High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

PubMed

Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

2014-05-01

The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

Application of ToxCast High-Throughput Screening and ...

EPA Pesticide Factsheets

Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

PubMed Central

Sexton, Jonathan Z; Danshina, Polina V; Lamson, David R; Hughes, Mark; House, Alan J; Yeh, Li-An; O’Brien, Deborah A; Williams, Kevin P

2011-01-01

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents. PMID:21760877

20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

PubMed

Chokhawala, Harshal A; Huang, Shengshu; Lau, Kam; Yu, Hai; Cheng, Jiansong; Thon, Vireak; Hurtado-Ziola, Nancy; Guerrero, Juan A; Varki, Ajit; Chen, Xi

2008-09-19

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.

A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics

NASA Astrophysics Data System (ADS)

Lagus, Todd P.; Edd, Jon F.

2013-03-01

Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.

High-throughput screening (HTS) and modeling of the retinoid ...

EPA Pesticide Factsheets

Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

PubMed

Liu, Yong-Juan; Chen, Xu-Sheng; Zhao, Jun-Jie; Pan, Long; Mao, Zhong-Gui

2017-12-01

ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

Plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT; the details are in the attached slide presentation presentation on plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT, given at the OECD meeting on June 23, 2016

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

PubMed

Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

2015-01-01

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

Compound Transfer by Acoustic Droplet Ejection Promotes Quality and Efficiency in Ultra-High-Throughput Screening Campaigns.

PubMed

Dawes, Timothy D; Turincio, Rebecca; Jones, Steven W; Rodriguez, Richard A; Gadiagellan, Dhireshan; Thana, Peter; Clark, Kevin R; Gustafson, Amy E; Orren, Linda; Liimatta, Marya; Gross, Daniel P; Maurer, Till; Beresini, Maureen H

2016-02-01

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed. © 2015 Society for Laboratory Automation and Screening.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

PubMed

Terekhov, Stanislav S; Smirnov, Ivan V; Stepanova, Anastasiya V; Bobik, Tatyana V; Mokrushina, Yuliana A; Ponomarenko, Natalia A; Belogurov, Alexey A; Rubtsova, Maria P; Kartseva, Olga V; Gomzikova, Marina O; Moskovtsev, Alexey A; Bukatin, Anton S; Dubina, Michael V; Kostryukova, Elena S; Babenko, Vladislav V; Vakhitova, Maria T; Manolov, Alexander I; Malakhova, Maja V; Kornienko, Maria A; Tyakht, Alexander V; Vanyushkina, Anna A; Ilina, Elena N; Masson, Patrick; Gabibov, Alexander G; Altman, Sidney

2017-03-07

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus , and predicted which genera were associated with inhibitory activity.

High Throughput Screening For Hazard and Risk of Environmental Contaminants

EPA Science Inventory

High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis.

PubMed

Chen, Wei; Zhang, Shan; Jiang, Peixia; Yao, Jun; He, Yongzhi; Chen, Lincai; Gui, Xiwu; Dong, Zhiyang; Tang, Shuang-Yan

2015-07-01

Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

Biased ligand quantification in drug discovery: from theory to high throughput screening to identify new biased μ opioid receptor agonists

PubMed Central

Winpenny, David; Clark, Mellissa

2016-01-01

Background and Purpose Biased GPCR ligands are able to engage with their target receptor in a manner that preferentially activates distinct downstream signalling and offers potential for next generation therapeutics. However, accurate quantification of ligand bias in vitro is complex, and current best practice is not amenable for testing large numbers of compound. We have therefore sought to apply ligand bias theory to an industrial scale screening campaign for the identification of new biased μ receptor agonists. Experimental Approach μ receptor assays with appropriate dynamic range were developed for both Gαi‐dependent signalling and β‐arrestin2 recruitment. Δlog(Emax/EC50) analysis was validated as an alternative for the operational model of agonism in calculating pathway bias towards Gαi‐dependent signalling. The analysis was applied to a high throughput screen to characterize the prevalence and nature of pathway bias among a diverse set of compounds with μ receptor agonist activity. Key Results A high throughput screening campaign yielded 440 hits with greater than 10‐fold bias relative to DAMGO. To validate these results, we quantified pathway bias of a subset of hits using the operational model of agonism. The high degree of correlation across these biased hits confirmed that Δlog(Emax/EC50) was a suitable method for identifying genuine biased ligands within a large collection of diverse compounds. Conclusions and Implications This work demonstrates that using Δlog(Emax/EC50), drug discovery can apply the concept of biased ligand quantification on a large scale and accelerate the deliberate discovery of novel therapeutics acting via this complex pharmacology. PMID:26791140

ToxCast Workflow: High-throughput screening assay data processing, analysis and management (SOT)

EPA Science Inventory

US EPA’s ToxCast program is generating data in high-throughput screening (HTS) and high-content screening (HCS) assays for thousands of environmental chemicals, for use in developing predictive toxicity models. Currently the ToxCast screening program includes over 1800 unique c...

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.

PubMed

Hily, Jean-Michel; Liu, Zongrang

2009-03-01

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.

Metabolomics Approach for Toxicity Screening of Volatile Substances

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However, the ch...

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

EPA Science Inventory

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2017-11-01

SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

PubMed

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

PubMed Central

Sharlow, Elizabeth R.; Lyda, Todd A.; Dodson, Heidi C.; Mustata, Gabriela; Morris, Meredith T.; Leimgruber, Stephanie S.; Lee, Kuo-Hsiung; Kashiwada, Yoshiki; Close, David; Lazo, John S.; Morris, James C.

2010-01-01

Background The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay. Methodology/Principal Findings Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC50<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics. Conclusions/Significance The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome. PMID:20405000

Review of microfluidic microbioreactor technology for high-throughput submerged microbiological cultivation

PubMed Central

Hegab, Hanaa M.; ElMekawy, Ahmed; Stakenborg, Tim

2013-01-01

Microbial fermentation process development is pursuing a high production yield. This requires a high throughput screening and optimization of the microbial strains, which is nowadays commonly achieved by applying slow and labor-intensive submerged cultivation in shake flasks or microtiter plates. These methods are also limited towards end-point measurements, low analytical data output, and control over the fermentation process. These drawbacks could be overcome by means of scaled-down microfluidic microbioreactors (μBR) that allow for online control over cultivation data and automation, hence reducing cost and time. This review goes beyond previous work not only by providing a detailed update on the current μBR fabrication techniques but also the operation and control of μBRs is compared to large scale fermentation reactors. PMID:24404006

Lessons from high-throughput protein crystallization screening: 10 years of practical experience

PubMed Central

JR, Luft; EH, Snell; GT, DeTitta

2011-01-01

Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Microfluidics for cell-based high throughput screening platforms - A review.

PubMed

Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

PubMed Central

Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

2012-01-01

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System#

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the cha...

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2016-09-01

Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY CLASSIFICATION OF: 17...developed a method to perform fragment based screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor...cancer, cell-cycle inhibition, activation, modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex.

PubMed

Hao, Ge-Fei; Wang, Fu; Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A; Yang, Guang-Fu

2012-07-11

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.

Droplet microfluidic technology for single-cell high-throughput screening.

PubMed

Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

2009-08-25

We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.

PubMed

Livingstone, Mark; Larsson, Ola; Sukarieh, Rami; Pelletier, Jerry; Sonenberg, Nahum

2009-12-24

The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens. Copyright 2009 Elsevier Ltd. All rights reserved.

From screen to structure with a harvestable microfluidic device.

PubMed

Stojanoff, Vivian; Jakoncic, Jean; Oren, Deena A; Nagarajan, V; Poulsen, Jens-Christian Navarro; Adams-Cioaba, Melanie A; Bergfors, Terese; Sommer, Morten O A

2011-08-01

Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

EPA Science Inventory

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limi...

Evaluating the Impact of Uncertainties in Clearance and Exposure When Prioritizing Chemicals Screened in High-Throughput Assays

EPA Science Inventory

The toxicity-testing paradigm has evolved to include high-throughput (HT) methods for addressing the increasing need to screen hundreds to thousands of chemicals rapidly. Approaches that involve in vitro screening assays, in silico predictions of exposure concentrations, and phar...

Primers of glycosaminoglycan biosynthesis from Peruvian rain forest plants.

PubMed

Taylor, W H; Sinha, A; Khan, I A; McDaniel, S T; Esko, J D

1998-08-28

We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.

Materials Screening for the Discovery of New Half-Heuslers: Machine Learning versus ab Initio Methods.

PubMed

Legrain, Fleur; Carrete, Jesús; van Roekeghem, Ambroise; Madsen, Georg K H; Mingo, Natalio

2018-01-18

Machine learning (ML) is increasingly becoming a helpful tool in the search for novel functional compounds. Here we use classification via random forests to predict the stability of half-Heusler (HH) compounds, using only experimentally reported compounds as a training set. Cross-validation yields an excellent agreement between the fraction of compounds classified as stable and the actual fraction of truly stable compounds in the ICSD. The ML model is then employed to screen 71 178 different 1:1:1 compositions, yielding 481 likely stable candidates. The predicted stability of HH compounds from three previous high-throughput ab initio studies is critically analyzed from the perspective of the alternative ML approach. The incomplete consistency among the three separate ab initio studies and between them and the ML predictions suggests that additional factors beyond those considered by ab initio phase stability calculations might be determinant to the stability of the compounds. Such factors can include configurational entropies and quasiharmonic contributions.

High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

EPA Science Inventory

Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

Uncertainty Quantification in High Throughput Screening: Applications to Models of Endocrine Disruption, Cytotoxicity, and Zebrafish Development (GRC Drug Safety)

EPA Science Inventory

Using uncertainty quantification, we aim to improve the quality of modeling data from high throughput screening assays for use in risk assessment. ToxCast is a large-scale screening program that analyzes thousands of chemicals using over 800 assays representing hundreds of bioche...

High-Throughput Screening and Quantitative Chemical Ranking for Sodium Iodide Symporter Inhibitors in ToxCast Phase 1 Chemical Library

EPA Science Inventory

The U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) and Office of Research and Development (ORD) are currently developing high throughput assays to screen chemicals that may alter the thyroid hormone pathway. One potential target in this pathway is the sodium iodide...

High-Throughput Screening and Quantitative Chemical Ranking for Sodium Iodide Symporter Inhibitors in ToxCast Phase 1 Chemical Library

EPA Science Inventory

The U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) and Office of Research and Development (ORD) are currently developing high throughput assays to screen chemicals that may alter the thyroid hormone pathway. One potential target in this pathway is the sodium iodide sympo...

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR library

PubMed Central

Zhu, Shiyou; Li, Wei; Liu, Jingze; Chen, Chen-Hao; Liao, Qi; Xu, Ping; Xu, Han; Xiao, Tengfei; Cao, Zhongzheng; Peng, Jingyu; Yuan, Pengfei; Brown, Myles; Liu, Xiaole Shirley; Wei, Wensheng

2017-01-01

CRISPR/Cas9 screens have been widely adopted to analyse coding gene functions, but high throughput screening of non-coding elements using this method is more challenging, because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. Herein, we report a high throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We individually validated 9 lncRNAs using CRISPR/Cas9-mediated genomic deletion and functional rescue, CRISPR activation or inhibition, and gene expression profiling. Our high-throughput pgRNA genome deletion method should enable rapid identification of functional mammalian non-coding elements. PMID:27798563

A high-throughput screening approach to discovering good forms of biologically inspired visual representation.

PubMed

Pinto, Nicolas; Doukhan, David; DiCarlo, James J; Cox, David D

2009-11-01

While many models of biological object recognition share a common set of "broad-stroke" properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model--e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit) is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct "parts" have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor). In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision.

A High-Throughput Screening Approach to Discovering Good Forms of Biologically Inspired Visual Representation

PubMed Central

Pinto, Nicolas; Doukhan, David; DiCarlo, James J.; Cox, David D.

2009-01-01

While many models of biological object recognition share a common set of “broad-stroke” properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model—e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit) is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct “parts” have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor). In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision. PMID:19956750

Selecting, Acquiring, and Using Small Molecule Libraries for High-Throughput Screening

PubMed Central

Dandapani, Sivaraman; Rosse, Gerard; Southall, Noel; Salvino, Joseph M.; Thomas, Craig J.

2015-01-01

The selection, acquisition and use of high quality small molecule libraries for screening is an essential aspect of drug discovery and chemical biology programs. Screening libraries continue to evolve as researchers gain a greater appreciation of the suitability of small molecules for specific biological targets, processes and environments. The decisions surrounding the make-up of any given small molecule library is informed by a multitude of variables and opinions vary on best-practices. The fitness of any collection relies upon upfront filtering to avoiding problematic compounds, assess appropriate physicochemical properties, install the ideal level of structural uniqueness and determine the desired extent of molecular complexity. These criteria are under constant evaluation and revision as academic and industrial organizations seek out collections that yield ever improving results from their screening portfolios. Practical questions including cost, compound management, screening sophistication and assay objective also play a significant role in the choice of library composition. This overview attempts to offer advice to all organizations engaged in small molecule screening based upon current best practices and theoretical considerations in library selection and acquisition. PMID:26705509

Selecting, Acquiring, and Using Small Molecule Libraries for High-Throughput Screening.

PubMed

Dandapani, Sivaraman; Rosse, Gerard; Southall, Noel; Salvino, Joseph M; Thomas, Craig J

The selection, acquisition and use of high quality small molecule libraries for screening is an essential aspect of drug discovery and chemical biology programs. Screening libraries continue to evolve as researchers gain a greater appreciation of the suitability of small molecules for specific biological targets, processes and environments. The decisions surrounding the make-up of any given small molecule library is informed by a multitude of variables and opinions vary on best-practices. The fitness of any collection relies upon upfront filtering to avoiding problematic compounds, assess appropriate physicochemical properties, install the ideal level of structural uniqueness and determine the desired extent of molecular complexity. These criteria are under constant evaluation and revision as academic and industrial organizations seek out collections that yield ever improving results from their screening portfolios. Practical questions including cost, compound management, screening sophistication and assay objective also play a significant role in the choice of library composition. This overview attempts to offer advice to all organizations engaged in small molecule screening based upon current best practices and theoretical considerations in library selection and acquisition.

Establishment of integrated protocols for automated high throughput kinetic chlorophyll fluorescence analyses.

PubMed

Tschiersch, Henning; Junker, Astrid; Meyer, Rhonda C; Altmann, Thomas

2017-01-01

Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (Φ PSII ). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of Φ PSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

High-throughput differentiation of heparin from other glycosaminoglycans by pyrolysis mass spectrometry.

PubMed

Nemes, Peter; Hoover, William J; Keire, David A

2013-08-06

Sensors with high chemical specificity and enhanced sample throughput are vital to screening food products and medical devices for chemical or biochemical contaminants that may pose a threat to public health. For example, the rapid detection of oversulfated chondroitin sulfate (OSCS) in heparin could prevent reoccurrence of heparin adulteration that caused hundreds of severe adverse events including deaths worldwide in 2007-2008. Here, rapid pyrolysis is integrated with direct analysis in real time (DART) mass spectrometry to rapidly screen major glycosaminoglycans, including heparin, chondroitin sulfate A, dermatan sulfate, and OSCS. The results demonstrate that, compared to traditional liquid chromatography-based analyses, pyrolysis mass spectrometry achieved at least 250-fold higher sample throughput and was compatible with samples volume-limited to about 300 nL. Pyrolysis yielded an abundance of fragment ions (e.g., 150 different m/z species), many of which were specific to the parent compound. Using multivariate and statistical data analysis models, these data enabled facile differentiation of the glycosaminoglycans with high throughput. After method development was completed, authentically contaminated samples obtained during the heparin crisis by the FDA were analyzed in a blinded manner for OSCS contamination. The lower limit of differentiation and detection were 0.1% (w/w) OSCS in heparin and 100 ng/μL (20 ng) OSCS in water, respectively. For quantitative purposes the linear dynamic range spanned approximately 3 orders of magnitude. Moreover, this chemical readout was successfully employed to find clues in the manufacturing history of the heparin samples that can be used for surveillance purposes. The presented technology and data analysis protocols are anticipated to be readily adaptable to other chemical and biochemical agents and volume-limited samples.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

PubMed

Yoshii, Yukie; Furukawa, Takako; Waki, Atsuo; Okuyama, Hiroaki; Inoue, Masahiro; Itoh, Manabu; Zhang, Ming-Rong; Wakizaka, Hidekatsu; Sogawa, Chizuru; Kiyono, Yasushi; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2015-05-01

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis of d‐ and l‐Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process†

PubMed Central

Parmeggiani, Fabio; Lovelock, Sarah L.; Weise, Nicholas J.; Ahmed, Syed T.

2015-01-01

Abstract The synthesis of substituted d‐phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one‐pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high‐throughput solid‐phase screening method has also been developed to identify PALs with higher rates of formation of non‐natural d‐phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d‐configured product. Furthermore, the system was extended to the preparation of those l‐phenylalanines which are obtained with a low ee value using PAL amination. PMID:27478261

Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process.

PubMed

Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

2015-04-07

The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee  value using PAL amination.

Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process**

PubMed Central

Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

2015-01-01

The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination. PMID:25728350

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Tox21 Enricher: Web-based Chemical/Biological Functional Annotation Analysis Tool Based on Tox21 Toxicity Screening Platform.

PubMed

Hur, Junguk; Danes, Larson; Hsieh, Jui-Hua; McGregor, Brett; Krout, Dakota; Auerbach, Scott

2018-05-01

The US Toxicology Testing in the 21st Century (Tox21) program was established to develop more efficient and human-relevant toxicity assessment methods. The Tox21 program screens >10,000 chemicals using quantitative high-throughput screening (qHTS) of assays that measure effects on toxicity pathways. To date, more than 70 assays have yielded >12 million concentration-response curves. The patterns of activity across assays can be used to define similarity between chemicals. Assuming chemicals with similar activity profiles have similar toxicological properties, we may infer toxicological properties based on its neighbourhood. One approach to inference is chemical/biological annotation enrichment analysis. Here, we present Tox21 Enricher, a web-based chemical annotation enrichment tool for the Tox21 toxicity screening platform. Tox21 Enricher identifies over-represented chemical/biological annotations among lists of chemicals (neighbourhoods), facilitating the identification of the toxicological properties and mechanisms in the chemical set. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops.

PubMed

Wasson, A P; Richards, R A; Chatrath, R; Misra, S C; Prasad, S V Sai; Rebetzke, G J; Kirkegaard, J A; Christopher, J; Watt, M

2012-05-01

Wheat yields globally will depend increasingly on good management to conserve rainfall and new varieties that use water efficiently for grain production. Here we propose an approach for developing new varieties to make better use of deep stored water. We focus on water-limited wheat production in the summer-dominant rainfall regions of India and Australia, but the approach is generally applicable to other environments and root-based constraints. Use of stored deep water is valuable because it is more predictable than variable in-season rainfall and can be measured prior to sowing. Further, this moisture is converted into grain with twice the efficiently of in-season rainfall since it is taken up later in crop growth during the grain-filling period when the roots reach deeper layers. We propose that wheat varieties with a deeper root system, a redistribution of branch root density from the surface to depth, and with greater radial hydraulic conductivity at depth would have higher yields in rainfed systems where crops rely on deep water for grain fill. Developing selection systems for mature root system traits is challenging as there are limited high-throughput phenotyping methods for roots in the field, and there is a risk that traits selected in the lab on young plants will not translate into mature root system traits in the field. We give an example of a breeding programme that combines laboratory and field phenotyping with proof of concept evaluation of the trait at the beginning of the selection programme. This would greatly enhance confidence in a high-throughput laboratory or field screen, and avoid investment in screens without yield value. This approach requires careful selection of field sites and years that allow expression of deep roots and increased yield. It also requires careful selection and crossing of germplasm to allow comparison of root expression among genotypes that are similar for other traits, especially flowering time and disease and toxicity resistances. Such a programme with field and laboratory evaluation at the outset will speed up delivery of varieties with improved root systems for higher yield.

