase inhibition was done.A375 and G-361 cells were used for in vitro cellular analysis to determine antiproliferative effects,annexin V binding,nuclear fragmentation and cell cycle analysis.Computational screening of small molecules indicates compound CB-006-3 selectively targets PI3KCG(gamma subunit),PI3KCD(delta subunit)and BRAF^(V600E).Molecular dynamics simulation and MMPBSA bases binding free energy calculations predict a stable binding of CB-006-3 to the active sites of PI3K and BRAF^(V600E).The compound effectively inhibited PI3KCG,PI3KCD and BRAF^(V600E)kinases with respective IC50 values of 75.80,160.10 and 70.84 nM.CB-006-3 controlled the proliferation of A375 and G-361 cells with GI50 values of 223.3 and 143.6 nM,respectively.A dose dependent increase in apoptotic cell population and sub G0/G1 phase of cell cycle were also observed with the compound treatment in addition to observed nuclear fragmentation in these cells.Furthermore,CB-006-3 inhibited BRAF^(V600E),PI3KCD and PI3KCG in both melanoma cells.Collectively,based on the computational modeling and in vitro validations,we propose CB-006-3 as a lead candidate for selectively targeting PI3K and mutant BRAF^(V600E) to inhibit melanoma cell proliferation.Further experimental validations,including pharmacokinetic evaluations in mouse models will identify the druggability of the proposed lead candidate for further development as a therapeutic agent for treating melanoma.

Designing solar-cell absorber materials through computational high-throughput screening

Although the efficiency of CH3 NH3 PI3 has been refreshed to 25.2%,stability and toxicity remain the main challenges for its applications.The search for novel solar-cell absorbers that are highly stable,non-toxic,inexpensive,and highly efficient is now a viable research focus.In this review,we summarize our recent research into the high-throughput screening and materials design of solar-cell absorbers,including single perovskites,double perovskites,and materials beyond Perovskites.BazrS3(single perovskite),Ba2 BiNbS6(double perovskite),HgAl2 Se4(spinel),and IrSb3(skutterudite)were discovered to be potential candidates in terms of their high stabilities,appropriate bandgaps,small carrier effective masses,and strong optical absorption.

High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]-1H-isoindole-1,3(2H)-dione(C3),as a novel EGFR—HER2 dual inhibitor in gastric tumors

Gastric cancers are caused primarily due to the activation and amplification of the EGFR or HER2 kinases resulting in cell proliferation,adhesion,angiogenesis,and metastasis.Conventional therapies are ineffective due to the intra-tumoral heterogeneity and concomitant genetic mutations.Hence,dual inhibition strategies are recommended to increase potency and reduce cytotoxicity.In this study,we have conducted computational high-throughput screening of the ChemBridge library followed by in vitro assays and identified novel selective inhibitors that have a dual impediment of EGFR/HER2 kinase activities.Diversity-based High-throughput Virtual Screening(D-HTVS)was used to screen the whole ChemBridge small molecular library against EGFR and HER2.The atomistic molecular dynamic simulation was conducted to understand the dynamics and stability of the protein-ligand complexes.EGFR/HER2 kinase enzymes,KATOIII,and Snu-5 cells were used for in vitro validations.The atomistic Molecular Dynamics simulations followed by solvent-based Gibbs binding free energy calculation of top molecules,identified compound C3(5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]-1H-isoindole-1,3(2H)-dione)to have a good affinity for both EGFR and HER2.The predicted compound,C3,was promising with better binding energy,good binding pose,and optimum interactions with the EGFR and HER2 residues.C3 inhibited EGFR and HER2 kinases with IC50 values of 37.24 and 45.83 nM,respectively.The GI50 values of C3 to inhibit KATOIII and Snu-5 cells were 84.76 and 48.26 nM,respectively.Based on these findings,we conclude that the identified compound C3 showed a conceivable dual inhibitory activity on EGFR/HER2 kinase,and therefore can be considered as a plausible lead-like molecule for treating gastric cancers with minimal side effects,though testing in higher models with pharmacokinetic approach is required.

