Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

Background:In-feed antibiotics are being phased out in livestock production worldwide.Alternatives to antibiotics are urgently needed to maintain animal health and production performance.Host defense peptides(HDPs)are known for their broad-spectrum antimicrobial and immunomodulatory capabilities.Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.Methods:To identify natural products with an ability to stimulate the synthesis of endogenous HDPs,we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc.The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells,3D4/31 macrophages,and jejunal explants were verified using RT-qPCR.Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium(enterotoxigenic E.coli)and a Gram-positive bacterium(Staphylococcus aureus)were further confirmed with four selected HDP-inducing compounds.Results:A total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening,with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment.Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression,showing a minimum 10-fold increase in IPEC-J2,3D4/31 cells,and jejunal explants.Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants,but not 3D4/31 cells.In addition to pBD3,other porcine HDP genes such as pBD2,PG1-5,and pEP2C were induced to different magnitudes by xanthohumol,deoxyshikonin,isorhapontigenin,and calycosin,although clear gene-and cell type-specific patterns of regulation were observed.Desirably,these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes.Furthermore,when used at HDP-inducing concentrations,these compounds showed no obvious direct antibacterial activity,but significantly augmented the antibacterial activity of 3D4/31 macrophages(P<0.05)against both Gram-negative and Gram-positive bacteria.Conclusions:Our results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

High throughput screening of single atomic catalysts with optimized local structures for the electrochemical oxygen reduction by machine learning

Single atomic catalysts(SACs),especially metal-nitrogen doped carbon(M-NC)catalysts,have been extensively explored for the electrochemical oxygen reduction reaction(ORR),owing to their high activity and atomic utilization efficiency.However,there is still a lack of systematic screening and optimization of local structures surrounding active centers of SACs for ORR as the local coordination has an essential impact on their electronic structures and catalytic performance.Herein,we systematic study the ORR catalytic performance of M-NC SACs with different central metals and environmental atoms in the first and second coordination sphere by using density functional theory(DFT)calculation and machine learning(ML).The geometric and electronic informed overpotential model(GEIOM)based on random forest algorithm showed the highest accuracy,and its R^(2) and root mean square errors(RMSE)were 0.96 and 0.21,respectively.30 potential high-performance catalysts were screened out by GEIOM,and the RMSE of the predicted result was only 0.12 V.This work not only helps us fast screen high-performance catalysts,but also provides a low-cost way to improve the accuracy of ML models.

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.

High-throughput computational screening and design of nanoporous materials for methane storage and carbon dioxide capture

The globally increasing concentrations of greenhouse gases in atmosphere after combustion of coal-or petroleum-based fuels give rise to tremendous interest in searching for porous materials to efficiently capture carbon dioxide(CO_2) and store methane(CH4), where the latter is a kind of clean energy source with abundant reserves and lower CO_2 emission. Hundreds of thousands of porous materials can be enrolled on the candidate list, but how to quickly identify the really promising ones, or even evolve materials(namely, rational design high-performing candidates) based on the large database of present porous materials? In this context, high-throughput computational techniques, which have emerged in the past few years as powerful tools, make the targets of fast evaluation of adsorbents and evolving materials for CO_2 capture and CH_4 storage feasible. This review provides an overview of the recent computational efforts on such related topics and discusses the further development in this field.

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.

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.

Self-encoding Functional Resin Applying for Combinatorial Chemistry and High Throughput Screening

A novel solid phase organic synthesis resin was synthesized for combinatorial high-throughput screening,which based on FTIR spectra self-encoding functional resin technology. A new deconvolution strategy termed position encoding deconvolution had illustrated and was compared with some popular combinatorial deconvolution strategies in efficiency and information content. The mimic high throughput screening of hexapeptide library successfully proved the applying of the self-encoding functional resin technology and the position encoding deconvolution strategy.

A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.

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 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.

Xenobiotic effects in cancer related pathways-high throughput screening and proof of concept in animal models

Xenobiotic drugs and chemicals directly interact with DNA,proteins,or other biomolecules in cells. These direct interactions with molecular targets may trigger a series of downstream effects on metabolic-biochemical and regulatory-signaling networks that can invoke cellular consequences leading to adaptive homeostatic or adverse pathological responses. Regulators for drug and chemicals safety have therefore since long required the testing of toxicity in animal models before drugs and pesticides can enter the market. The US National Research Council of the National Academy of Sciences,in its report,Toxicity Testing in the 21st Century: a Vision and a Strategy [1] ,proposed that toxicity testing should become less reliant on apical endpoints from whole animal tests and eventually rely instead on quantitative,doseresponse models based on information from in vitro assays and in vivo biomarkers,which can be used to screen large numbers of chemicals. The present paper reports about a combination of HTS in vitro assays that can be used to study the potential tumorigenic effect of xenobiotics ( drug targets,environmental chemicals) via a set of"sentinel"genes [2] that are functionally interrelated based on evidence weighted functional linkage network ( FLN ) log-likelihood scores ( Linghu et al [3] ) .