AOPs & Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making.

EPA Science Inventory

As high throughput screening (HTS) approaches play a larger role in toxicity testing, computational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models for this purpose are becoming increasingly more sophisticated...

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization

PubMed Central

Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; Wan, Frank; Sheraden, Paige N.; Broecker, Jana; Ernst, Oliver P.; Gennis, Robert B.

2017-01-01

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å. PMID:28469762

Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

PubMed

Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

2015-01-01

Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies.

High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

PubMed Central

Rebbeck, Robyn T.; Essawy, Maram M.; Nitu, Florentin R.; Grant, Benjamin D.; Gillispie, Gregory D.; Thomas, David D.; Bers, Donald M.; Cornea, Razvan L.

2017-01-01

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3SD. Dose-response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent z′-factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators. PMID:27760856

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers*

PubMed Central

Faries, Kaitlyn M.; Kressel, Lucas L.; Wander, Marc J.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine; Hanson, Deborah K.

2012-01-01

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization. PMID:22247556

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Robust high-throughput batch screening method in 384-well format with optical in-line resin quantification.

PubMed

Kittelmann, Jörg; Ottens, Marcel; Hubbuch, Jürgen

2015-04-15

High-throughput batch screening technologies have become an important tool in downstream process development. Although continuative miniaturization saves time and sample consumption, there is yet no screening process described in the 384-well microplate format. Several processes are established in the 96-well dimension to investigate protein-adsorbent interactions, utilizing between 6.8 and 50 μL resin per well. However, as sample consumption scales with resin volumes and throughput scales with experiments per microplate, they are limited in costs and saved time. In this work, a new method for in-well resin quantification by optical means, applicable in the 384-well format, and resin volumes as small as 0.1 μL is introduced. A HTS batch isotherm process is described, utilizing this new method in combination with optical sample volume quantification for screening of isotherm parameters in 384-well microplates. Results are qualified by confidence bounds determined by bootstrap analysis and a comprehensive Monte Carlo study of error propagation. This new approach opens the door to a variety of screening processes in the 384-well format on HTS stations, higher quality screening data and an increase in throughput. Copyright © 2015 Elsevier B.V. All rights reserved.

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

PubMed

Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

2018-05-01

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

Quality control methodology for high-throughput protein-protein interaction screening.

PubMed

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast

EPA Science Inventory

High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

Development of a thyroperoxidase inhibition assay for high-throughput screening

EPA Science Inventory

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

High-throughput screening, predictive modeling and computational embryology - Abstract

EPA Science Inventory

High-throughput screening (HTS) studies are providing a rich source of data that can be applied to chemical profiling to address sensitivity and specificity of molecular targets, biological pathways, cellular and developmental processes. EPA’s ToxCast project is testing 960 uniq...

Picking Cell Lines for High-Throughput Transcriptomic Toxicity Screening (SOT)

EPA Science Inventory

High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captu...

Quantitative description on structure-property relationships of Li-ion battery materials for high-throughput computations

NASA Astrophysics Data System (ADS)

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-12-01

Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure-property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure-property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure-property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials.

Quantitative description on structure-property relationships of Li-ion battery materials for high-throughput computations.

PubMed

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-01-01

Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure-property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure-property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure-property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials.

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

PubMed

Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

2017-06-01

The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

PubMed

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Advanced phenotyping and phenotype data analysis for the study of plant growth and development.

PubMed

Rahaman, Md Matiur; Chen, Dijun; Gillani, Zeeshan; Klukas, Christian; Chen, Ming

2015-01-01

Due to an increase in the consumption of food, feed, fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to breed high yielding crops that can adapt to the future climate changes, particularly in developing countries. To solve these global challenges, novel approaches are required to identify quantitative phenotypes and to explain the genetic basis of agriculturally important traits. These advances will facilitate the screening of germplasm with high performance characteristics in resource-limited environments. Recently, plant phenomics has offered and integrated a suite of new technologies, and we are on a path to improve the description of complex plant phenotypes. High-throughput phenotyping platforms have also been developed that capture phenotype data from plants in a non-destructive manner. In this review, we discuss recent developments of high-throughput plant phenotyping infrastructure including imaging techniques and corresponding principles for phenotype data analysis.

Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology.

PubMed

Wang, Jing; Fang, Pengfei; Chase, Peter; Tshori, Sagi; Razin, Ehud; Spicer, Timothy P; Scampavia, Louis; Hodder, Peter; Guo, Min

2017-01-01

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z' factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms.

PubMed

Kosa, Gergely; Vuoristo, Kiira S; Horn, Svein Jarle; Zimmermann, Boris; Afseth, Nils Kristian; Kohler, Achim; Shapaval, Volha

2018-06-01

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet

EPA Pesticide Factsheets

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet (Presented by Dr. Marilyn J. Aardema, Chief Scientific Advisor, Toxicology, Dr. Leon Stankowski, et. al. (6/28/2012)

Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

EPA Science Inventory

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multip...

Environmental Impact on Vascular Development Predicted by High Throughput Screening

EPA Science Inventory

Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High throughput screening (HTS) in EPA’s ToxCastTM project provides vast d...

AOPs and Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making

EPA Science Inventory

As high throughput screening (HTS) plays a larger role in toxicity testing, camputational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models designed to quantify potential adverse effects based on HTS data will b...

tcpl: The ToxCast Pipeline for High-Throughput Screening Data

EPA Science Inventory

Motivation: The large and diverse high-throughput chemical screening efforts carried out by the US EPAToxCast program requires an efficient, transparent, and reproducible data pipeline.Summary: The tcpl R package and its associated MySQL database provide a generalized platform fo...

High-throughput screening, predictive modeling and computational embryology

EPA Science Inventory

High-throughput screening (HTS) studies are providing a rich source of data that can be applied to profile thousands of chemical compounds for biological activity and potential toxicity. EPA’s ToxCast™ project, and the broader Tox21 consortium, in addition to projects worldwide,...

Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

EPA Science Inventory

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, c...

Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model

NASA Astrophysics Data System (ADS)

Mondal, Sudip; Hegarty, Evan; Martin, Chris; Gökçe, Sertan Kutal; Ghorashian, Navid; Ben-Yakar, Adela

2016-10-01

Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ~4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ~100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ~1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

High-throughput screening and small animal models, where are we?

PubMed Central

Giacomotto, Jean; Ségalat, Laurent

2010-01-01

Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day. PMID:20423335

Developing and validating predictive decision tree models from mining chemical structural fingerprints and high-throughput screening data in PubChem.

PubMed

Han, Lianyi; Wang, Yanli; Bryant, Stephen H

2008-09-25

Recent advances in high-throughput screening (HTS) techniques and readily available compound libraries generated using combinatorial chemistry or derived from natural products enable the testing of millions of compounds in a matter of days. Due to the amount of information produced by HTS assays, it is a very challenging task to mine the HTS data for potential interest in drug development research. Computational approaches for the analysis of HTS results face great challenges due to the large quantity of information and significant amounts of erroneous data produced. In this study, Decision Trees (DT) based models were developed to discriminate compound bioactivities by using their chemical structure fingerprints provided in the PubChem system http://pubchem.ncbi.nlm.nih.gov. The DT models were examined for filtering biological activity data contained in four assays deposited in the PubChem Bioassay Database including assays tested for 5HT1a agonists, antagonists, and HIV-1 RT-RNase H inhibitors. The 10-fold Cross Validation (CV) sensitivity, specificity and Matthews Correlation Coefficient (MCC) for the models are 57.2 approximately 80.5%, 97.3 approximately 99.0%, 0.4 approximately 0.5 respectively. A further evaluation was also performed for DT models built for two independent bioassays, where inhibitors for the same HIV RNase target were screened using different compound libraries, this experiment yields enrichment factor of 4.4 and 9.7. Our results suggest that the designed DT models can be used as a virtual screening technique as well as a complement to traditional approaches for hits selection.

Identification of Novel Synthetic Toll-like Receptor 2 Agonists by High Throughput Screening*

PubMed Central

Guan, Yue; Omueti-Ayoade, Katherine; Mutha, Sarita K.; Hergenrother, Paul J.; Tapping, Richard I.

2010-01-01

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit. PMID:20504771

Evaluation of Compatibility of ToxCast High-Throughput/High-Content Screening Assays with Engineered Nanomaterials

EPA Science Inventory

High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

EPA Science Inventory

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

EPA Science Inventory

Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

PubMed

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

PubMed

Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

2018-01-01

Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation

PubMed Central

Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G.; Garraway, Levi A.; Struhl, Kevin

2015-01-01

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment. PMID:25902495

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

PubMed

Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G; Garraway, Levi A; Struhl, Kevin

2015-05-05

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Synthesis of D- and L-phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process.

PubMed

Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

2015-04-07

The synthesis of substituted D-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural D-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D-configured product. Furthermore, the system was extended to the preparation of those L-phenylalanines which are obtained with a low ee value using PAL amination. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

PubMed Central

Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

2014-01-01

The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

High Throughput, High Content Screening for Novel Pigmentation Regulators Using a Keratinocyte/Melanocyte Co-culture System

PubMed Central

Lee, Ju Hee; Chen, Hongxiang; Kolev, Vihren; Aull, Katherine H.; Jung, Inhee; Wang, Jun; Miyamoto, Shoko; Hosoi, Junichi; Mandinova, Anna; Fisher, David E.

2014-01-01

Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of MITF and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4,000 screened compounds including zoxazolamine, 3-methoxycatechol, and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors, and are worthy of further evaluation for potential translation to clinical use. PMID:24438532

Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges

PubMed Central

Montanez-Sauri, Sara I.; Beebe, David J.; Sung, Kyung Eun

2015-01-01

The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g. spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments. PMID:25274061

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitablymore » designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.« less

Identifying Toxicity Pathways with ToxCast High-Throughput Screening and Applications to Predicting Developmental Toxicity

EPA Science Inventory

Results from rodent and non-rodent prenatal developmental toxicity tests for over 300 chemicals have been curated into the relational database ToxRefDB. These same chemicals have been run in concentration-response format through over 500 high-throughput screening assays assessin...

SeqAPASS to evaluate conservation of high-throughput screening targets across non-mammalian species

EPA Science Inventory

Cell-based high-throughput screening (HTS) and computational technologies are being applied as tools for toxicity testing in the 21st century. The U.S. Environmental Protection Agency (EPA) embraced these technologies and created the ToxCast Program in 2007, which has served as a...

Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

EPA Science Inventory

There are thousands of chemicals that are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The ToxCast high-throughput screening (HTS) program has evaluated over 1,800 chemicals in concentration-response across ~8...

Application of Physiologically-Based Pharmacokinetic/Pharmacodynamic Model for Interpretation of High-throughput Screening Assay for Thyroperoxidase Inhibition

EPA Science Inventory

In vitro based assays are used to identify potential endocrine disrupting chemicals. Thyroperoxidase (TPO), an enzyme essential for thyroid hormone (TH) synthesis, is a target site for disruption of the thyroid axis for which a high-throughput screening (HTPS) assay has recently ...

Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

EPA Science Inventory

The EPA ToxCast research program uses high throughput screening for bioactivity profiling and predicting the toxicity of large numbers of chemicals. ToxCast Phase‐I tested 309 well‐characterized chemicals in over 500 assays for a wide range of molecular targets and cellular respo...

Neural Progenitor Cells as Models for High-Throughput Screens of Developmental Neurotoxicity: State of the Science

EPA Science Inventory

In vitro, high-throughput approaches have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramificat...

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

EPA Science Inventory

AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

EPA Science Inventory

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants.

PubMed

Buckner, Diana; Wilson, Suzanne; Kurk, Sandra; Hardy, Michele; Miessner, Nicole; Jutila, Mark A

2006-09-01

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.

Novel Acoustic Loading of a Mass Spectrometer: Toward Next-Generation High-Throughput MS Screening.

PubMed

Sinclair, Ian; Stearns, Rick; Pringle, Steven; Wingfield, Jonathan; Datwani, Sammy; Hall, Eric; Ghislain, Luke; Majlof, Lars; Bachman, Martin

2016-02-01

High-throughput, direct measurement of substrate-to-product conversion by label-free detection, without the need for engineered substrates or secondary assays, could be considered the "holy grail" of drug discovery screening. Mass spectrometry (MS) has the potential to be part of this ultimate screening solution, but is constrained by the limitations of existing MS sample introduction modes that cannot meet the throughput requirements of high-throughput screening (HTS). Here we report data from a prototype system (Echo-MS) that uses acoustic droplet ejection (ADE) to transfer femtoliter-scale droplets in a rapid, precise, and accurate fashion directly into the MS. The acoustic source can load samples into the MS from a microtiter plate at a rate of up to three samples per second. The resulting MS signal displays a very sharp attack profile and ions are detected within 50 ms of activation of the acoustic transducer. Additionally, we show that the system is capable of generating multiply charged ion species from simple peptides and large proteins. The combination of high speed and low sample volume has significant potential within not only drug discovery, but also other areas of the industry. © 2015 Society for Laboratory Automation and Screening.

Break-up of droplets in a concentrated emulsion flowing through a narrow constriction

NASA Astrophysics Data System (ADS)

Kim, Minkyu; Rosenfeld, Liat; Tang, Sindy; Tang Lab Team

2014-11-01

Droplet microfluidics has enabled a wide range of high throughput screening applications. Compared with other technologies such as robotic screening technology, droplet microfluidics has 1000 times higher throughput, which makes the technology one of the most promising platforms for the ultrahigh throughput screening applications. Few studies have considered the throughput of the droplet interrogation process, however. In this research, we show that the probability of break-up increases with increasing flow rate, entrance angle to the constriction, and size of the drops. Since single drops do not break at the highest flow rate used in the system, break-ups occur primarily from the interactions between highly packed droplets close to each other. Moreover, the probabilistic nature of the break-up process arises from the stochastic variations in the packing configuration. Our results can be used to calculate the maximum throughput of the serial interrogation process. For 40 pL-drops, the highest throughput with less than 1% droplet break-up was measured to be approximately 7,000 drops per second. In addition, the results are useful for understanding the behavior of concentrated emulsions in applications such as mobility control in enhanced oil recovery.

Quantitative description on structure–property relationships of Li-ion battery materials for high-throughput computations

PubMed Central

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-01-01

Abstract Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure–property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure–property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure–property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials. PMID:28458737

Creation of a small high-throughput screening facility.

PubMed

Flak, Tod

2009-01-01

The creation of a high-throughput screening facility within an organization is a difficult task, requiring a substantial investment of time, money, and organizational effort. Major issues to consider include the selection of equipment, the establishment of data analysis methodologies, and the formation of a group having the necessary competencies. If done properly, it is possible to build a screening system in incremental steps, adding new pieces of equipment and data analysis modules as the need grows. Based upon our experience with the creation of a small screening service, we present some guidelines to consider in planning a screening facility.

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

PubMed

Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

2015-10-01

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

Diastereoselective synthesis of L: -threo-3,4-dihydroxyphenylserine by low-specific L: -threonine aldolase mutants.

PubMed

Gwon, Hui-Jeong; Baik, Sang-Ho

2010-01-01

Diastereoselectivity-enhanced mutants of L: -threonine aldolase (L: -TA) for L: -threo-3,4-dihydroxyphenylserine (L: -threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type L: -TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg L: -threo-DOPS/ml.

Low-cost fluorimetric determination of radicals based on fluorogenic dimerization of the natural phenol sesamol.

PubMed

Makino, Yumi; Uchiyama, Seiichi; Ohno, Ken-ichi; Arakawa, Hidetoshi

2010-02-15

A novel fluorimetric method for determining radicals using the natural phenol sesamol as a fluorogenic reagent is reported. In this assay, sesamol was reacted with aqueous radicals to yield one isomer of a sesamol dimer exclusively. The dimer emitted purple fluorescence near 400 nm around neutral pH, where it assumed the monoanionic form. This method was applied to the straightforward detection of radical nitric oxide (NO). The ready availability of sesamol should enable rapid implementation of applications utilizing this new assay, particularly in high-throughput analysis or screening.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

tcpl: the ToxCast pipeline for high-throughput screening data.

PubMed

Filer, Dayne L; Kothiya, Parth; Setzer, R Woodrow; Judson, Richard S; Martin, Matthew T

2017-02-15

Large high-throughput screening (HTS) efforts are widely used in drug development and chemical toxicity screening. Wide use and integration of these data can benefit from an efficient, transparent and reproducible data pipeline. Summary: The tcpl R package and its associated MySQL database provide a generalized platform for efficiently storing, normalizing and dose-response modeling of large high-throughput and high-content chemical screening data. The novel dose-response modeling algorithm has been tested against millions of diverse dose-response series, and robustly fits data with outliers and cytotoxicity-related signal loss. tcpl is freely available on the Comprehensive R Archive Network under the GPL-2 license. martin.matt@epa.gov. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.

Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

EPA Science Inventory

Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

An industrial engineering approach to laboratory automation for high throughput screening

PubMed Central

Menke, Karl C.

2000-01-01

Across the pharmaceutical industry, there are a variety of approaches to laboratory automation for high throughput screening. At Sphinx Pharmaceuticals, the principles of industrial engineering have been applied to systematically identify and develop those automated solutions that provide the greatest value to the scientists engaged in lead generation. PMID:18924701

Evaluation of High-throughput Genotoxicity Assays Used in Profiling the US EPA ToxCast Chemicals

EPA Science Inventory

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Pha...

Collaborative Core Research Program for Chemical-Biological Warfare Defense

DTIC Science & Technology

2015-01-04

Discovery through High Throughput Screening (HTS) and Fragment-Based Drug Design (FBDD...Discovery through High Throughput Screening (HTS) and Fragment-Based Drug Design (FBDD) Current pharmaceutical approaches involving drug discovery...structural analysis and docking program generally known as fragment based drug design (FBDD). The main advantage of using these approaches is that

PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

EPA Science Inventory

Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

Detecting and overcoming systematic bias in high-throughput screening technologies: a comprehensive review of practical issues and methodological solutions.

PubMed

Caraus, Iurie; Alsuwailem, Abdulaziz A; Nadon, Robert; Makarenkov, Vladimir

2015-11-01

Significant efforts have been made recently to improve data throughput and data quality in screening technologies related to drug design. The modern pharmaceutical industry relies heavily on high-throughput screening (HTS) and high-content screening (HCS) technologies, which include small molecule, complementary DNA (cDNA) and RNA interference (RNAi) types of screening. Data generated by these screening technologies are subject to several environmental and procedural systematic biases, which introduce errors into the hit identification process. We first review systematic biases typical of HTS and HCS screens. We highlight that study design issues and the way in which data are generated are crucial for providing unbiased screening results. Considering various data sets, including the publicly available ChemBank data, we assess the rates of systematic bias in experimental HTS by using plate-specific and assay-specific error detection tests. We describe main data normalization and correction techniques and introduce a general data preprocessing protocol. This protocol can be recommended for academic and industrial researchers involved in the analysis of current or next-generation HTS data. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

PubMed

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Medicinal chemistry inspired fragment-based drug discovery.