From the perspective of experimental practice: High-throughput computational screening in photocatalysis

Photocatalysis,a critical strategy for harvesting sunlight to address energy demand and environmental concerns,is underpinned by the discovery of high-performance photocatalysts,thereby how to design photocatalysts is now generating widespread interest in boosting the conversion effi-ciency of solar energy.In the past decade,computational technologies and theoretical simulations have led to a major leap in the development of high-throughput computational screening strategies for novel high-efficiency photocatalysts.In this viewpoint,we started with introducing the challenges of photocatalysis from the view of experimental practice,especially the inefficiency of the traditional“trial and error”method.Sub-sequently,a cross-sectional comparison between experimental and high-throughput computational screening for photocatalysis is presented and discussed in detail.On the basis of the current experimental progress in photocatalysis,we also exemplified the various challenges associated with high-throughput computational screening strategies.Finally,we offered a preferred high-throughput computational screening procedure for pho-tocatalysts from an experimental practice perspective(model construction and screening,standardized experiments,assessment and revision),with the aim of a better correlation of high-throughput simulations and experimental practices,motivating to search for better descriptors.

High-throughput screening of novel TFEB agonists in protecting against acetaminopheninduced liver injury in mice

Macroautophagy(referred to as autophagy hereafter)is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles.Previous studies showed that autophagy protects against acetaminophen(APAP)-induced injury(AILI)via selective removal of damaged mitochondria and APAP protein adducts.The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes.In the present study,we showed that transcription factor EB(TFEB),a master transcription factor for lysosomal biogenesis,was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers.Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI,respectively.Mechanistically,overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2(NRF2)activation to protect against AILI.We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists.Among these agonists,salinomycin,an anticoccidial and antibacterial agent,activated TFEB and protected against AILI in mice.In conclusion,genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

High-throughput screening of single-atom catalysts confined in monolayer black phosphorus for efficient nitrogen reduction reaction

The discovery of metal-nitrogen centers as the active sites for electrolysis has aroused significant interest in utilizing single-atom catalysts for nitrogen reduction reaction(NRR).Properly designed nanostructured catalysts that strongly interact with nitrogen molecules(N_(2))can promote adsorption and activation,thereby resulting in efficient catalysts with high stability,activity,and selectivity.In this study,using density functional theory calculations,we selected monolayer black phosphorus(BP)as the substrate and screened a series of single-atom transition metals confined in tri-coordinated and tetra-coordinated active centers(without and with N dopants)to electro-catalyze NRR.As a result,we have identified two promising candidates(Hf_(1)-N_(1)P_(2)-1 and Tc_(1)-N_(4)),which exhibit not only low overpotentials of 0.56 and 0.49 V but also high thermodynamic and electrochemical stability,as well as good selectivity towards NRR over the competing hydrogen evolution reaction.We also demonstrate the ability of Hf_(1)-N_(1)P_(2)-1 and Tc_(1)-N_(4) to activate and hydrogenate N_(2) by donating electrons and regulating charge transfer.This study not only predicts new BP-based promising catalysts but also provides guidance for the rational design of high-performance NRR electrocatalysts under ambient conditions.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people,leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment.Here,we screened about 1.8 million small molecules against the main protease(M^(pro))and papain like protease(PL^(pro)),two major proteases in severe acute respiratory syndrome-coronavirus 2 genome,and identified 1851M^(pro)inhibitors and 205 PL^(pro)inhibitors with low nmol/l activity of the best hits.Among these inhibitors,eight small molecules showed dual inhibition effects on both M^(pro)and PL^(pro),exhibiting potential as better candidates for COVID-19 treatment.The best inhibitors of each protease were tested in antiviral assay,with over 40%of M^(pro)inhibitors and over 20%of PL^(pro)inhibitors showing high potency in viral inhibition with low cytotoxicity.The X-ray crystal structure of SARS-CoV-2 M^(pro)in complex with its potent inhibitor 4a was determined at 1.8Åresolution.Together with docking assays,our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4–SMAD3–DNA complex

The transforming growth factor-beta(TGFβ)signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment,making anti-TGFβagents a significant area of interest in cancer immunotherapy.However,the clinical translation of current anti-TGFβagents that target upstream cytokines and receptors remains challenging.Therefore,the development of small-molecule inhibitors specifically targeting SMAD4,the downstream master regulator of the TGFβpathway,would offer an alternative approach with significant therapeutic potential for anti-TGFβsignaling.In this study,we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer(TR-FRET)assay in an ultrahigh-throughput screening(uHTS)1536-well plate format.This assay enables simultaneous monitoring of the protein–protein interaction between SMAD4 and SMAD3,as well as the protein–DNA interaction between SMADs and their consensus DNA-binding motif.The multiplexed TR-FRET assay exhibits high sensitivity,allowing the dynamic analysis of the SMAD4–SMAD3–DNA complex at single-amino acid resolution.Moreover,the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors.Through a pilot screening of an FDA-approved bioactive compound library,we identified gambogic acid and gambogenic acid as potential hit compounds.These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4–SMAD3–DNA complex as novel antiTGFβsignaling agents.