Screening and Identification of Myxobacterias with Antifungal Activity

[ Objective] The paper was to screen myxobacteria with antifungal activity, and identify its species. [ Method] 237 myxobacteria were isolated and pu- rified from 200 copies of soil samples collected from 23 provinces in China. The strains were fermented in four different media, and the fermentation products were extracted by methanol to obtain metabolites. With Candida albicans as the model, the secondary metabolites were carried out high-throughput screening. [ Result ] A strain of myxohacteria numbered ZJ2 was obtained after secondary screening, its secondary metabolites had activities against G. albicans. 16S rDNA cloning and sequencing results showed that ZJ2 was Myxococcus fidvus. [ Conclusion ] The secondary metabolites of ZJ2 strain had bioactive substances with significant inhibi- tion effect against the growth of C. albicans, which had potential pharmaceutical research and development value.

基于HTS-ELISA的单克隆抗体分泌杂交瘤细胞筛选技术进展

基于单克隆抗体的各类免疫分析技术是现代食品安全快速检测技术发展的主要方向之一,因此,高活性单克隆抗体的制备方法自然为众多研究者所关注。通常单克隆抗体的制备过程涉及到很多技术环节,包括抗原制备、免疫剂量设计、细胞融合、活性杂交瘤细胞筛选以及抗体纯化等,其中分泌单克隆抗体杂交瘤细胞的筛选环节是单克隆抗体制备的关键一环。为此,本文主要介绍了一种基于HTS-ELISA的高活性单克隆抗体分泌杂交瘤细胞的筛选技术,重点是对融合细胞的低密度培养、特殊仪器设备和在筛选过程中如何依据拖孔数(Trailing)来判断杂交瘤细胞的活性等相关的原理和技术,旨在为我国高活性单克隆抗体制备技术的发展及加快食品安全快速检测技术的发展提供一个参考方法。

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research,the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems.Recently,researchers have been actively developing and evaluating three-dimensional(3D)cell culture-based platforms using microfluidic technologies,such as organ-on-a-chip and organoid-on-a-chip platforms,and they have achieved promising breakthroughs in stem cell engineering.In this review,we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery.In a subsequent section,we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research.In addition,some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

Computational screening of doping schemes for LiTi2(PO4)3 as cathode coating materials

In all-solid-state lithium batteries,the impedance at the cathode/electrolyte interface shows close relationship with the cycle performance.Cathode coatings are helpful to reduce the impedance and increase the stability at the interface effectively.LiTi2(PO4)3(LTP),a fast ion conductor with high ionic conductivity approaching 10^(-3)S·cm^(-1),is adopted as the coating materials in this study.The crystal and electronic structures,as well as the Li^+ion migration properties are evaluated for LTP and its doped derivatives based on density functional theory(DFT)and bond valence(BV)method.Substituting part of Ti sites with element Mn,Fe,or Mg in LTP can improve the electronic conductivity of LTP while does not decrease its high ionic conductivity.In this way,the coating materials with both high ionic conductivities and electronic conductivities can be prepared for all-solid-state lithium batteries to improve the ion and electron transport properties at the interface.

High-throughput theoretical design of lithium battery materials

The rapid evolution of high-throughput theoretical design schemes to discover new lithium battery materials is re- viewed, including fiigh-capacity cathodes, low-strain cathodes, anodes, solid state eleclrolytes, and electrolyte additives. With tfie development of efficient theoretical methods and inexpensive computers, high-throughput theoretical calculations have played an increasingly important role in the discovery of new malerials. With the help of automatic simnlation flow, many types of materials can be screened, optimized and designed from a structural database according to specific search criteria. In advanced cell technology, new materials for next generation lithium batteries are of great significance to achieve perlbmmnce, and some representative criteria are： higher energy density, better safety, and faster charge/discharge speed.

Dynamic microphysiological system chip platform for high-throughput,customizable,and multi-dimensional drug screening

Spheroids and organoids have attracted significant attention as innovative models for disease modeling and drug screening.By employing diverse types of spheroids or organoids,it is feasible to establish microphysiological systems that enhance the precision of disease modeling and offer more dependable and comprehensive drug screening.High-throughput microphysiological systems that support optional,parallel testing of multiple drugs have promising applications in personalized medical treatment and drug research.However,establishing such a system is highly challenging and requires a multidisciplinary approach.This study introduces a dynamic Microphysiological System Chip Platform(MSCP)with multiple functional microstructures that encompass the mentioned advantages.We developed a high-throughput lung cancer spheroids model and an intestine-liverheart-lung cancer microphysiological system for conducting parallel testing on four anti-lung cancer drugs,demonstrating the feasibility of the MSCP.This microphysiological system combines microscale and macroscale biomimetics to enable a comprehensive assessment of drug efficacy and side effects.Moreover,the microphysiological system enables evaluation of the real pharmacological effect of drug molecules reaching the target lesion after absorption by normal organs through fluid-based physiological communication.The MSCP could serves as a valuable platform for microphysiological system research,making significant contributions to disease modeling,drug development,and personalized medical treatment.