PubMed

Lanter, James; Zhang, Xuqing; Sui, Zhihua

2011-01-01

Lead generation can be a very challenging phase of the drug discovery process. The two principal methods for this stage of research are blind screening and rational design. Among the rational or semirational design approaches, fragment-based drug discovery (FBDD) has emerged as a useful tool for the generation of lead structures. It is particularly powerful as a complement to high-throughput screening approaches when the latter failed to yield viable hits for further development. Engagement of medicinal chemists early in the process can accelerate the progression of FBDD efforts by incorporating drug-friendly properties in the earliest stages of the design process. Medium-chain acyl-CoA synthetase 2b and ketohexokinase are chosen as examples to illustrate the importance of close collaboration of medicinal chemists, crystallography, and modeling. Copyright © 2011 Elsevier Inc. All rights reserved.

False negative rates in Drosophila cell-based RNAi screens: a case study

PubMed Central

2011-01-01

Background High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. Results We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. Conclusions RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully. PMID:21251254

Rapid Screening of Ergot Alkaloids in Sclerotia by MALDI-TOF Mass Spectrometry.

PubMed

Sivagnanam, Kumaran; Komatsu, Emy; Patrick, Susan; Rampitsch, Christoph; Perreault, Hélène; Gräfenhan, Tom

2016-07-01

Ergot is a common disease of wheat and other cereal grains that is predominantly caused by Claviceps purpurea in the field, often affecting crop yield in addition to the environment. Infected grain can be contaminated with dark sclerotia, which contain fungal metabolites such as ergot alkaloids. The occurrence of ergot alkaloids in cereal grain is a major health concern for humans and livestock. Effective and rapid screening of these mycotoxins is crucial for producers, processors, and consumers of cereal-based food and feed grain. Established methods of ergot alkaloid screening based on LC-MS or GC-MS require laborious processes. A novel method using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS was developed to identify four ergot alkaloids. Using dihydroxybenzoic acid as the matrix, ergosine, ergocornine, ergocryptine, and ergocristine were readily detected in individual sclerotia of C. purpurea. The accuracy of the identified ergot alkaloids was further confirmed by tandem MS analysis. MALDI-TOF MS is suitable for high-throughput screening of ergot alkaloids because it permits rapid and accurate identification, simple sample preparation, and no derivatization or chromatographic separation.

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

PubMed Central

Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

2010-01-01

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

Development of a colourimetric assay for glycosynthases.

PubMed

Hayes, Marc R; Bochinsky, Kevin A; Seibt, Lisa S; Elling, Lothar; Pietruszka, Jörg

2017-09-10

The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus. Copyright © 2017 Elsevier B.V. All rights reserved.

A Perspective on the Future of High-Throughput RNAi Screening: Will CRISPR Cut Out the Competition or Can RNAi Help Guide the Way?

PubMed

Taylor, Jessica; Woodcock, Simon

2015-09-01

For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA-Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery. © 2015 Society for Laboratory Automation and Screening.

High throughput and miniaturised systems for biodegradability assessments.

PubMed

Cregut, Mickael; Jouanneau, Sulivan; Brillet, François; Durand, Marie-José; Sweetlove, Cyril; Chenèble, Jean-Charles; L'Haridon, Jacques; Thouand, Gérald

2014-01-01

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

Expert Opinion Editorial Tissue Engineered Blood Vessels as Promising Tools for Testing Drug Toxicity

PubMed Central

Truskey, George A.; Fernandez, Cristina E.

2015-01-01

Drug-induced vascular injury (DIVI) is a serious problem in preclinical studies of vasoactive molecules and for survivors of pediatric cancers. DIVI is often observed in rodents and some larger animals, primarily with drugs affecting vascular tone, but not in humans; however, DIVI observed in animal studies often precludes a drug candidate from continuing along the development pipeline. Thus, there is great interest by the pharmaceutical industry to identify quantifiable human biomarkers of DIVI. Small scale endothelialized tissue-engineered blood vessels using human cells represent a promising approach to screen drug candidates and developed alternatives to cancer therapeutics in vitro. We identify several technical challenges that remain to be addressed, including high throughput systems to screen large numbers of candidates, identification of suitable cell sources, and establishing and maintaining a differentiated state of the vessel wall cells. Adequately addressing these challenges should yield novel platforms to screen drugs and develop new therapeutics to treat cardiovascular disease. PMID:26028128

A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia

PubMed Central

Bray, Walter M.; White, Pamela M.; Ruybal, Jordan; Lokey, R. Scott; Debec, Alain; Sullivan, William

2012-01-01

Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. PMID:23028321

Genome-Wide RNAi Ionomics Screen Reveals New Genes and Regulation of Human Trace Element Metabolism

PubMed Central

Malinouski, Mikalai; Hasan, Nesrin M.; Zhang, Yan; Seravalli, Javier; Lin, Jie; Avanesov, Andrei; Lutsenko, Svetlana; Gladyshev, Vadim N.

2017-01-01

Trace elements are essential for human metabolism and dysregulation of their homeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires machinery involved in protein trafficking and posttranslational modifications; and the iron levels are influenced by iron import and expression of the iron/heme-containing enzymes. Our approach can be applied to a variety of disease models and/or nutritional conditions, and the generated dataset opens new directions for studies of human trace element metabolism. PMID:24522796

Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.

PubMed

Mei, Feng; Fancy, Stephen P J; Shen, Yun-An A; Niu, Jianqin; Zhao, Chao; Presley, Bryan; Miao, Edna; Lee, Seonok; Mayoral, Sonia R; Redmond, Stephanie A; Etxeberria, Ainhoa; Xiao, Lan; Franklin, Robin J M; Green, Ari; Hauser, Stephen L; Chan, Jonah R

2014-08-01

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

Large-scale Topographical Screen for Investigation of Physical Neural-Guidance Cues

NASA Astrophysics Data System (ADS)

Li, Wei; Tang, Qing Yuan; Jadhav, Amol D.; Narang, Ankit; Qian, Wei Xian; Shi, Peng; Pang, Stella W.

2015-03-01

A combinatorial approach was used to present primary neurons with a large library of topographical features in the form of micropatterned substrate for high-throughput screening of physical neural-guidance cues that can effectively promote different aspects of neuronal development, including axon and dendritic outgrowth. Notably, the neuronal-guidance capability of specific features was automatically identified using a customized image processing software, thus significantly increasing the screening throughput with minimal subjective bias. Our results indicate that the anisotropic topographies promote axonal and in some cases dendritic extension relative to the isotropic topographies, while dendritic branching showed preference to plain substrates over the microscale features. The results from this work can be readily applied towards engineering novel biomaterials with precise surface topography that can serve as guidance conduits for neuro-regenerative applications. This novel topographical screening strategy combined with the automated processing capability can also be used for high-throughput screening of chemical or genetic regulatory factors in primary neurons.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

PubMed

Weimar, M R; Cheung, J; Dey, D; McSweeney, C; Morrison, M; Kobayashi, Y; Whitman, W B; Carbone, V; Schofield, L R; Ronimus, R S; Cook, G M

2017-08-01

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions. IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions. Copyright © 2017 American Society for Microbiology.

Improvement of High-throughput Genotype Analysis After Implementation of a Dual-curve Sybr Green I-based Quantification and Normalization Procedure

USDA-ARS?s Scientific Manuscript database

The ability to rapidly screen a large number of individuals is the key to any successful plant breeding program. One of the primary bottlenecks in high throughput screening is the preparation of DNA samples, particularly the quantification and normalization of samples for downstream processing. A ...

High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

EPA Science Inventory

Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

Use of FRTL-5 Cell Line as a Complementary Assay for Chemicals Identified During High-Throughput Screening as Sodium/Iodide Symporter (NIS) Inhibitors

EPA Science Inventory

Confirmation of Test Chemicals Identified by a High-Throughput Screen (HTPS) as Sodium Iodide Symporter (NIS) Inhibitors in FRTL-5 Model S. Laws1, A. Buckalew1, J. Wang2, D. Hallinger1, A. Murr1, and T. Stoker1. 1Endocrin...

Computational Toxicology as Implemented by the U.S. EPA: Providing High Throughput Decision Support Tools for Screening and Assessing Chemical Exposure, Hazard and Risk

EPA Science Inventory

Computational toxicology is the application of mathematical and computer models to help assess chemical hazards and risks to human health and the environment. Supported by advances in informatics, high-throughput screening (HTS) technologies, and systems biology, the U.S. Environ...

Use of Threshold of Toxicological Concern (TTC) with High Throughput Exposure Predictions as a Risk-Based Screening Approach of Several Thousand Commodity Chemicals (SOT Poster)

EPA Science Inventory

Although progress has been made with HTS (high throughput screening) in profiling biological activity (e.g., EPA’s ToxCast™), challenges arise interpreting HTS results in the context of adversity & converting HTS assay concentrations to equivalent human doses for the broad domain...

Probe molecules (PrM) approach in adverse outcome pathway (AOP) based high throughput screening (HTS): in vivo discovery for developing in vitro target methods

EPA Science Inventory

Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by rel...

Using adverse outcome pathway analysis to guide development of high-throughput screening assays for thyroid-disruptors

EPA Science Inventory

Using Adverse Outcome Pathway Analysis to Guide Development of High-Throughput Screening Assays for Thyroid-Disruptors Katie B. Paul1,2, Joan M. Hedge2, Daniel M. Rotroff4, Kevin M. Crofton4, Michael W. Hornung3, Steven O. Simmons2 1Oak Ridge Institute for Science Education Post...

Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries.

PubMed

Jiang, Rongzhong

2007-07-01

An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10 to 20 mAcm(2). The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150 mAcm(2), respectively.

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening.

PubMed

Brengdahl, Johan; Fowler, Christopher J

2006-12-01

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.

Multi-pathway exposure modeling of chemicals in cosmetics with application to shampoo.

PubMed

Ernstoff, Alexi S; Fantke, Peter; Csiszar, Susan A; Henderson, Andrew D; Chung, Susie; Jolliet, Olivier

2016-01-01

We present a novel multi-pathway, mass balance based, fate and exposure model compatible with life cycle and high-throughput screening assessments of chemicals in cosmetic products. The exposures through product use as well as post-use emissions and environmental media were quantified based on the chemical mass originally applied via a product, multiplied by the product intake fractions (PiF, the fraction of a chemical in a product that is taken in by exposed persons) to yield intake rates. The average PiFs for the evaluated chemicals in shampoo ranged from 3×10(-4) up to 0.3 for rapidly absorbed ingredients. Average intake rates ranged between nano- and micrograms per kilogram bodyweight per day; the order of chemical prioritization was strongly affected by the ingredient concentration in shampoo. Dermal intake and inhalation (for 20% of the evaluated chemicals) during use dominated exposure, while the skin permeation coefficient dominated the estimated uncertainties. The fraction of chemical taken in by a shampoo user often exceeded, by orders of magnitude, the aggregated fraction taken in by the population through post-use environmental emissions. Chemicals with relatively high octanol-water partitioning and/or volatility, and low molecular weight tended to have higher use stage exposure. Chemicals with low intakes during use (<1%) and subsequent high post-use emissions, however, may yield comparable intake for a member of the general population. The presented PiF based framework offers a novel and critical advancement for life cycle assessments and high-throughput exposure screening of chemicals in cosmetic products demonstrating the importance of consistent consideration of near- and far-field multi-pathway exposures. Copyright © 2016 Elsevier Ltd. All rights reserved.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

High-Throughput RT-PCR for small-molecule screening assays

PubMed Central

Bittker, Joshua A.

2012-01-01

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni

PubMed Central

Gardner, J. Mark F.; Bell, Andrew S.; Parkinson, Tanya; Bickle, Quentin

2016-01-01

An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium Tuberculosis Infections

PubMed Central

Ndungu, John Maina; Suponitsky-Kroyter, Irena; Cavett, Valerie J.; McEnaney, Patrick J.; MacConnell, Andrew B.; Doran, Todd. M.; Ronacher, Katharina; Stanley, Kim; Utset, Ofelia; Walzl, Gerhard; Paegel, Brian M.; Kodadek, Thomas

2017-01-01

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover “epitope surrogates” (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448k unique compounds) using fluorescent anti-human IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens. PMID:27957856

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Fluorescence imaging technology (FI) for high-throughput screening of selenide-modified nano-TiO2 catalysts.

PubMed

Wang, Liping; Lee, Jianchao; Zhang, Meijuan; Duan, Qiannan; Zhang, Jiarui; Qi, Hailang

2016-02-18

A high-throughput screening (HTS) method based on fluorescence imaging (FI) was implemented to evaluate the catalytic performance of selenide-modified nano-TiO2. Chemical ink-jet printing (IJP) technology was reformed to fabricate a catalyst library comprising 1405 (Ni(a)Cu(b)Cd(c)Ce(d)In(e)Y(f))Se(x)/TiO2 (M6Se/Ti) composite photocatalysts. Nineteen M6Se/Tis were screened out from the 1405 candidates efficiently.

Fast Optimization of LiMgMnOx/La2O3 Catalysts for the Oxidative Coupling of Methane.

PubMed

Li, Zhinian; He, Lei; Wang, Shenliang; Yi, Wuzhong; Zou, Shihui; Xiao, Liping; Fan, Jie

2017-01-09

The development of efficient catalyst for oxidative coupling of methane (OCM) reaction represents a grand challenge in direct conversion of methane into other useful products. Here, we reported that a newly developed combinatorial approach can be used for ultrafast optimization of La 2 O 3 -based multicomponent metal oxide catalysts in OCM reaction. This new approach integrated inkjet printing assisted synthesis (IJP-A) with multidimensional group testing strategy (m-GT) tactfully takes the place of conventionally high-throughput synthesis-and-screen experiment. Just within a week, 2048 formulated LiMgMnO x -La 2 O 3 catalysts in a 64·8·8·8·8 = 262 144 compositional space were fabricated by IJP-A in a four-round synthesis-and-screen process, and an optimized formulation has been successfully identified through only 4·8 = 32 times of tests via m-GT screening strategy. The screening process identifies the most promising ternary composition region is Li 0-0.48 Mg 0-6.54 Mn 0-0.62 -La 100 O x with an external C 2 yield of 10.87% at 700 °C. The yield of C 2 is two times as high as the pure nano-La 2 O 3 . The good performance of the optimized catalyst formulation has been validated by the manual preparation, which further prove the effectiveness of the new combinatorial methodology in fast discovery of heterogeneous catalyst.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

NASA Astrophysics Data System (ADS)

Beneyton, Thomas; Wijaya, I. Putu Mahendra; Postros, Prexilia; Najah, Majdi; Leblond, Pascal; Couvent, Angélique; Mayot, Estelle; Griffiths, Andrew D.; Drevelle, Antoine

2016-06-01

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

EPA Science Inventory

Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

Advanced phenotyping and phenotype data analysis for the study of plant growth and development

PubMed Central

Rahaman, Md. Matiur; Chen, Dijun; Gillani, Zeeshan; Klukas, Christian; Chen, Ming

2015-01-01

Due to an increase in the consumption of food, feed, fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to breed high yielding crops that can adapt to the future climate changes, particularly in developing countries. To solve these global challenges, novel approaches are required to identify quantitative phenotypes and to explain the genetic basis of agriculturally important traits. These advances will facilitate the screening of germplasm with high performance characteristics in resource-limited environments. Recently, plant phenomics has offered and integrated a suite of new technologies, and we are on a path to improve the description of complex plant phenotypes. High-throughput phenotyping platforms have also been developed that capture phenotype data from plants in a non-destructive manner. In this review, we discuss recent developments of high-throughput plant phenotyping infrastructure including imaging techniques and corresponding principles for phenotype data analysis. PMID:26322060

High-throughput in Vitro Data To Inform Prioritization of Ambient Water Monitoring and Testing for Endocrine Active Chemicals.

PubMed

Heiger-Bernays, Wendy J; Wegner, Susanna; Dix, David J

2018-01-16

The presence of industrial chemicals, consumer product chemicals, and pharmaceuticals is well documented in waters in the U.S. and globally. Most of these chemicals lack health-protective guidelines and many have been shown to have endocrine bioactivity. There is currently no systematic or national prioritization for monitoring waters for chemicals with endocrine disrupting activity. We propose ambient water bioactivity concentrations (AWBCs) generated from high throughput data as a health-based screen for endocrine bioactivity of chemicals in water. The U.S. EPA ToxCast program has screened over 1800 chemicals for estrogen receptor (ER) and androgen receptor (AR) pathway bioactivity. AWBCs are calculated for 110 ER and 212 AR bioactive chemicals using high throughput ToxCast data from in vitro screening assays and predictive pathway models, high-throughput toxicokinetic data, and data-driven assumptions about consumption of water. Chemical-specific AWBCs are compared with measured water concentrations in data sets from the greater Denver area, Minnesota lakes, and Oregon waters, demonstrating a framework for identifying endocrine bioactive chemicals. This approach can be used to screen potential cumulative endocrine activity in drinking water and to inform prioritization of future monitoring, chemical testing and pollution prevention efforts.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

web cellHTS2: a web-application for the analysis of high-throughput screening data.

PubMed

Pelz, Oliver; Gilsdorf, Moritz; Boutros, Michael

2010-04-12

The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2. The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats. The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

PubMed

Zheng, S

2016-01-01

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. © 2016 Elsevier Inc. All rights reserved.

Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations.

PubMed

Erickson, Heidi S

2012-09-28

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling. Copyright © 2012 John Wiley & Sons, Ltd.

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

PubMed

Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

2016-02-01

Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. © 2015 Society for Laboratory Automation and Screening.

Fluorescence-based high-throughput screening of dicer cleavage activity.

PubMed

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design.

PubMed

Islam, R S; Tisi, D; Levy, M S; Lye, G J

2007-01-01

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takamiya, Mari; Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama; Sakurai, Masaaki

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed amore » RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive {sup 14}C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of its high-throughput and accuracy. • A combination of fluorescent and RF-MS assays is effective for Elovl6 inhibitors.« less

Quantification of a bacterial secondary metabolite by SERS combined with SLM extraction for bioprocess monitoring.

PubMed

Morelli, Lidia; Andreasen, Sune Zoëga; Jendresen, Christian Bille; Nielsen, Alex Toftgaard; Emnéus, Jenny; Zór, Kinga; Boisen, Anja

2017-11-20

During the last few decades, great advances have been reached in high-throughput design and building of genetically engineered microbial strains, leading to a need for fast and reliable screening methods. We developed and optimized a microfluidic supported liquid membrane (SLM) extraction device and combined it with surface enhanced Raman scattering (SERS) sensing for the screening of a biological process, namely for the quantification of a bacterial secondary metabolite, p-coumaric acid (pHCA), produced by Escherichia coli. The microfluidic device proved to be robust and reusable, enabling efficient removal of interfering compounds from the real samples, reaching more than 13-fold up-concentration of the donor at 10 μL min -1 flow rate. With this method, we quantified pHCA directly from the bacterial supernatant, distinguishing between various culture conditions based on the pHCA production yield. The obtained data showed good correlation with HPLC analysis.

Methodology for enabling high-throughput simultaneous saccharification and fermentation screening of yeast using solid biomass as a substrate.

PubMed

Elliston, Adam; Wood, Ian P; Soucouri, Marie J; Tantale, Rachelle J; Dicks, Jo; Roberts, Ian N; Waldron, Keith W

2015-01-01

High-throughput (HTP) screening is becoming an increasingly useful tool for collating biological data which would otherwise require the employment of excessive resources. Second generation biofuel production is one such process. HTP screening allows the investigation of large sample sets to be undertaken with increased speed and cost effectiveness. This paper outlines a methodology that will enable solid lignocellulosic substrates to be hydrolyzed and fermented at a 96-well plate scale, facilitating HTP screening of ethanol production, whilst maintaining repeatability similar to that achieved at a larger scale. The results showed that utilizing sheets of biomass of consistent density (handbills), for paper, and slurries of pretreated biomass that could be pipetted allowed standardized and accurate transfers to 96-well plates to be achieved (±3.1 and 1.7%, respectively). Processing these substrates by simultaneous saccharification and fermentation (SSF) at various volumes showed no significant difference on final ethanol yields, either at standard shake flask (200 mL), universal bottle (10 mL) or 96-well plate (1 mL) scales. Substrate concentrations of up to 10% (w/v) were trialed successfully for SSFs at 1 mL volume. The methodology was successfully tested by showing the effects of steam explosion pretreatment on both oilseed rape and wheat straws. This methodology could be used to replace large shake flask reactions with comparatively fast 96-well plate SSF assays allowing for HTP experimentation. Additionally this method is compatible with a number of standardized assay techniques such as simple colorimetric, High-performance liquid chromatography (HPLC) and Nuclear magnetic resonance (NMR) spectroscopy. Furthermore this research has practical uses in the biorefining of biomass substrates for second generation biofuels and novel biobased chemicals by allowing HTP SSF screening, which should allow selected samples to be scaled up or studied in more detail.

A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

PubMed Central

Bhattacharyya, Nisan; Hu, Xin; Chen, Catherine Z.; Mathews Griner, Lesley A.; Zheng, Wei; Inglese, James; Austin, Christopher P.; Marugan, Juan J.; Southall, Noel; Neumann, Susanne; Northup, John K.; Ferrer, Marc; Collins, Michael T.

2014-01-01

Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses. PMID:24667240

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

Rapid Catalyst Screening by a Continuous-Flow Microreactor Interfaced with Ultra High Pressure Liquid Chromatography

PubMed Central

Fang, Hui; Xiao, Qing; Wu, Fanghui; Floreancig, Paul E.; Weber, Stephen G.

2010-01-01

A high-throughput screening system for homogeneous catalyst discovery has been developed by integrating a continuous-flow capillary-based microreactor with ultra-high pressure liquid chromatography (UHPLC) for fast online analysis. Reactions are conducted in distinct and stable zones in a flow stream that allows for time and temperature regulation. UHPLC detection at high temperature allows high throughput online determination of substrate, product, and byproduct concentrations. We evaluated the efficacies of a series of soluble acid catalysts for an intramolecular Friedel-Crafts addition into an acyliminium ion intermediate within one day and with minimal material investment. The effects of catalyst loading, reaction time, and reaction temperature were also screened. This system exhibited high reproducibility for high-throughput catalyst screening and allowed several acid catalysts for the reaction to be identified. Major side products from the reactions were determined through off-line mass spectrometric detection. Er(OTf)3, the catalyst that showed optimal efficiency in the screening, was shown to be effective at promoting the cyclization reaction on a preparative scale. PMID:20666502

HTS-Net: An integrated regulome-interactome approach for establishing network regulation models in high-throughput screenings

PubMed Central

Rioualen, Claire; Da Costa, Quentin; Chetrit, Bernard; Charafe-Jauffret, Emmanuelle; Ginestier, Christophe

2017-01-01

High-throughput RNAi screenings (HTS) allow quantifying the impact of the deletion of each gene in any particular function, from virus-host interactions to cell differentiation. However, there has been less development for functional analysis tools dedicated to RNAi analyses. HTS-Net, a network-based analysis program, was developed to identify gene regulatory modules impacted in high-throughput screenings, by integrating transcription factors-target genes interaction data (regulome) and protein-protein interaction networks (interactome) on top of screening z-scores. HTS-Net produces exhaustive HTML reports for results navigation and exploration. HTS-Net is a new pipeline for RNA interference screening analyses that proves better performance than simple gene rankings by z-scores, by re-prioritizing genes and replacing them in their biological context, as shown by the three studies that we reanalyzed. Formatted input data for the three studied datasets, source code and web site for testing the system are available from the companion web site at http://htsnet.marseille.inserm.fr/. We also compared our program with existing algorithms (CARD and hotnet2). PMID:28949986

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

PubMed Central

Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

2015-01-01

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

PubMed Central

ANANTHAN, SUBRAMANIAM; FAALEOLEA, ELLEN R.; GOLDMAN, ROBERT C.; HOBRATH, JUDITH V.; KWONG, CECIL D.; LAUGHON, BARBARA E.; MADDRY, JOSEPH A.; MEHTA, ALKA; RASMUSSEN, LYNN; REYNOLDS, ROBERT C.; SECRIST, JOHN A.; SHINDO, NICE; SHOWE, DUSTIN N.; SOSA, MELINDA I.; SULING, WILLIAM J.; WHITE, E. LUCILE

2009-01-01

SUMMARY There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein. PMID:19758845

High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

DOEpatents

Kim, Sung-Hou [Moraga, CA; Kim, Rosalind [Moraga, CA; Jancarik, Jamila [Walnut Creek, CA

2012-01-31

An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

High-throughput screening for bioactive components from traditional Chinese medicine.

PubMed

Zhu, Yanhui; Zhang, Zhiyun; Zhang, Meng; Mais, Dale E; Wang, Ming-Wei

2010-12-01

Throughout the centuries, traditional Chinese medicine has been a rich resource in the development of new drugs. Modern drug discovery, which relies increasingly on automated high throughput screening and quick hit-to-lead development, however, is confronted with the challenges of the chemical complexity associated with natural products. New technologies for biological screening as well as library building are in great demand in order to meet the requirements. Here we review the developments in these techniques under the perspective of their applicability in natural product drug discovery. Methods in library building, component characterizing, biological evaluation, and other screening methods including NMR and X-ray diffraction are discussed.

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

High-throughput screening (HTS) generates in vitro data for characterizing potential chemical hazard. TK models are needed to allow in vitro to in vivo extrapolation (IVIVE) to real world situations. The U.S. EPA has created a public tool (R package “httk” for high throughput tox...

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Detection of COPB2 as a KRAS synthetic lethal partner through integration of functional genomics screens

PubMed Central

Christodoulou, Eleni G.; Yang, Hai; Lademann, Franziska; Pilarsky, Christian; Beyer, Andreas; Schroeder, Michael

2017-01-01

Mutated KRAS plays an important role in many cancers. Although targeting KRAS directly is difficult, indirect inactivation via synthetic lethal partners (SLPs) is promising. Yet to date, there are no SLPs from high-throughput RNAi screening, which are supported by multiple screens. Here, we address this problem by aggregating and ranking data over three independent high-throughput screens. We integrate rankings by minimizing the displacement and by considering established methods such as RIGER and RSA. Our meta analysis reveals COPB2 as a potential SLP of KRAS with good support from all three screens. COPB2 is a coatomer subunit and its knock down has already been linked to disabled autophagy and reduced tumor growth. We confirm COPB2 as SLP in knock down experiments on pancreas and colorectal cancer cell lines. Overall, consistent integration of high throughput data can generate candidate synthetic lethal partners, which individual screens do not uncover. Concretely, we reveal and confirm that COPB2 is a synthetic lethal partner of KRAS and hence a promising cancer target. Ligands inhibiting COPB2 may, therefore, be promising new cancer drugs. PMID:28415695

A high-throughput screening approach for the optoelectronic properties of conjugated polymers.

PubMed

Wilbraham, Liam; Berardo, Enrico; Turcani, Lukas; Jelfs, Kim E; Zwijnenburg, Martijn A

2018-06-25

We propose a general high-throughput virtual screening approach for the optical and electronic properties of conjugated polymers. This approach makes use of the recently developed xTB family of low-computational-cost density functional tight-binding methods from Grimme and co-workers, calibrated here to (TD-)DFT data computed for a representative diverse set of (co-)polymers. Parameters drawn from the resulting calibration using a linear model can then be applied to the xTB derived results for new polymers, thus generating near DFT-quality data with orders of magnitude reduction in computational cost. As a result, after an initial computational investment for calibration, this approach can be used to quickly and accurately screen on the order of thousands of polymers for target applications. We also demonstrate that the (opto)electronic properties of the conjugated polymers show only a very minor variation when considering different conformers and that the results of high-throughput screening are therefore expected to be relatively insensitive with respect to the conformer search methodology applied.

Integrating modelling and phenotyping approaches to identify and screen complex traits - Illustration for transpiration efficiency in cereals.

PubMed

Chenu, K; van Oosterom, E J; McLean, G; Deifel, K S; Fletcher, A; Geetika, G; Tirfessa, A; Mace, E S; Jordan, D R; Sulman, R; Hammer, G L

2018-02-21

Following advances in genetics, genomics, and phenotyping, trait selection in breeding is limited by our ability to understand interactions within the plants and with their environments, and to target traits of most relevance for the target population of environments. We propose an integrated approach that combines insights from crop modelling, physiology, genetics, and breeding to identify traits valuable for yield gain in the target population of environments, develop relevant high-throughput phenotyping platforms, and identify genetic controls and their values in production environments. This paper uses transpiration efficiency (biomass produced per unit of water used) as an example of a complex trait of interest to illustrate how the approach can guide modelling, phenotyping, and selection in a breeding program. We believe that this approach, by integrating insights from diverse disciplines, can increase the resource use efficiency of breeding programs for improving yield gains in target populations of environments.

Multitrait, random regression, or simple repeatability model in high-throughput phenotyping data improve genomic prediction for wheat grain yield

USDA-ARS?s Scientific Manuscript database

High-throughput phenotyping (HTP) platforms can be used to measure traits that are genetically correlated with wheat (Triticum aestivum L.) grain yield across time. Incorporating such secondary traits in the multivariate pedigree and genomic prediction models would be desirable to improve indirect s...

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

A catalog of putative adverse outcome pathways (AOPs) that ...

EPA Pesticide Factsheets

A number of putative AOPs for several distinct MIEs of thyroid disruption have been formulated for amphibian metamorphosis and fish swim bladder inflation. These have been entered into the AOP knowledgebase on the OECD WIKI. The EDSP has been actively advancing high-throughput screening for chemical activity toward estrogen, androgen and thyroid targets. However, it has been recently identified that coverage for thyroid-related targets is lagging behind estrogen and androgen assay coverage. As thyroid-related medium-high throughput assays are actively being developed for inclusion in the ToxCast chemical screening program, a parallel effort is underway to characterize putative adverse outcome pathways (AOPs) specific to these thyroid-related targets. This effort is intended to provide biological and ecological context that will enhance the utility of ToxCast high throughput screening data for hazard identification.

Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics.

PubMed

Szigeti, Marton; Lew, Clarence; Roby, Keith; Guttman, Andras

2016-04-01

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences. © 2015 Society for Laboratory Automation and Screening.

Profiling Cholinesterase Adduction: A High-Throughput Prioritization Method for Organophosphate Exposure Samples

PubMed Central

Carter, Melissa D.; Crow, Brian S.; Pantazides, Brooke G.; Watson, Caroline M.; deCastro, B. Rey; Thomas, Jerry D.; Blake, Thomas A.; Johnson, Rudolph C.

2017-01-01

A high-throughput prioritization method was developed for use with a validated confirmatory method detecting organophosphorus nerve agent exposure by immunomagnetic separation-HPLC-MS/MS. A ballistic gradient was incorporated into this analytical method in order to profile unadducted butyrylcholinesterase (BChE) in clinical samples. With Zhang, et al. 1999’s Z′-factor of 0.88 ± 0.01 (SD) of control analytes and Z-factor of 0.25 ± 0.06 (SD) of serum samples, the assay is rated an “excellent assay” for the synthetic peptide controls used and a “double assay” when used to prioritize clinical samples. Hits, defined as samples containing BChE Ser-198 adducts or no BChE present, were analyzed in a confirmatory method for identification and quantitation of the BChE adduct, if present. The ability to prioritize samples by highest exposure for confirmatory analysis is of particular importance in an exposure to cholinesterase inhibitors such as organophosphorus nerve agents where a large number of clinical samples may be collected. In an initial blind screen, 67 out of 70 samples were accurately identified giving an assay accuracy of 96% and yielded no false negatives. The method is the first to provide a high-throughput prioritization assay for profiling adduction of Ser-198 BChE in clinical samples. PMID:23954929

ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discovery at high-throughput rates

PubMed Central

White, David T; Eroglu, Arife Unal; Wang, Guohua; Zhang, Liyun; Sengupta, Sumitra; Ding, Ding; Rajpurohit, Surendra K; Walker, Steven L; Ji, Hongkai; Qian, Jiang; Mumm, Jeff S

2017-01-01

The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed ‘ARQiv-HTS’. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. We also outline what we see as best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery, and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, we provide a detailed protocol for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models. ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives. PMID:27831568

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

PubMed Central

Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

2015-01-01

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties ofmore » suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.« less

Integrative data mining of high-throughput in vitro screens, in vivo data, and disease information to identify Adverse Outcome Pathway (AOP) signatures:ToxCast high-throughput screening data and Comparative Toxicogenomics Database (CTD) as a case study.

EPA Science Inventory

The Adverse Outcome Pathway (AOP) framework provides a systematic way to describe linkages between molecular and cellular processes and organism or population level effects. The current AOP assembly methods however, are inefficient. Our goal is to generate computationally-pr...

High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants.

PubMed

McClure, Sean M; Ahl, Patrick L; Blue, Jeffrey T

2018-03-05

The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® - a commercially available fluorescent rotor dye - for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements. CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®. Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening. DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions - including surfactants -with standard, plate based rT-PCR instrumentation.

Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer.

PubMed

Ellingson, Sally R; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C; Baudry, Jerome

2014-04-25

In this paper we give the current state of high-throughput virtual screening. We describe a case study of using a task-parallel MPI (Message Passing Interface) version of Autodock4 [1], [2] to run a virtual high-throughput screen of one-million compounds on the Jaguar Cray XK6 Supercomputer at Oak Ridge National Laboratory. We include a description of scripts developed to increase the efficiency of the predocking file preparation and postdocking analysis. A detailed tutorial, scripts, and source code for this MPI version of Autodock4 are available online at http://www.bio.utk.edu/baudrylab/autodockmpi.htm.

Enhancing high throughput toxicology - development of putative adverse outcome pathways linking US EPA ToxCast screening targets to relevant apical hazards.

EPA Science Inventory

High throughput toxicology programs, such as ToxCast and Tox21, have provided biological effects data for thousands of chemicals at multiple concentrations. Compared to traditional, whole-organism approaches, high throughput assays are rapid and cost-effective, yet they generall...

Evaluation of High-Throughput Chemical Exposure Models via Analysis of Matched Environmental and Biological Media Measurements

EPA Science Inventory

The U.S. EPA, under its ExpoCast program, is developing high-throughput near-field modeling methods to estimate human chemical exposure and to provide real-world context to high-throughput screening (HTS) hazard data. These novel modeling methods include reverse methods to infer ...

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

PubMed Central

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

High-Throughput Toxicity Testing: New Strategies for ...

EPA Pesticide Factsheets

In recent years, the food industry has made progress in improving safety testing methods focused on microbial contaminants in order to promote food safety. However, food industry toxicologists must also assess the safety of food-relevant chemicals including pesticides, direct additives, and food contact substances. With the rapidly growing use of new food additives, as well as innovation in food contact substance development, an interest in exploring the use of high-throughput chemical safety testing approaches has emerged. Currently, the field of toxicology is undergoing a paradigm shift in how chemical hazards can be evaluated. Since there are tens of thousands of chemicals in use, many of which have little to no hazard information and there are limited resources (namely time and money) for testing these chemicals, it is necessary to prioritize which chemicals require further safety testing to better protect human health. Advances in biochemistry and computational toxicology have paved the way for animal-free (in vitro) high-throughput screening which can characterize chemical interactions with highly specific biological processes. Screening approaches are not novel; in fact, quantitative high-throughput screening (qHTS) methods that incorporate dose-response evaluation have been widely used in the pharmaceutical industry. For toxicological evaluation and prioritization, it is the throughput as well as the cost- and time-efficient nature of qHTS that makes it

Automated recycling of chemistry for virtual screening and library design.

PubMed

Vainio, Mikko J; Kogej, Thierry; Raubacher, Florian

2012-07-23

An early stage drug discovery project needs to identify a number of chemically diverse and attractive compounds. These hit compounds are typically found through high-throughput screening campaigns. The diversity of the chemical libraries used in screening is therefore important. In this study, we describe a virtual high-throughput screening system called Virtual Library. The system automatically "recycles" validated synthetic protocols and available starting materials to generate a large number of virtual compound libraries, and allows for fast searches in the generated libraries using a 2D fingerprint based screening method. Virtual Library links the returned virtual hit compounds back to experimental protocols to quickly assess the synthetic accessibility of the hits. The system can be used as an idea generator for library design to enrich the screening collection and to explore the structure-activity landscape around a specific active compound.

A kinase-focused compound collection: compilation and screening strategy.

PubMed

Sun, Dongyu; Chuaqui, Claudio; Deng, Zhan; Bowes, Scott; Chin, Donovan; Singh, Juswinder; Cullen, Patrick; Hankins, Gretchen; Lee, Wen-Cherng; Donnelly, Jason; Friedman, Jessica; Josiah, Serene

2006-06-01

Lead identification by high-throughput screening of large compound libraries has been supplemented with virtual screening and focused compound libraries. To complement existing approaches for lead identification at Biogen Idec, a kinase-focused compound collection was designed, developed and validated. Two strategies were adopted to populate the compound collection: a ligand shape-based virtual screening and a receptor-based approach (structural interaction fingerprint). Compounds selected with the two approaches were cherry-picked from an existing high-throughput screening compound library, ordered from suppliers and supplemented with specific medicinal compounds from internal programs. Promising hits and leads have been generated from the kinase-focused compound collection against multiple kinase targets. The principle of the collection design and screening strategy was validated and the use of the kinase-focused compound collection for lead identification has been added to existing strategies.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

PubMed Central

Lucanic, Mark; Garrett, Theo; Gill, Matthew S.; Lithgow, Gordon J.

2018-01-01

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening. PMID:29630057

The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology.

PubMed

McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

2011-07-01

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory.

Rapid identification and validation of novel targeted approaches for Glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.

PubMed

Daher, Ahmad; de Groot, John

2018-01-01

Tumor heterogeneity is a major factor in glioblastoma's poor response to therapy and seemingly inevitable recurrence. Only two glioblastoma drugs have received Food and Drug Administration approval since 1998, highlighting the urgent need for new therapies. Profiling "omics" analyses have helped characterize glioblastoma molecularly and have thus identified multiple molecular targets for precision medicine. These molecular targets have influenced clinical trial design; many "actionable" mutation-focused trials are underway, but because they have not yet led to therapeutic breakthroughs, new strategies for treating glioblastoma, especially those with a pharmacological functional component, remain in high demand. In that regard, high-throughput screening that allows for expedited preclinical drug testing and the use of GBM models that represent tumor heterogeneity more accurately than traditional cancer cell lines is necessary to maximize the successful translation of agents into the clinic. High-throughput screening has been successfully used in the testing, discovery, and validation of potential therapeutics in various cancer models, but it has not been extensively utilized in glioblastoma models. In this report, we describe the basic aspects of high-throughput screening and propose a modified high-throughput screening model in which ex vivo and in vivo drug testing is complemented by post-screening pharmacological, pan-omic analysis to expedite anti-glioma drugs' preclinical testing and develop predictive biomarker datasets that can aid in personalizing glioblastoma therapy and inform clinical trial design. Copyright © 2017 Elsevier Inc. All rights reserved.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials

PubMed Central

Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

2008-01-01

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

High-throughput phenotyping (HTP) identifies seedling root traits linked to variation in seed yield and nutrient capture in field-grown oilseed rape (Brassica napus L.)

PubMed Central

Thomas, C. L.; Graham, N. S.; Hayden, R.; Meacham, M. C.; Neugebauer, K.; Nightingale, M.; Dupuy, L. X.; Hammond, J. P.; White, P. J.; Broadley, M. R.

2016-01-01

Background and Aims Root traits can be selected for crop improvement. Techniques such as soil excavations can be used to screen root traits in the field, but are limited to genotypes that are well-adapted to field conditions. The aim of this study was to compare a low-cost, high-throughput root phenotyping (HTP) technique in a controlled environment with field performance, using oilseed rape (OSR; Brassica napus) varieties. Methods Primary root length (PRL), lateral root length and lateral root density (LRD) were measured on 14-d-old seedlings of elite OSR varieties (n = 32) using a ‘pouch and wick’ HTP system (∼40 replicates). Six field experiments were conducted using the same varieties at two UK sites each year for 3 years. Plants were excavated at the 6- to 8-leaf stage for general vigour assessments of roots and shoots in all six experiments, and final seed yield was determined. Leaves were sampled for mineral composition from one of the field experiments. Key Results Seedling PRL in the HTP system correlated with seed yield in four out of six (r = 0·50, 0·50, 0·33, 0·49; P < 0·05) and with emergence in three out of five (r = 0·59, 0·22, 0·49; P < 0·05) field experiments. Seedling LRD correlated positively with leaf concentrations of some minerals, e.g. calcium (r = 0·46; P < 0·01) and zinc (r = 0·58; P < 0·001), but did not correlate with emergence, general early vigour or yield in the field. Conclusions Associations between PRL and field performance are generally related to early vigour. These root traits might therefore be of limited additional selection value, given that vigour can be measured easily on shoots/canopies. In contrast, LRD cannot be assessed easily in the field and, if LRD can improve nutrient uptake, then it may be possible to use HTP systems to screen this trait in both elite and more genetically diverse, non-field-adapted OSR. PMID:27052342

Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

Developing science gateways for drug discovery in a grid environment.

PubMed

Pérez-Sánchez, Horacio; Rezaei, Vahid; Mezhuyev, Vitaliy; Man, Duhu; Peña-García, Jorge; den-Haan, Helena; Gesing, Sandra

2016-01-01

Methods for in silico screening of large databases of molecules increasingly complement and replace experimental techniques to discover novel compounds to combat diseases. As these techniques become more complex and computationally costly we are faced with an increasing problem to provide the research community of life sciences with a convenient tool for high-throughput virtual screening on distributed computing resources. To this end, we recently integrated the biophysics-based drug-screening program FlexScreen into a service, applicable for large-scale parallel screening and reusable in the context of scientific workflows. Our implementation is based on Pipeline Pilot and Simple Object Access Protocol and provides an easy-to-use graphical user interface to construct complex workflows, which can be executed on distributed computing resources, thus accelerating the throughput by several orders of magnitude.

Microfluidic cell chips for high-throughput drug screening

PubMed Central

Chi, Chun-Wei; Ahmed, AH Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

2016-01-01

The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell–drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

High-Throughput Screening of Therapeutic Neural Stimulation Targets: Toward Principles of Preventing and Treating Post-Traumatic Stress Disorder

DTIC Science & Technology

2009-09-01

onset and averaged across all excited units tested (mean ± SE). 7 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Virus design and production...to baseline level 355 ± 505 ms later. The level of post -light firing did not vary with repeated light exposure (p > 0.7, paired t- test comparing...High-Throughput Screening of Therapeutic Neural Stimulation Targets: Toward Principles of Preventing and Treating Post - Traumatic Stress Disorder

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

PubMed

Jacques, Philippe; Béchet, Max; Bigan, Muriel; Caly, Delphine; Chataigné, Gabrielle; Coutte, François; Flahaut, Christophe; Heuson, Egon; Leclère, Valérie; Lecouturier, Didier; Phalip, Vincent; Ravallec, Rozenn; Dhulster, Pascal; Froidevaux, Rénato

2017-02-01

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening.

PubMed

Neto, A I; Correia, C R; Oliveira, M B; Rial-Hermida, M I; Alvarez-Lorenzo, C; Reis, R L; Mano, J F

2015-04-01

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

PubMed

Jacobs, K R; Guillemin, G J; Lovejoy, D B

2018-02-01

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

PubMed Central

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-01-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

PubMed

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-10-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

PubMed

Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

2016-01-01

Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

Selection and optimization of hits from a high-throughput phenotypic screen against Trypanosoma cruzi.

PubMed

Keenan, Martine; Alexander, Paul W; Chaplin, Jason H; Abbott, Michael J; Diao, Hugo; Wang, Zhisen; Best, Wayne M; Perez, Catherine J; Cornwall, Scott M J; Keatley, Sarah K; Thompson, R C Andrew; Charman, Susan A; White, Karen L; Ryan, Eileen; Chen, Gong; Ioset, Jean-Robert; von Geldern, Thomas W; Chatelain, Eric

2013-10-01

Inhibitors of Trypanosoma cruzi with novel mechanisms of action are urgently required to diversify the current clinical and preclinical pipelines. Increasing the number and diversity of hits available for assessment at the beginning of the discovery process will help to achieve this aim. We report the evaluation of multiple hits generated from a high-throughput screen to identify inhibitors of T. cruzi and from these studies the discovery of two novel series currently in lead optimization. Lead compounds from these series potently and selectively inhibit growth of T. cruzi in vitro and the most advanced compound is orally active in a subchronic mouse model of T. cruzi infection. High-throughput screening of novel compound collections has an important role to play in diversifying the trypanosomatid drug discovery portfolio. A new T. cruzi inhibitor series with good drug-like properties and promising in vivo efficacy has been identified through this process.

High-throughput fabrication and screening improves gold nanoparticle chemiresistor sensor performance.

PubMed

Hubble, Lee J; Cooper, James S; Sosa-Pintos, Andrea; Kiiveri, Harri; Chow, Edith; Webster, Melissa S; Wieczorek, Lech; Raguse, Burkhard

2015-02-09

Chemiresistor sensor arrays are a promising technology to replace current laboratory-based analysis instrumentation, with the advantage of facile integration into portable, low-cost devices for in-field use. To increase the performance of chemiresistor sensor arrays a high-throughput fabrication and screening methodology was developed to assess different organothiol-functionalized gold nanoparticle chemiresistors. This high-throughput fabrication and testing methodology was implemented to screen a library consisting of 132 different organothiol compounds as capping agents for functionalized gold nanoparticle chemiresistor sensors. The methodology utilized an automated liquid handling workstation for the in situ functionalization of gold nanoparticle films and subsequent automated analyte testing of sensor arrays using a flow-injection analysis system. To test the methodology we focused on the discrimination and quantitation of benzene, toluene, ethylbenzene, p-xylene, and naphthalene (BTEXN) mixtures in water at low microgram per liter concentration levels. The high-throughput methodology identified a sensor array configuration consisting of a subset of organothiol-functionalized chemiresistors which in combination with random forests analysis was able to predict individual analyte concentrations with overall root-mean-square errors ranging between 8-17 μg/L for mixtures of BTEXN in water at the 100 μg/L concentration. The ability to use a simple sensor array system to quantitate BTEXN mixtures in water at the low μg/L concentration range has direct and significant implications to future environmental monitoring and reporting strategies. In addition, these results demonstrate the advantages of high-throughput screening to improve the performance of gold nanoparticle based chemiresistors for both new and existing applications.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

PubMed

Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

2014-09-01

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

PubMed

Le Saux, Thomas; Hisamoto, Hideaki; Terabe, Shigeru

2006-02-03

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

PubMed

Jones, Stuart; Ahmet, Jonathan; Ayton, Kelly; Ball, Matthew; Cockerill, Mark; Fairweather, Emma; Hamilton, Nicola; Harper, Paul; Hitchin, James; Jordan, Allan; Levy, Colin; Lopez, Ruth; McKenzie, Eddie; Packer, Martin; Plant, Darren; Simpson, Iain; Simpson, Peter; Sinclair, Ian; Somervaille, Tim C P; Small, Helen; Spencer, Gary J; Thomson, Graeme; Tonge, Michael; Waddell, Ian; Walsh, Jarrod; Waszkowycz, Bohdan; Wigglesworth, Mark; Wiseman, Daniel H; Ogilvie, Donald

2016-12-22

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

A QSAR Model for Thyroperoxidase Inhibition and Screening ...

EPA Pesticide Factsheets

Thyroid hormones (THs) are critical modulators of a wide range of biological processes from neurodevelopment to metabolism. Well regulated levels of THs are critical during development and even moderate changes in maternal or fetal TH levels produce irreversible neurological deficits in children. The enzyme thyroperoxidase (TPO) plays a key role in the synthesis of THs. Inhibition of TPO by xenobiotics leads to decreased TH synthesis and, depending on the degree of synthesis inhibition, may result in adverse developmental outcomes. Recently, a high-throughput screening assay for TPO inhibition (AUR-TPO) was developed and used to screen the ToxCast Phase I and II chemicals. In the present study, we used the results from the AUR-TPO screening to develop a Quantitative Structure-Activity Relationship (QSAR) model for TPO inhibition in Leadscope®. The training set consisted of 898 discrete organic chemicals: 134 positive and 764 negative for TPO inhibition. A 10 times two-fold 50% cross-validation of the model was performed, yielding a balanced accuracy of 78.7% within its defined applicability domain. More recently, an additional ~800 chemicals from the US EPA Endocrine Disruption Screening Program (EDSP21) were screened using the AUR-TPO assay. This data was used for external validation of the QSAR model, demonstrating a balanced accuracy of 85.7% within its applicability domain. Overall, the cross- and external validations indicate a model with a high predictiv

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiba, Takuya, E-mail: takuya@nagasaki-u.ac.jp; Tsuchiya, Tomoshi; Komatsu, Toshimitsu

2010-10-15

Research highlights: {yields} We identified four sequence motifs lying upstream of putative pro-longevity genes. {yields} One of these motifs binds to HNF-4{alpha}. {yields} HNF-4{alpha}/PGC-1{alpha} could up-regulate the transcription of a reporter gene linked to this motif. {yields} The reporter system described here could be used to screen candidate anti-aging molecules. -- Abstract: Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulatedmore » by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4{alpha} (HNF-4{alpha}), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4{alpha} and its regulator peroxisome proliferator-activated receptor (PPAR) {gamma} coactivator-1{alpha} (PGC-1{alpha}), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.« less

Rational Methods for the Selection of Diverse Screening Compounds

PubMed Central

Huggins, David J.; Venkitaraman, Ashok R.; Spring, David R.

2016-01-01

Traditionally a pursuit of large pharmaceutical companies, high-throughput screening assays are becoming increasingly common within academic and government laboratories. This shift has been instrumental in enabling projects that have not been commercially viable, such as chemical probe discovery and screening against high risk targets. Once an assay has been prepared and validated, it must be fed with screening compounds. Crafting a successful collection of small molecules for screening poses a significant challenge. An optimized collection will minimize false positives whilst maximizing hit rates of compounds that are amenable to lead generation and optimization. Without due consideration of the relevant protein targets and the downstream screening assays, compound filtering and selection can fail to explore the great extent of chemical diversity and eschew valuable novelty. Herein, we discuss the different factors to be considered and methods that may be employed when assembling a structurally diverse compound screening collection. Rational methods for selecting diverse chemical libraries are essential for their effective use in high-throughput screens. PMID:21261294

Plate-based diversity subset screening: an efficient paradigm for high throughput screening of a large screening file.

PubMed

Bell, Andrew S; Bradley, Joseph; Everett, Jeremy R; Knight, Michelle; Loesel, Jens; Mathias, John; McLoughlin, David; Mills, James; Sharp, Robert E; Williams, Christine; Wood, Terence P

2013-05-01

The screening files of many large companies, including Pfizer, have grown considerably due to internal chemistry efforts, company mergers and acquisitions, external contracted synthesis, or compound purchase schemes. In order to screen the targets of interest in a cost-effective fashion, we devised an easy-to-assemble, plate-based diversity subset (PBDS) that represents almost the entire computed chemical space of the screening file whilst comprising only a fraction of the plates in the collection. In order to create this file, we developed new design principles for the quality assessment of screening plates: the Rule of 40 (Ro40) and a plate selection process that insured excellent coverage of both library chemistry and legacy chemistry space. This paper describes the rationale, design, construction, and performance of the PBDS, that has evolved into the standard paradigm for singleton (one compound per well) high-throughput screening in Pfizer since its introduction in 2006.

A Robotic Platform for Quantitative High-Throughput Screening

PubMed Central

Michael, Sam; Auld, Douglas; Klumpp, Carleen; Jadhav, Ajit; Zheng, Wei; Thorne, Natasha; Austin, Christopher P.; Inglese, James

2008-01-01

Abstract High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation. PMID:19035846

Diving deeper into Zebrafish development of social behavior: analyzing high resolution data.

PubMed

Buske, Christine; Gerlai, Robert

2014-08-30

Vertebrate model organisms have been utilized in high throughput screening but only with substantial cost and human capital investment. The zebrafish is a vertebrate model species that is a promising and cost effective candidate for efficient high throughput screening. Larval zebrafish have already been successfully employed in this regard (Lessman, 2011), but adult zebrafish also show great promise. High throughput screening requires the use of a large number of subjects and collection of substantial amount of data. Collection of data is only one of the demanding aspects of screening. However, in most screening approaches that involve behavioral data the main bottleneck that slows throughput is the time consuming aspect of analysis of the collected data. Some automated analytical tools do exist, but often they only work for one subject at a time, eliminating the possibility of fully utilizing zebrafish as a screening tool. This is a particularly important limitation for such complex phenotypes as social behavior. Testing multiple fish at a time can reveal complex social interactions but it may also allow the identification of outliers from a group of mutagenized or pharmacologically treated fish. Here, we describe a novel method using a custom software tool developed within our laboratory, which enables tracking multiple fish, in combination with a sophisticated analytical approach for summarizing and analyzing high resolution behavioral data. This paper focuses on the latter, the analytic tool, which we have developed using the R programming language and environment for statistical computing. We argue that combining sophisticated data collection methods with appropriate analytical tools will propel zebrafish into the future of neurobehavioral genetic research. Copyright © 2014. Published by Elsevier B.V.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinzon, NM; Aukema, KG; Gralnick, JA

A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.« less

Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

PubMed

Wu, Szu-Huei; Yao, Chun-Hsu; Hsieh, Chieh-Jui; Liu, Yu-Wei; Chao, Yu-Sheng; Song, Jen-Shin; Lee, Jinq-Chyi

2015-07-10

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 μM by high-throughput screening of 41,000 compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

PubMed

Tigabu, Bersabeh; Rasmussen, Lynn; White, E Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W; Noah, James W

2014-04-01

Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

High-Throughput Screening for a Moderately Halophilic Phenol-Degrading Strain and Its Salt Tolerance Response

PubMed Central

Lu, Zhi-Yan; Guo, Xiao-Jue; Li, Hui; Huang, Zhong-Zi; Lin, Kuang-Fei; Liu, Yong-Di

2015-01-01

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg·L−1 starting concentration) over a range of 3%–10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process. PMID:26020478

A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

PubMed Central

Tigabu, Bersabeh; Rasmussen, Lynn; White, E. Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.

2014-01-01

Abstract Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses. PMID:24735442

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

DOE PAGES

Kang, Aram; Meadows, Corey W.; Canu, Nicolas; ...

2017-04-05

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonatemore » monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.« less

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Aram; Meadows, Corey W.; Canu, Nicolas

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonatemore » monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.« less

Evaluating Rapid Models for High-Throughput Exposure Forecasting (SOT)

EPA Science Inventory

High throughput exposure screening models can provide quantitative predictions for thousands of chemicals; however these predictions must be systematically evaluated for predictive ability. Without the capability to make quantitative, albeit uncertain, forecasts of exposure, the ...

The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology

PubMed Central

McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

2011-01-01

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

On-chip Magnetic Separation and Cell Encapsulation in Droplets

NASA Astrophysics Data System (ADS)

Chen, A.; Byvank, T.; Bharde, A.; Miller, B. L.; Chalmers, J. J.; Sooryakumar, R.; Chang, W.-J.; Bashir, R.

2012-02-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment could prevent cross-contamination, provide high recovery yield and ability to study biological traits at a single cell level These advantages of on-chip biological experiments contrast to conventional methods, which require bulk samples that provide only averaged information on cell metabolism. We report on a device that integrates microfluidic technology with a magnetic tweezers array to combine the functionality of separation and encapsulation of objects such as immunomagnetically labeled cells or magnetic beads into pico-liter droplets on the same chip. The ability to control the separation throughput that is independent of the hydrodynamic droplet generation rate allows the encapsulation efficiency to be optimized. The device can potentially be integrated with on-chip labeling and/or bio-detection to become a powerful single-cell analysis device.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

PubMed

Akeroyd, Michiel; Olsthoorn, Maurien; Gerritsma, Jort; Gutker-Vermaas, Diana; Ekkelkamp, Laurens; van Rij, Tjeerd; Klaassen, Paul; Plugge, Wim; Smit, Ed; Strupat, Kerstin; Wenzel, Thibaut; van Tilborg, Marcel; van der Hoeven, Rob

2013-03-10

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery. Here, we present a sensitive, generic workflow for high throughput screening of successful microbial protein over-expression in microtiter plates containing a complex matrix based on mass spectrometry techniques. MALDI-LTQ-Orbitrap screening followed by principal component analysis and peptide mass fingerprinting was developed to obtain a throughput of ∼12,000 samples per week. Alternatively, a UHPLC-MS(2) approach including MS(2) protein identification was developed for microorganisms with a complex protein secretome with a throughput of ∼2000 samples per week. TCA-induced protein precipitation enhanced by addition of bovine serum albumin is used for protein purification prior to MS detection. We show that this generic workflow can effectively reduce large expression libraries from fungi and bacteria to their minimal size by detection of successful protein over-expression using MS. Copyright © 2012 Elsevier B.V. All rights reserved.

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions.

PubMed

De Diego, Nuria; Fürst, Tomáš; Humplík, Jan F; Ugena, Lydia; Podlešáková, Kateřina; Spíchal, Lukáš

2017-01-01

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidops is as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance.

Noise Reduction in High-Throughput Gene Perturbation Screens

USDA-ARS?s Scientific Manuscript database

Motivation: Accurate interpretation of perturbation screens is essential for a successful functional investigation. However, the screened phenotypes are often distorted by noise, and their analysis requires specialized statistical analysis tools. The number and scope of statistical methods available...

Expedient Caution: Approximating Exposure and Dosimetry to Understand Chemical Risk (OSU EMT Research Day keynote presentation)

EPA Science Inventory

I describe research on high throughput exposure and toxicokinetics. These tools provide context for data generated by high throughput toxicity screening to allow risk-based prioritization of thousands of chemicals.

MIPHENO: Data normalization for high throughput metabolic analysis.

EPA Science Inventory

High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course...

High-Throughput Pharmacokinetics for Environmental Chemicals (SOT)

EPA Science Inventory

High throughput screening (HTS) promises to allow prioritization of thousands of environmental chemicals with little or no in vivo information. For bioactivity identified by HTS, toxicokinetic (TK) models are essential to predict exposure thresholds below which no significant bio...

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density

DOE PAGES

Teplitsky, Ella; Joshi, Karan; Ericson, Daniel L.; ...

2015-07-01

We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5 nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5 nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using thismore » system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. Moreover, a fragment mini-library was screened to observe two known lysozyme We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5 nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5 nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5 nL of each component.ds using both co-crystallization and soaking. We used a A similar approach to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5 nL of each component.« less

Predicting human liver microsomal stability with machine learning techniques.

PubMed

Sakiyama, Yojiro; Yuki, Hitomi; Moriya, Takashi; Hattori, Kazunari; Suzuki, Misaki; Shimada, Kaoru; Honma, Teruki

2008-02-01

To ensure a continuing pipeline in pharmaceutical research, lead candidates must possess appropriate metabolic stability in the drug discovery process. In vitro ADMET (absorption, distribution, metabolism, elimination, and toxicity) screening provides us with useful information regarding the metabolic stability of compounds. However, before the synthesis stage, an efficient process is required in order to deal with the vast quantity of data from large compound libraries and high-throughput screening. Here we have derived a relationship between the chemical structure and its metabolic stability for a data set of in-house compounds by means of various in silico machine learning such as random forest, support vector machine (SVM), logistic regression, and recursive partitioning. For model building, 1952 proprietary compounds comprising two classes (stable/unstable) were used with 193 descriptors calculated by Molecular Operating Environment. The results using test compounds have demonstrated that all classifiers yielded satisfactory results (accuracy > 0.8, sensitivity > 0.9, specificity > 0.6, and precision > 0.8). Above all, classification by random forest as well as SVM yielded kappa values of approximately 0.7 in an independent validation set, slightly higher than other classification tools. These results suggest that nonlinear/ensemble-based classification methods might prove useful in the area of in silico ADME modeling.

Screening_mgmt: a Python module for managing screening data.

PubMed

Helfenstein, Andreas; Tammela, Päivi

2015-02-01

High-throughput screening is an established technique in drug discovery and, as such, has also found its way into academia. High-throughput screening generates a considerable amount of data, which is why specific software is used for its analysis and management. The commercially available software packages are often beyond the financial limits of small-scale academic laboratories and, furthermore, lack the flexibility to fulfill certain user-specific requirements. We have developed a Python module, screening_mgmt, which is a lightweight tool for flexible data retrieval, analysis, and storage for different screening assays in one central database. The module reads custom-made analysis scripts and plotting instructions, and it offers a graphical user interface to import, modify, and display the data in a uniform manner. During the test phase, we used this module for the management of 10,000 data points of various origins. It has provided a practical, user-friendly tool for sharing and exchanging information between researchers. © 2014 Society for Laboratory Automation and Screening.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Prioritizing Environmental Chemicals for Obesity and Diabetes Outcomes Research: A Screening Approach Using ToxCast™ High-Throughput Data

PubMed Central

Auerbach, Scott; Filer, Dayne; Reif, David; Walker, Vickie; Holloway, Alison C.; Schlezinger, Jennifer; Srinivasan, Supriya; Svoboda, Daniel; Judson, Richard; Bucher, John R.; Thayer, Kristina A.

2016-01-01

Background: Diabetes and obesity are major threats to public health in the United States and abroad. Understanding the role that chemicals in our environment play in the development of these conditions is an emerging issue in environmental health, although identifying and prioritizing chemicals for testing beyond those already implicated in the literature is challenging. This review is intended to help researchers generate hypotheses about chemicals that may contribute to diabetes and to obesity-related health outcomes by summarizing relevant findings from the U.S. Environmental Protection Agency (EPA) ToxCast™ high-throughput screening (HTS) program. Objectives: Our aim was to develop new hypotheses around environmental chemicals of potential interest for diabetes- or obesity-related outcomes using high-throughput screening data. Methods: We identified ToxCast™ assay targets relevant to several biological processes related to diabetes and obesity (insulin sensitivity in peripheral tissue, pancreatic islet and β cell function, adipocyte differentiation, and feeding behavior) and presented chemical screening data against those assay targets to identify chemicals of potential interest. Discussion: The results of this screening-level analysis suggest that the spectrum of environmental chemicals to consider in research related to diabetes and obesity is much broader than indicated by research papers and reviews published in the peer-reviewed literature. Testing hypotheses based on ToxCast™ data will also help assess the predictive utility of this HTS platform. Conclusions: More research is required to put these screening-level analyses into context, but the information presented in this review should facilitate the development of new hypotheses. Citation: Auerbach S, Filer D, Reif D, Walker V, Holloway AC, Schlezinger J, Srinivasan S, Svoboda D, Judson R, Bucher JR, Thayer KA. 2016. Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast™ high-throughput data. Environ Health Perspect 124:1141–1154; http://dx.doi.org/10.1289/ehp.1510456 PMID:26978842

Prioritizing Environmental Chemicals for Obesity and Diabetes Outcomes Research: A Screening Approach Using ToxCast™ High-Throughput Data.

PubMed

Auerbach, Scott; Filer, Dayne; Reif, David; Walker, Vickie; Holloway, Alison C; Schlezinger, Jennifer; Srinivasan, Supriya; Svoboda, Daniel; Judson, Richard; Bucher, John R; Thayer, Kristina A

2016-08-01

Diabetes and obesity are major threats to public health in the United States and abroad. Understanding the role that chemicals in our environment play in the development of these conditions is an emerging issue in environmental health, although identifying and prioritizing chemicals for testing beyond those already implicated in the literature is challenging. This review is intended to help researchers generate hypotheses about chemicals that may contribute to diabetes and to obesity-related health outcomes by summarizing relevant findings from the U.S. Environmental Protection Agency (EPA) ToxCast™ high-throughput screening (HTS) program. Our aim was to develop new hypotheses around environmental chemicals of potential interest for diabetes- or obesity-related outcomes using high-throughput screening data. We identified ToxCast™ assay targets relevant to several biological processes related to diabetes and obesity (insulin sensitivity in peripheral tissue, pancreatic islet and β cell function, adipocyte differentiation, and feeding behavior) and presented chemical screening data against those assay targets to identify chemicals of potential interest. The results of this screening-level analysis suggest that the spectrum of environmental chemicals to consider in research related to diabetes and obesity is much broader than indicated by research papers and reviews published in the peer-reviewed literature. Testing hypotheses based on ToxCast™ data will also help assess the predictive utility of this HTS platform. More research is required to put these screening-level analyses into context, but the information presented in this review should facilitate the development of new hypotheses. Auerbach S, Filer D, Reif D, Walker V, Holloway AC, Schlezinger J, Srinivasan S, Svoboda D, Judson R, Bucher JR, Thayer KA. 2016. Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast™ high-throughput data. Environ Health Perspect 124:1141-1154; http://dx.doi.org/10.1289/ehp.1510456.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

PubMed

Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

2016-01-01

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σS).

PubMed

Campos-Gomez, Javier; Benitez, Jorge A

2018-07-01

RNA polymerase containing the stress response regulator σ S subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification and validation of vesicant therapeutic targets using a high, throughput siRNA screening approach

DTIC Science & Technology

2014-12-24

toxlet.2011.04.007 Rogers JV, Choi YW, Kiser RC et al (2004) Microarray analysis of gene expression in murine skin exposed to sulfur mustard. J Bio...Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard. Inflam- mation 21(2...Project ID Number CBM.CUTOC.04.10. RC 00114. ABSTRACT See reprint. 15. SUBJECT TERMS sulfur mustard, cutaneous injury, siRNA, high-throughput screening

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

PubMed

Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

2016-04-01

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. © 2016 Society for Laboratory Automation and Screening.

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

PubMed Central

Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

2011-01-01

Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening. PMID:21643507

Ultra High Throughput Screening of Natural Product Extracts to Identify Pro-apoptotic Inhibitors of Bcl-2 Family Proteins

PubMed Central

Hassig, Christian A.; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W.; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J.; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E.; Brown, Susan G.; Baire, Beeraiah; Michel, Andrew R.; Hoye, Thomas R.; Francis, Subhashree; Georg, Gunda I.; Walters, Michael A.; Divlianska, Daniela B.; Roth, Gregory P.; Wright, Amy E.; Reed, John C.

2015-01-01

Anti-apoptotic Bcl-2 family proteins are validated cancer targets comprised of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). While several isoform-selective inhibitors have been developed using structure-based design or high throughput screening (HTS) of synthetic chemical libraries, no large scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six anti-apoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally-relevant PPIs. The screens were conducted in 1,536-well format and displayed satisfactory overall HTS statistics, with Z’-factor values ranging from 0.72 to 0.83, and a hit confirmation rate between 16-64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra high throughput screening using natural product sources and highlight some of the challenges associated with this approach. PMID:24870016

Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen

PubMed Central

Adissu, Hibret A.; Estabel, Jeanne; Sunter, David; Tuck, Elizabeth; Hooks, Yvette; Carragher, Damian M.; Clarke, Kay; Karp, Natasha A.; Project, Sanger Mouse Genetics; Newbigging, Susan; Jones, Nora; Morikawa, Lily; White, Jacqueline K.; McKerlie, Colin

2014-01-01

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice. PMID:24652767

Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development.

PubMed

Goldberg, Deborah S; Lewus, Rachael A; Esfandiary, Reza; Farkas, David C; Mody, Neil; Day, Katrina J; Mallik, Priyanka; Tracka, Malgorzata B; Sealey, Smita K; Samra, Hardeep S

2017-08-01

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

PubMed

Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

2017-11-21

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

High throughput imaging and analysis for biological interpretation of agricultural plants and environmental interaction

NASA Astrophysics Data System (ADS)

Hong, Hyundae; Benac, Jasenka; Riggsbee, Daniel; Koutsky, Keith

2014-03-01

High throughput (HT) phenotyping of crops is essential to increase yield in environments deteriorated by climate change. The controlled environment of a greenhouse offers an ideal platform to study the genotype to phenotype linkages for crop screening. Advanced imaging technologies are used to study plants' responses to resource limitations such as water and nutrient deficiency. Advanced imaging technologies coupled with automation make HT phenotyping in the greenhouse not only feasible, but practical. Monsanto has a state of the art automated greenhouse (AGH) facility. Handling of the soil, pots water and nutrients are all completely automated. Images of the plants are acquired by multiple hyperspectral and broadband cameras. The hyperspectral cameras cover wavelengths from visible light through short wave infra-red (SWIR). Inhouse developed software analyzes the images to measure plant morphological and biochemical properties. We measure phenotypic metrics like plant area, height, and width as well as biomass. Hyperspectral imaging allows us to measure biochemcical metrics such as chlorophyll, anthocyanin, and foliar water content. The last 4 years of AGH operations on crops like corn, soybean, and cotton have demonstrated successful application of imaging and analysis technologies for high throughput plant phenotyping. Using HT phenotyping, scientists have been showing strong correlations to environmental conditions, such as water and nutrient deficits, as well as the ability to tease apart distinct differences in the genetic backgrounds of crops.

Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.

PubMed

Gerrard, Gareth; Valgañón, Mikel; Foong, Hui En; Kasperaviciute, Dalia; Iskander, Deena; Game, Laurence; Müller, Michael; Aitman, Timothy J; Roberts, Irene; de la Fuente, Josu; Foroni, Letizia; Karadimitris, Anastasios

2013-08-01

Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA. © 2013 John Wiley & Sons Ltd.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells.

PubMed

Efthymiou, Anastasia; Shaltouki, Atossa; Steiner, Joseph P; Jha, Balendu; Heman-Ackah, Sabrina M; Swistowski, Andrzej; Zeng, Xianmin; Rao, Mahendra S; Malik, Nasir

2014-01-01

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Screening Chemicals for Estrogen Receptor Bioactivity Using a Computational Model.

PubMed

Browne, Patience; Judson, Richard S; Casey, Warren M; Kleinstreuer, Nicole C; Thomas, Russell S

2015-07-21

The U.S. Environmental Protection Agency (EPA) is considering high-throughput and computational methods to evaluate the endocrine bioactivity of environmental chemicals. Here we describe a multistep, performance-based validation of new methods and demonstrate that these new tools are sufficiently robust to be used in the Endocrine Disruptor Screening Program (EDSP). Results from 18 estrogen receptor (ER) ToxCast high-throughput screening assays were integrated into a computational model that can discriminate bioactivity from assay-specific interference and cytotoxicity. Model scores range from 0 (no activity) to 1 (bioactivity of 17β-estradiol). ToxCast ER model performance was evaluated for reference chemicals, as well as results of EDSP Tier 1 screening assays in current practice. The ToxCast ER model accuracy was 86% to 93% when compared to reference chemicals and predicted results of EDSP Tier 1 guideline and other uterotrophic studies with 84% to 100% accuracy. The performance of high-throughput assays and ToxCast ER model predictions demonstrates that these methods correctly identify active and inactive reference chemicals, provide a measure of relative ER bioactivity, and rapidly identify chemicals with potential endocrine bioactivities for additional screening and testing. EPA is accepting ToxCast ER model data for 1812 chemicals as alternatives for EDSP Tier 1 ER binding, ER transactivation, and uterotrophic assays.

Staged anticonvulsant screening for chronic epilepsy.

PubMed

Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung-Il; Roach, Bonnie; Pouliot, Wendy; Lu, Kimberly; Swiercz, Waldemar; Dudek, F Edward; Staley, Kevin J

2016-12-01

Current anticonvulsant screening programs are based on seizures evoked in normal animals. One-third of epileptic patients do not respond to the anticonvulsants discovered with these models. We evaluated a tiered program based on chronic epilepsy and spontaneous seizures, with compounds advancing from high-throughput in vitro models to low-throughput in vivo models. Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and lactate dehydrogenase release into culture media as rapid assays for seizure-like activity and cell death, respectively. Compounds that reduced these biochemical measures were retested with in vitro electrophysiological confirmation (i.e., second stage). The third stage involved crossover testing in the kainate model of chronic epilepsy, with blinded analysis of spontaneous seizures after continuous electrographic recordings. We screened 407 compound-concentration combinations. The cyclooxygenase inhibitor, celecoxib, had no effect on seizures evoked in normal brain tissue but demonstrated robust antiseizure activity in all tested models of chronic epilepsy. The use of organotypic hippocampal cultures, where epileptogenesis occurs on a compressed time scale, and where seizure-like activity and seizure-induced cell death can be easily quantified with biomarker assays, allowed us to circumvent the throughput limitations of in vivo chronic epilepsy models. Ability to rapidly screen compounds in a chronic model of epilepsy allowed us to find an anticonvulsant that would be missed by screening in acute models.

Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

EPA Science Inventory

High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

Zebrafish Development: High-throughput Test Systems to Assess Developmental Toxicity

EPA Science Inventory

Abstract Because of its developmental concordance, ease of handling and rapid development, the small teleost, zebrafish (Danio rerio), is frequently promoted as a vertebrate model for medium-throughput developmental screens. This present chapter discusses zebrafish as an altern...

Enabling Large-Scale Design, Synthesis and Validation of Small Molecule Protein-Protein Antagonists

PubMed Central

Koes, David; Khoury, Kareem; Huang, Yijun; Wang, Wei; Bista, Michal; Popowicz, Grzegorz M.; Wolf, Siglinde; Holak, Tad A.; Dömling, Alexander; Camacho, Carlos J.

2012-01-01

Although there is no shortage of potential drug targets, there are only a handful known low-molecular-weight inhibitors of protein-protein interactions (PPIs). One problem is that current efforts are dominated by low-yield high-throughput screening, whose rigid framework is not suitable for the diverse chemotypes present in PPIs. Here, we developed a novel pharmacophore-based interactive screening technology that builds on the role anchor residues, or deeply buried hot spots, have in PPIs, and redesigns these entry points with anchor-biased virtual multicomponent reactions, delivering tens of millions of readily synthesizable novel compounds. Application of this approach to the MDM2/p53 cancer target led to high hit rates, resulting in a large and diverse set of confirmed inhibitors, and co-crystal structures validate the designed compounds. Our unique open-access technology promises to expand chemical space and the exploration of the human interactome by leveraging in-house small-scale assays and user-friendly chemistry to rationally design ligands for PPIs with known structure. PMID:22427896

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

PubMed Central

Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain. PMID:28472077

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening.

PubMed

Fan, Xiaoguang; Wu, Heyun; Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5'-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

PubMed

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

Novel method for high-throughput colony PCR screening in nanoliter-reactors

PubMed Central

Walser, Marcel; Pellaux, Rene; Meyer, Andreas; Bechtold, Matthias; Vanderschuren, Herve; Reinhardt, Richard; Magyar, Joseph; Panke, Sven; Held, Martin

2009-01-01

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. PMID:19282448

A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

PubMed

Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

2013-02-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

Effect-size measures as descriptors of assay quality in high-content screening: A brief review of some available methodologies

USDA-ARS?s Scientific Manuscript database

The field of high-content screening (HCS) typically uses measures of screen quality conceived for fairly straightforward high-throughput screening (HTS) scenarios. However, in contrast to HTS, image-based HCS systems rely on multidimensional readouts reporting biological responses associated with co...

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries

PubMed Central

Watt, Eric D.; Hornung, Michael W.; Hedge, Joan M.; Judson, Richard S.; Crofton, Kevin M.; Houck, Keith A.; Simmons, Steven O.

2016-01-01

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use. PMID:26884060

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

PubMed

Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

2017-12-01

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

Polymer-Based Dense Fluidic Networks for High Throughput Screening with Ultrasensitive Fluorescence Detection

PubMed Central

Okagbare, Paul I.; Soper, Steven A.

2011-01-01

Microfluidics represents a viable platform for performing High Throughput Screening (HTS) due to its ability to automate fluid handling and generate fluidic networks with high number densities over small footprints appropriate for the simultaneous optical interrogation of many screening assays. While most HTS campaigns depend on fluorescence, readers typically use point detection and serially address the assay results significantly lowering throughput or detection sensitivity due to a low duty cycle. To address this challenge, we present here the fabrication of a high density microfluidic network packed into the imaging area of a large field-of-view (FoV) ultrasensitive fluorescence detection system. The fluidic channels were 1, 5 or 10 μm (width), 1 μm (depth) with a pitch of 1–10 μm and each fluidic processor was individually addressable. The fluidic chip was produced from a molding tool using hot embossing and thermal fusion bonding to enclose the fluidic channels. A 40X microscope objective (numerical aperture = 0.75) created a FoV of 200 μm, providing the ability to interrogate ~25 channels using the current fluidic configuration. An ultrasensitive fluorescence detection system with a large FoV was used to transduce fluorescence signals simultaneously from each fluidic processor onto the active area of an electron multiplying charge-coupled device (EMCCD). The utility of these multichannel networks for HTS was demonstrated by carrying out the high throughput monitoring of the activity of an enzyme, APE1, used as a model screening assay. PMID:20872611

High Throughput Assays and Exposure Science (ISES annual meeting)

EPA Science Inventory

High throughput screening (HTS) data characterizing chemical-induced biological activity has been generated for thousands of environmentally-relevant chemicals by the US inter-agency Tox21 and the US EPA ToxCast programs. For a limited set of chemicals, bioactive concentrations r...

High Throughput Exposure Estimation Using NHANES Data (SOT)

EPA Science Inventory

In the ExpoCast project, high throughput (HT) exposure models enable rapid screening of large numbers of chemicals for exposure potential. Evaluation of these models requires empirical exposure data and due to the paucity of human metabolism/exposure data such evaluations includ...

EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES

EPA Science Inventory

Embryonic vascular disruption is an important adverse outcome pathway (AOP) given the knowledge that chemical disruption of early cardiovascular system development leads to broad prenatal defects. High throughput screening (HTS) assays provide potential building blocks for AOP d...

Accounting For Uncertainty in The Application Of High Throughput Datasets

EPA Science Inventory

The use of high throughput screening (HTS) datasets will need to adequately account for uncertainties in the data generation process and propagate these uncertainties through to ultimate use. Uncertainty arises at multiple levels in the construction of predictors using in vitro ...

20180312 - Uncertainty and Variability in High-Throughput Toxicokinetics for Risk Prioritization (SOT)

EPA Science Inventory

Streamlined approaches that use in vitro experimental data to predict chemical toxicokinetics (TK) are increasingly being used to perform risk-based prioritization based upon dosimetric adjustment of high-throughput screening (HTS) data across thousands of chemicals. However, ass...

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.

PubMed

Myers, Stephanie M; Bawn, Ruth H; Bisset, Louise C; Blackburn, Timothy J; Cottyn, Betty; Molyneux, Lauren; Wong, Ai-Ching; Cano, Celine; Clegg, William; Harrington, Ross W; Leung, Hing; Rigoreau, Laurent; Vidot, Sandrine; Golding, Bernard T; Griffin, Roger J; Hammonds, Tim; Newell, David R; Hardcastle, Ian R

2016-08-08

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Sensitive high-throughput screening for the detection of reducing sugars.

PubMed

Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

2012-01-01

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Candidiasis and the impact of flow cytometry on antifungal drug discovery.

PubMed

Ku, Tsun Sheng N; Bernardo, Stella; Walraven, Carla J; Lee, Samuel A

2017-11-01

Invasive candidiasis continues to be associated with significant morbidity and mortality as well as substantial health care costs nationally and globally. One of the contributing factors is the development of resistance to antifungal agents that are already in clinical use. Moreover, there are known treatment limitations with all of the available antifungal agents. Since traditional techniques in novel drug discovery are time consuming, high-throughput screening using flow cytometry presents as a potential tool to identify new antifungal agents that would be useful in the management of these patients. Areas covered: In this review, the authors discuss the use of automated high-throughput screening assays based upon flow cytometry to identify potential antifungals from a library comprised of a large number of bioactive compounds. They also review studies that employed the use of this research methodology that has identified compounds with antifungal activity. Expert opinion: High-throughput screening using flow cytometry has substantially decreased the processing time necessary for screening thousands of compounds, and has helped enhance our understanding of fungal pathogenesis. Indeed, the authors see this technology as a powerful tool to help scientists identify new antifungal agents that can be added to the clinician's arsenal in their fight against invasive candidiasis.

The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, C.A.; Cohen, A.E.

2009-05-26

The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screenedmore » in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.« less

Introducing Bayesian thinking to high-throughput screening for false-negative rate estimation.

PubMed

Wei, Xin; Gao, Lin; Zhang, Xiaolei; Qian, Hong; Rowan, Karen; Mark, David; Peng, Zhengwei; Huang, Kuo-Sen

2013-10-01

High-throughput screening (HTS) has been widely used to identify active compounds (hits) that bind to biological targets. Because of cost concerns, the comprehensive screening of millions of compounds is typically conducted without replication. Real hits that fail to exhibit measurable activity in the primary screen due to random experimental errors will be lost as false-negatives. Conceivably, the projected false-negative rate is a parameter that reflects screening quality. Furthermore, it can be used to guide the selection of optimal numbers of compounds for hit confirmation. Therefore, a method that predicts false-negative rates from the primary screening data is extremely valuable. In this article, we describe the implementation of a pilot screen on a representative fraction (1%) of the screening library in order to obtain information about assay variability as well as a preliminary hit activity distribution profile. Using this training data set, we then developed an algorithm based on Bayesian logic and Monte Carlo simulation to estimate the number of true active compounds and potential missed hits from the full library screen. We have applied this strategy to five screening projects. The results demonstrate that this method produces useful predictions on the numbers of false negatives.

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

A high throughput screen for biomining cellulase activity from metagenomic libraries.

PubMed

Mewis, Keith; Taupp, Marcus; Hallam, Steven J

2011-02-01

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

Application of 2,4-Dinitrophenylhydrazine (DNPH) in High-Throughput Screening for Microorganism Mutants Accumulating 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD)

PubMed Central

Liu, Yang; Cao, Fei; Xiong, Hui; Shen, Yanbing; Wang, Min

2016-01-01

To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9α-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application. PMID:27706217

Application of 2,4-Dinitrophenylhydrazine (DNPH) in High-Throughput Screening for Microorganism Mutants Accumulating 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD).

PubMed

Liu, Yang; Cao, Fei; Xiong, Hui; Shen, Yanbing; Wang, Min

2016-01-01

To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9α-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application.

iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

PubMed

Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

2015-09-01

High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. © 2014 Society for Laboratory Automation and Screening.

A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

PubMed

Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

2016-05-09

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Alkylation sensitivity screens reveal a conserved cross-species functionome

PubMed Central

Svilar, David; Dyavaiah, Madhu; Brown, Ashley R.; Tang, Jiang-bo; Li, Jianfeng; McDonald, Peter R.; Shun, Tong Ying; Braganza, Andrea; Wang, Xiao-hong; Maniar, Salony; St Croix, Claudette M.; Lazo, John S.; Pollack, Ian F.; Begley, Thomas J.; Sobol, Robert W.

2013-01-01

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. PMID:23038810

Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine.

PubMed

Loren, Bradley P; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang; Nagy, Zoltan K; Thompson, David H; Cooks, R Graham

2017-06-01

A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis.

High Throughput, Polymeric Aqueous Two-Phase Printing of Tumor Spheroids

PubMed Central

Atefi, Ehsan; Lemmo, Stephanie; Fyffe, Darcy; Luker, Gary D.; Tavana, Hossein

2014-01-01

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the “patterning” phase containing cells into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models. PMID:25411577

Improving the quality of physician communication with rapid-throughput analysis and report cards.

PubMed

Farrell, Michael H; Christopher, Stephanie A; La Pean Kirschner, Alison; Roedl, Sara J; O'Tool, Faith O; Ahmad, Nadia Y; Farrell, Philip M

2014-11-01

Problems with clinician-patient communication negatively impact newborn screening, genetics, and all of healthcare. Training programs teach communication, but educational methods are not feasible for entire populations of clinicians. To address this healthcare quality gap, we developed a Communication Quality Assurance intervention. Child health providers volunteered for a randomized controlled trial of assessment and a report card. Participants provided telephone counseling to a standardized parent regarding a newborn screening result showing heterozygous status for cystic fibrosis or sickle cell disease. Our rapid-throughput timeline allows individualized feedback within a week. Two encounters were recorded (baseline and after a random sample received the report card) and abstracted for four groups of communication quality indicators. 92 participants finished both counseling encounters within our rapid-throughput time limits. Participants randomized to receive the report card improved communication behaviors more than controls, including request for teach-back (p<0.01), opening behaviors (p=0.01), anticipate/validate emotion (p<0.001) and the ratio of explained to unexplained jargon words (p<0.03). The rapid-throughput report card is effective at improving specific communication behaviors. Communication can be taught, but this project shows how healthcare organizations can assure communication quality everywhere. Further implementation could improve newborn screening, genetics, and healthcare in general. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Fun with High Throughput Toxicokinetics (CalEPA webinar)

EPA Science Inventory

Thousands of chemicals have been profiled by high-throughput screening (HTS) programs such as ToxCast and Tox21. These chemicals are tested in part because there are limited or no data on hazard, exposure, or toxicokinetics (TK). TK models aid in predicting tissue concentrations ...

HTTK: R Package for High-Throughput Toxicokinetics

EPA Science Inventory

Thousands of chemicals have been profiled by high-throughput screening programs such as ToxCast and Tox21; these chemicals are tested in part because most of them have limited or no data on hazard, exposure, or toxicokinetics. Toxicokinetic models aid in predicting tissue concent...

High-throughput screening for combinatorial thin-film library of thermoelectric materials.

PubMed

Watanabe, Masaki; Kita, Takuji; Fukumura, Tomoteru; Ohtomo, Akira; Ueno, Kazunori; Kawasaki, Masashi

2008-01-01

A high-throughput method has been developed to evaluate the Seebeck coefficient and electrical resistivity of combinatorial thin-film libraries of thermoelectric materials from room temperature to 673 K. Thin-film samples several millimeters in size were deposited on an integrated Al2O3 substrate with embedded lead wires and local heaters for measurement of the thermopower under a controlled temperature gradient. An infrared camera was used for real-time observation of the temperature difference Delta T between two electrical contacts on the sample to obtain the Seebeck coefficient. The Seebeck coefficient and electrical resistivity of constantan thin films were shown to be almost identical to standard data for bulk constantan. High-throughput screening was demonstrated for a thermoelectric Mg-Si-Ge combinatorial library.

Correction of Microplate Data from High-Throughput Screening.

PubMed

Wang, Yuhong; Huang, Ruili

2016-01-01

High-throughput screening (HTS) makes it possible to collect cellular response data from a large number of cell lines and small molecules in a timely and cost-effective manner. The errors and noises in the microplate-formatted data from HTS have unique characteristics, and they can be generally grouped into three categories: run-wise (temporal, multiple plates), plate-wise (background pattern, single plate), and well-wise (single well). In this chapter, we describe a systematic solution for identifying and correcting such errors and noises, mainly basing on pattern recognition and digital signal processing technologies.

Detecting and removing multiplicative spatial bias in high-throughput screening technologies.

PubMed

Caraus, Iurie; Mazoure, Bogdan; Nadon, Robert; Makarenkov, Vladimir

2017-10-15

Considerable attention has been paid recently to improve data quality in high-throughput screening (HTS) and high-content screening (HCS) technologies widely used in drug development and chemical toxicity research. However, several environmentally- and procedurally-induced spatial biases in experimental HTS and HCS screens decrease measurement accuracy, leading to increased numbers of false positives and false negatives in hit selection. Although effective bias correction methods and software have been developed over the past decades, almost all of these tools have been designed to reduce the effect of additive bias only. Here, we address the case of multiplicative spatial bias. We introduce three new statistical methods meant to reduce multiplicative spatial bias in screening technologies. We assess the performance of the methods with synthetic and real data affected by multiplicative spatial bias, including comparisons with current bias correction methods. We also describe a wider data correction protocol that integrates methods for removing both assay and plate-specific spatial biases, which can be either additive or multiplicative. The methods for removing multiplicative spatial bias and the data correction protocol are effective in detecting and cleaning experimental data generated by screening technologies. As our protocol is of a general nature, it can be used by researchers analyzing current or next-generation high-throughput screens. The AssayCorrector program, implemented in R, is available on CRAN. makarenkov.vladimir@uqam.ca. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

A high-throughput screen for single gene activities: isolation of apoptosis inducers.

PubMed

Albayrak, Timur; Grimm, Stefan

2003-05-16

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression library into a separate population of cells in a high-throughput mode. The screen allows one to achieve a hitherto unattained sensitivity in expression cloning which was exploited in a first read-out to clone apoptosis-inducing genes. This led to the isolation of several genes whose proteins induce distinct phenotypes of apoptosis in 293T cells. One of the isolated genes is the tumor suppressor cytochrome b(L) (cybL), a component of the respiratory chain complex II, that diminishes the activity of this complex for apoptosis induction. This gene is more efficient and specific for causing cell death than a drug with the same activity. These results suggest further applications, both of the isolated genes and the screen.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

PubMed

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Recent advances in quantitative high throughput and high content data analysis.

PubMed

Moutsatsos, Ioannis K; Parker, Christian N

2016-01-01

High throughput screening has become a basic technique with which to explore biological systems. Advances in technology, including increased screening capacity, as well as methods that generate multiparametric readouts, are driving the need for improvements in the analysis of data sets derived from such screens. This article covers the recent advances in the analysis of high throughput screening data sets from arrayed samples, as well as the recent advances in the analysis of cell-by-cell data sets derived from image or flow cytometry application. Screening multiple genomic reagents targeting any given gene creates additional challenges and so methods that prioritize individual gene targets have been developed. The article reviews many of the open source data analysis methods that are now available and which are helping to define a consensus on the best practices to use when analyzing screening data. As data sets become larger, and more complex, the need for easily accessible data analysis tools will continue to grow. The presentation of such complex data sets, to facilitate quality control monitoring and interpretation of the results will require the development of novel visualizations. In addition, advanced statistical and machine learning algorithms that can help identify patterns, correlations and the best features in massive data sets will be required. The ease of use for these tools will be important, as they will need to be used iteratively by laboratory scientists to improve the outcomes of complex analyses.

Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase

PubMed Central

Babaoglu, Kerim; Simeonov, Anton; Irwin, John J.; Nelson, Michael E.; Feng, Brian; Thomas, Craig J.; Cancian, Laura; Costi, M. Paola; Maltby, David A.; Jadhav, Ajit; Inglese, James; Austin, Christopher P.; Shoichet, Brian K.

2009-01-01

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate “hit lists”; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against β-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 µM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens. PMID:18333608

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

PubMed

Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L; Merrick, B Alex; Teng, Christina T; Tice, Raymond R

2015-10-01

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library

PubMed Central

Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R.; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L.; Merrick, B. Alex; Teng, Christina T.; Tice, Raymond R.

2015-01-01

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase. PMID:26141389

High-Throughput Models for Exposure-Based Chemical Prioritization in the ExpoCast Project

EPA Science Inventory

The United States Environmental Protection Agency (U.S. EPA) must characterize potential risks to human health and the environment associated with manufacture and use of thousands of chemicals. High-throughput screening (HTS) for biological activity allows the ToxCast research pr...

Use of High-Throughput Testing and Approaches for Evaluating Chemical Risk-Relevance to Humans

EPA Science Inventory

ToxCast is profiling the bioactivity of thousands of chemicals based on high-throughput screening (HTS) and computational models that integrate knowledge of biological systems and in vivo toxicities. Many of these assays probe signaling pathways and cellular processes critical to...

High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization

EPA Science Inventory

The U.S. EPA must consider lists of hundreds to thousands of chemicals when allocating resources to identify risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro have allowed the ToxCastTM program to identify...

High Throughput Genotoxicity Profiling of the US EPA ToxCast Chemical Library

EPA Science Inventory

A key aim of the ToxCast project is to investigate modern molecular and genetic high content and high throughput screening (HTS) assays, along with various computational tools to supplement and perhaps replace traditional assays for evaluating chemical toxicity. Genotoxicity is a...

Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

EPA Science Inventory

Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

Developing a gene biomarker at the tipping point of adaptive and adverse responses in human bronchial epithelial cells

EPA Science Inventory

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk...

Novel screening techniques for ion channel targeting drugs

PubMed Central

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation. PMID:26556400

Novel screening techniques for ion channel targeting drugs.

PubMed

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

PubMed

Meng, Juncai; Lai, Ming-Tain; Munshi, Vandna; Grobler, Jay; McCauley, John; Zuck, Paul; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Adam, Gregory C

2015-06-01

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. © 2015 Society for Laboratory Automation and Screening.

Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities

PubMed Central

2010-01-01

Background Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups. Results We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects. Conclusions The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities. PMID:20482787

Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities.

PubMed

Tolopko, Andrew N; Sullivan, John P; Erickson, Sean D; Wrobel, David; Chiang, Su L; Rudnicki, Katrina; Rudnicki, Stewart; Nale, Jennifer; Selfors, Laura M; Greenhouse, Dara; Muhlich, Jeremy L; Shamu, Caroline E

2010-05-18

Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups. We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects. The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities.

20180312 - Evaluating the applicability of read-across tools and high throughput screening data for food relevant chemicals (SOT)

EPA Science Inventory

Alternative toxicity assessment methods to characterize the hazards of chemical substances have been proposed to reduce animal testing and screen thousands of chemicals in an efficient manner. Resources to accomplish these goals include utilizing large in vitro chemical screening...

The U.S. EPA's ToxCast Chemical Screening Program and Predictive Modeling of Toxicity

EPA Science Inventory

The ToxCast program was developed by the U.S. EPA's National Center for Computational Toxicology to provide cost-effective high-throughput screening for the potential toxicity of thousands of chemicals. Phase I screened 309 compounds in over 500 assays to evaluate concentration-...

A High-Throughput Screening Assay to Detect Thyroperoxidase Inhibitors (Teratology Society)

EPA Science Inventory

In support of the Endocrine Disruption Screening Program (EDSP21), the US EPA ToxCast program is developing assays to enable screening for chemicals that may disrupt thyroid hormone synthesis. Thyroperoxidase (TPO) is critical for TH synthesis and is a known target of thyroid-dis...

The ToxCast Pathway Database for Identifying Toxicity Signatures and Potential Modes of Action from Chemical Screening Data

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA), through its ToxCast program, is developing predictive toxicity approaches that will use in vitro high-throughput screening (HTS), high-content screening (HCS) and toxicogenomic data to predict in vivo toxicity phenotypes. There are ...

Using information from historical high-throughput screens to predict active compounds.

PubMed

Riniker, Sereina; Wang, Yuan; Jenkins, Jeremy L; Landrum, Gregory A

2014-07-28

Modern high-throughput screening (HTS) is a well-established approach for hit finding in drug discovery that is routinely employed in the pharmaceutical industry to screen more than a million compounds within a few weeks. However, as the industry shifts to more disease-relevant but more complex phenotypic screens, the focus has moved to piloting smaller but smarter chemically/biologically diverse subsets followed by an expansion around hit compounds. One standard method for doing this is to train a machine-learning (ML) model with the chemical fingerprints of the tested subset of molecules and then select the next compounds based on the predictions of this model. An alternative approach would be to take advantage of the wealth of bioactivity information contained in older (full-deck) screens using so-called HTS fingerprints, where each element of the fingerprint corresponds to the outcome of a particular assay, as input to machine-learning algorithms. We constructed HTS fingerprints using two collections of data: 93 in-house assays and 95 publicly available assays from PubChem. For each source, an additional set of 51 and 46 assays, respectively, was collected for testing. Three different ML methods, random forest (RF), logistic regression (LR), and naïve Bayes (NB), were investigated for both the HTS fingerprint and a chemical fingerprint, Morgan2. RF was found to be best suited for learning from HTS fingerprints yielding area under the receiver operating characteristic curve (AUC) values >0.8 for 78% of the internal assays and enrichment factors at 5% (EF(5%)) >10 for 55% of the assays. The RF(HTS-fp) generally outperformed the LR trained with Morgan2, which was the best ML method for the chemical fingerprint, for the majority of assays. In addition, HTS fingerprints were found to retrieve more diverse chemotypes. Combining the two models through heterogeneous classifier fusion led to a similar or better performance than the best individual model for all assays. Further validation using a pair of in-house assays and data from a confirmatory screen--including a prospective set of around 2000 compounds selected based on our approach--confirmed the good performance. Thus, the combination of machine-learning with HTS fingerprints and chemical fingerprints utilizes information from both domains and presents a very promising approach for hit expansion, leading to more hits. The source code used with the public data is provided.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.

PubMed

Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang, Leyu; Moore, Jennifer; Kuo, Ming-Shang T; LaMarr, William A; Ozbal, Can C; Bhat, B Ganesh

2008-10-03

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K(m)=10.5 microM). The assay was highly reproducible with an average Z' value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC(50) values of 0.88 and 0.12 microM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 microM--14% conformation rate). Of the confirmed hits 172 had IC(50) values of <10 microM, including 111 <1 microM and 48 <100 nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Environmental surveillance and monitoring the next frontier for pathway-based high throughput screening

EPA Science Inventory

In response to a proposed vision and strategy for toxicity testing in the 21st century nascent high throughput toxicology (HTT) programs have tested thousands of chemicals in hundreds of pathway-based biological assays. Although, to date, use of HTT data for safety assessment of ...

20180311 - Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells (SOT)

EPA Science Inventory

Increasing efficiency and declining cost of generating whole transcriptome profiles has made high-throughput transcriptomics a practical option for chemical bioactivity screening. The resulting data output provides information on the expression of thousands of genes and is amenab...

High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells

EPA Science Inventory

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2,060 chemical samples...

High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization (Annual Meeting of ISES)

EPA Science Inventory

The U.S. EPA must consider thousands of chemicals when allocating resources to assess risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro are being implemented in the ToxCastTM program to rapidly characteri...

Incorporating High-Throughput Exposure Predictions with Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

EPA Science Inventory

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast™ HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compare...

High Throughput Assays for Exposure Science (NIEHS OHAT Staff Meeting presentation)

EPA Science Inventory

High throughput screening (HTS) data that characterize chemically induced biological activity have been generated for thousands of chemicals by the US interagency Tox21 and the US EPA ToxCast programs. In many cases there are no data available for comparing bioactivity from HTS w...

Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells

EPA Science Inventory

Increasing efficiency and declining cost of generating whole transcriptome profiles has made high-throughput transcriptomics a practical option for chemical bioactivity screening. The resulting data output provides information on the expression of thousands of genes and is amenab...

Differentiating pathway-specific from nonspecific effects in high-throughput toxicity data: A foundation for prioritizing adverse outcome pathway development

EPA Science Inventory

The U.S. Environmental Protection Agency’s ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential ...

“httk”: EPA’s Tool for High Throughput Toxicokinetics (CompTox CoP)

EPA Science Inventory

Thousands of chemicals have been pro?led by high-throughput screening programs such as ToxCast and Tox21; these chemicals are tested in part because most of them have limited or no data on hazard, exposure, or toxicokinetics. Toxicokinetic models aid in predicting tissue concentr...

Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

EPA Science Inventory

In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

Complementing in vitro hazard assessment with exposure and pharmacokinetics considerations for chemical prioritization

EPA Science Inventory

Traditional toxicity testing involves a large investment in resources, often using low-throughput in vivo animal studies for limited numbers of chemicals. An alternative strategy is the emergence of high-throughput (HT) in vitro assays as a rapid, cost-efficient means to screen t...

Using Alternative Approaches to Prioritize Testing for the Universe of Chemicals with Potential for Human Exposure (WC9)

EPA Science Inventory

One use of alternative methods is to target animal use at only those chemicals and tests that are absolutely necessary. We discuss prioritization of testing based on high-throughput screening assays (HTS), QSAR modeling, high-throughput toxicokinetics (HTTK), and exposure modelin...

Harnessing High-Throughput Monitoring Methods to Strengthen 21st Century Risk-Based Evaluations (SETAC Presentation)

EPA Science Inventory

Over the past ten years, the US government has invested in high-throughput (HT) methods to screen chemicals for biological activity. Under the interagency Tox21 consortium and the US Environmental Protection Agency’s (EPA) ToxCast™ program, thousands of chemicals have...

A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus.

PubMed

Cheng, Han; Koning, Katie; O'Hearn, Aileen; Wang, Minxiu; Rumschlag-Booms, Emily; Varhegyi, Elizabeth; Rong, Lijun

2015-11-24

Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

PubMed

Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

2015-11-01

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

PubMed Central

Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

2015-01-01

Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

PubMed

Ehrenworth, Amy M; Claiborne, Tauris; Peralta-Yahya, Pamela

2017-10-17

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

PubMed Central

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, ChiHye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I.; Lee, Hoonkyung

2016-01-01

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10−3 bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc– or V–porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials. PMID:26902156

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide.

PubMed

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, ChiHye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I; Lee, Hoonkyung

2016-02-23

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10(-3) bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc- or V-porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials.

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

NASA Astrophysics Data System (ADS)

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, Chihye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I.; Lee, Hoonkyung

2016-02-01

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10-3 bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc- or V-porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials.

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.

PubMed

Morgens, David W; Wainberg, Michael; Boyle, Evan A; Ursu, Oana; Araya, Carlos L; Tsui, C Kimberly; Haney, Michael S; Hess, Gaelen T; Han, Kyuho; Jeng, Edwin E; Li, Amy; Snyder, Michael P; Greenleaf, William J; Kundaje, Anshul; Bassik, Michael C

2017-05-05

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

PubMed Central

Morgens, David W.; Wainberg, Michael; Boyle, Evan A.; Ursu, Oana; Araya, Carlos L.; Tsui, C. Kimberly; Haney, Michael S.; Hess, Gaelen T.; Han, Kyuho; Jeng, Edwin E.; Li, Amy; Snyder, Michael P.; Greenleaf, William J.; Kundaje, Anshul; Bassik, Michael C.

2017-01-01

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens. PMID:28474669

Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

PubMed

Yang, Bin; Lamb, Michelle L; Zhang, Tao; Hennessy, Edward J; Grewal, Gurmit; Sha, Li; Zambrowski, Mark; Block, Michael H; Dowling, James E; Su, Nancy; Wu, Jiaquan; Deegan, Tracy; Mikule, Keith; Wang, Wenxian; Kaspera, Rüdiger; Chuaqui, Claudio; Chen, Huawei

2014-12-11

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

Human Papillomavirus Biology, Pathogenesis, and Potential for Drug Discovery: A Literature Review for HIV Nurse Clinical Scientists.

PubMed

Walhart, Tara

2015-01-01

Persistent oncogenic human papillomavirus (HPV) infection increases the probability that precancerous anal high-grade squamous intraepithelial lesions will progress to invasive anal cancer. Anal neoplasia associated with HPV disproportionately affects HIV-infected individuals, especially men who have sex with men. Prevention is limited to HPV vaccine recommendations, highlighting the need for new treatments. The purpose of this review is to provide HIV information to nurse clinical scientists about HPV-related cancer to highlight the connection between: (a) HPV biology and pathogenesis and (b) the development of drugs and novel therapeutic methods using high-throughput screening. PubMed and CINAHL were used to search the literature to determine HPV-related epidemiology, biology, and use of high-throughput screening for drug discovery. Several events in the HPV life cycle have the potential to be developed into biologic targets for drug discovery using the high-throughput screening technique, which has been successfully used to identify compounds to inhibit HPV infections. Copyright © 2015 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.

A multilayer microdevice for cell-based high-throughput drug screening

NASA Astrophysics Data System (ADS)

Liu, Chong; Wang, Lei; Xu, Zheng; Li, Jingmin; Ding, Xiping; Wang, Qi; Chunyu, Li

2012-06-01

A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. PMID:21712420

Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor

PubMed Central

Wu, Yang; Tapia, Phillip H.; Jarvik, Jonathan; Waggoner, Alan S.; Sklar, Larry A.

2014-01-01

We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. PMID:24510772

Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory.

PubMed

Grandjean, Geoffrey; Graham, Ryan; Bartholomeusz, Geoffrey

2011-11-01

In recent years high throughput screening operations have become a critical application in functional and translational research. Although a seemingly unmanageable amount of data is generated by these high-throughput, large-scale techniques, through careful planning, an effective Laboratory Information Management System (LIMS) can be developed and implemented in order to streamline all phases of a workflow. Just as important as data mining and analysis procedures at the end of complex processes is the tracking of individual steps of applications that generate such data. Ultimately, the use of a customized LIMS will enable users to extract meaningful results from large datasets while trusting the robustness of their assays. To illustrate the design of a custom LIMS, this practical example is provided to highlight the important aspects of the design of a LIMS to effectively modulate all aspects of an siRNA screening service. This system incorporates inventory management, control of workflow, data handling and interaction with investigators, statisticians and administrators. All these modules are regulated in a synchronous manner within the LIMS. © 2011 Bentham Science Publishers

Entropy as a Gene-Like Performance Indicator Promoting Thermoelectric Materials.

PubMed

Liu, Ruiheng; Chen, Hongyi; Zhao, Kunpeng; Qin, Yuting; Jiang, Binbin; Zhang, Tiansong; Sha, Gang; Shi, Xun; Uher, Ctirad; Zhang, Wenqing; Chen, Lidong

2017-10-01

High-throughput explorations of novel thermoelectric materials based on the Materials Genome Initiative paradigm only focus on digging into the structure-property space using nonglobal indicators to design materials with tunable electrical and thermal transport properties. As the genomic units, following the biogene tradition, such indicators include localized crystal structural blocks in real space or band degeneracy at certain points in reciprocal space. However, this nonglobal approach does not consider how real materials differentiate from others. Here, this study successfully develops a strategy of using entropy as the global gene-like performance indicator that shows how multicomponent thermoelectric materials with high entropy can be designed via a high-throughput screening method. Optimizing entropy works as an effective guide to greatly improve the thermoelectric performance through either a significantly depressed lattice thermal conductivity down to its theoretical minimum value and/or via enhancing the crystal structure symmetry to yield large Seebeck coefficients. The entropy engineering using multicomponent crystal structures or other possible techniques provides a new avenue for an improvement of the thermoelectric performance beyond the current methods and approaches. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

PubMed

Chen, Wenjin; Wong, Chung; Vosburgh, Evan; Levine, Arnold J; Foran, David J; Xu, Eugenia Y

2014-07-08

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application - SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary "Manual Initialize" and "Hand Draw" tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

Accounting Artifacts in High-Throughput Toxicity Assays.

PubMed

Hsieh, Jui-Hua

2016-01-01

Compound activity identification is the primary goal in high-throughput screening (HTS) assays. However, assay artifacts including both systematic (e.g., compound auto-fluorescence) and nonsystematic (e.g., noise) complicate activity interpretation. In addition, other than the traditional potency parameter, half-maximal effect concentration (EC50), additional activity parameters (e.g., point-of-departure, POD) could be derived from HTS data for activity profiling. A data analysis pipeline has been developed to handle the artifacts and to provide compound activity characterization with either binary or continuous metrics. This chapter outlines the steps in the pipeline using Tox21 glucocorticoid receptor (GR) β-lactamase assays, including the formats to identify either agonists or antagonists, as well as the counter-screen assays for identifying artifacts as examples. The steps can be applied to other lower-throughput assays with concentration-response data.

An image analysis toolbox for high-throughput C. elegans assays

PubMed Central

Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H.; Riklin-Raviv, Tammy; Conery, Annie L.; O’Rourke, Eyleen J.; Sokolnicki, Katherine L.; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E.; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M.; Carpenter, Anne E.

2012-01-01

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available via the open-source CellProfiler project and enables objective scoring of whole-animal high-throughput image-based assays of C. elegans for the study of diverse biological pathways relevant to human disease. PMID:22522656

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files.

PubMed

Bell, Andrew S; Bradley, Joseph; Everett, Jeremy R; Loesel, Jens; McLoughlin, David; Mills, James; Peakman, Marie-Claire; Sharp, Robert E; Williams, Christine; Zhu, Hongyao

2016-11-01

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.

Microfluidics-assisted in vitro drug screening and carrier production

PubMed Central

Tsui, Jonathan H.; Lee, Woohyuk; Pun, Suzie H.; Kim, Jungkyu; Kim, Deok-Ho

2013-01-01

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development. PMID:23856409

The planning and establishment of a sample preparation laboratory for drug discovery

PubMed Central

Dufresne, Claude

2000-01-01

Nature has always been a productive source of new drugs. With the advent of high-throughput screening, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural product sources (micro-organisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts. As sample preparation protocols become more involved, automation can help to achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed. The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on solid-phase extraction cartridges, dry and weigh each fraction, re-dissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates. PMID:18924691

A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards

PubMed Central

Mordwinkin, Nicholas M.; Burridge, Paul W.; Wu, Joseph C.

2013-01-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach. PMID:23229562

An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

PubMed

Zhai, Yufeng; Chen, Kaisheng; Zhong, Yang; Zhou, Bin; Ainscow, Edward; Wu, Ying-Ta; Zhou, Yingyao

2016-09-01

The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-QC led to a noticeably stronger structure-activity relationship. The method was further tested in several independent HTS runs, where QC results were sampled for experimental validation. Significantly increased hit confirmation rates were obtained after the QC steps, confirming that the proposed method was effective in enriching true-positive hits. An implementation of the algorithm is available to the screening community. © 2016 Society for Laboratory Automation and Screening.

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

PubMed

Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

2014-05-22

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Harnessing Diversity in Wheat to Enhance Grain Yield, Climate Resilience, Disease and Insect Pest Resistance and Nutrition Through Conventional and Modern Breeding Approaches

PubMed Central

Mondal, Suchismita; Rutkoski, Jessica E.; Velu, Govindan; Singh, Pawan K.; Crespo-Herrera, Leonardo A.; Guzmán, Carlos; Bhavani, Sridhar; Lan, Caixia; He, Xinyao; Singh, Ravi P.

2016-01-01

Current trends in population growth and consumption patterns continue to increase the demand for wheat, a key cereal for global food security. Further, multiple abiotic challenges due to climate change and evolving pathogen and pests pose a major concern for increasing wheat production globally. Triticeae species comprising of primary, secondary, and tertiary gene pools represent a rich source of genetic diversity in wheat. The conventional breeding strategies of direct hybridization, backcrossing and selection have successfully introgressed a number of desirable traits associated with grain yield, adaptation to abiotic stresses, disease resistance, and bio-fortification of wheat varieties. However, it is time consuming to incorporate genes conferring tolerance/resistance to multiple stresses in a single wheat variety by conventional approaches due to limitations in screening methods and the lower probabilities of combining desirable alleles. Efforts on developing innovative breeding strategies, novel tools and utilizing genetic diversity for new genes/alleles are essential to improve productivity, reduce vulnerability to diseases and pests and enhance nutritional quality. New technologies of high-throughput phenotyping, genome sequencing and genomic selection are promising approaches to maximize progeny screening and selection to accelerate the genetic gains in breeding more productive varieties. Use of cisgenic techniques to transfer beneficial alleles and their combinations within related species also offer great promise especially to achieve durable rust resistance. PMID:27458472

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

PubMed

Hartwell, Kimberly A; Miller, Peter G; Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2013-12-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

PubMed

Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

2012-03-01

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

PubMed Central

Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2014-01-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening. PMID:24161946

A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases

PubMed Central

Guarnieri, Michael T.; Blagg, Brian S. J.

2011-01-01

Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069

In Vitro Toxicity Screening Technique for Volatile Substances ...

EPA Pesticide Factsheets

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for high throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules (e.g., signaling molecules nitric oxide (NO) and carbon monoxide (CO) in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are

High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

PubMed

Auray-Blais, Christiane; Maranda, Bruno; Lavoie, Pamela

2014-09-25

Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases. A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound. The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 μmol/l and from 1.0 to 20.9 μmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life. This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemiluminescence analyzer of NOx as a high-throughput screening tool in selective catalytic reduction of NO

PubMed Central

Oh, Kwang Seok; Woo, Seong Ihl

2011-01-01

A chemiluminescence-based analyzer of NOx gas species has been applied for high-throughput screening of a library of catalytic materials. The applicability of the commercial NOx analyzer as a rapid screening tool was evaluated using selective catalytic reduction of NO gas. A library of 60 binary alloys composed of Pt and Co, Zr, La, Ce, Fe or W on Al2O3 substrate was tested for the efficiency of NOx removal using a home-built 64-channel parallel and sequential tubular reactor. The NOx concentrations measured by the NOx analyzer agreed well with the results obtained using micro gas chromatography for a reference catalyst consisting of 1 wt% Pt on γ-Al2O3. Most alloys showed high efficiency at 275 °C, which is typical of Pt-based catalysts for selective catalytic reduction of NO. The screening with NOx analyzer allowed to select Pt-Ce(X) (X=1–3) and Pt–Fe(2) as the optimal catalysts for NOx removal: 73% NOx conversion was achieved with the Pt–Fe(2) alloy, which was much better than the results for the reference catalyst and the other library alloys. This study demonstrates a sequential high-throughput method of practical evaluation of catalysts for the selective reduction of NO. PMID:27877438

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

PubMed

Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

2008-12-01

Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Mass spectrometry-driven drug discovery for development of herbal medicine.

PubMed

Zhang, Aihua; Sun, Hui; Wang, Xijun

2018-05-01

Herbal medicine (HM) has made a major contribution to the drug discovery process with regard to identifying products compounds. Currently, more attention has been focused on drug discovery from natural compounds of HM. Despite the rapid advancement of modern analytical techniques, drug discovery is still a difficult and lengthy process. Fortunately, mass spectrometry (MS) can provide us with useful structural information for drug discovery, has been recognized as a sensitive, rapid, and high-throughput technology for advancing drug discovery from HM in the post-genomic era. It is essential to develop an efficient, high-quality, high-throughput screening method integrated with an MS platform for early screening of candidate drug molecules from natural products. We have developed a new chinmedomics strategy reliant on MS that is capable of capturing the candidate molecules, facilitating their identification of novel chemical structures in the early phase; chinmedomics-guided natural product discovery based on MS may provide an effective tool that addresses challenges in early screening of effective constituents of herbs against disease. This critical review covers the use of MS with related techniques and methodologies for natural product discovery, biomarker identification, and determination of mechanisms of action. It also highlights high-throughput chinmedomics screening methods suitable for lead compound discovery illustrated by recent successes. © 2016 Wiley Periodicals, Inc.

The Importance of Juvenile Root Traits for Crop Yields

NASA Astrophysics Data System (ADS)

White, Philip; Adu, Michael; Broadley, Martin; Brown, Lawrie; Dupuy, Lionel; George, Timothy; Graham, Neil; Hammond, John; Hayden, Rory; Neugebauer, Konrad; Nightingale, Mark; Ramsay, Gavin; Thomas, Catherine; Thompson, Jacqueline; Wishart, Jane; Wright, Gladys

2014-05-01

Genetic variation in root system architecture (RSA) is an under-exploited breeding resource. This is partly a consequence of difficulties in the rapid and accurate assessment of subterranean root systems. However, although the characterisation of root systems of large plants in the field are both time-consuming and labour-intensive, high-throughput (HTP) screens of root systems of juvenile plants can be performed in the field, glasshouse or laboratory. It is hypothesised that improving the root systems of juvenile plants can accelerate access to water and essential mineral elements, leading to rapid crop establishment and, consequently, greater yields. This presentation will illustrate how aspects of the juvenile root systems of potato (Solanum tuberosum L.) and oilseed rape (OSR; Brassica napus L.) correlate with crop yields and examine the reasons for such correlations. It will first describe the significant positive relationships between early root system development, phosphorus acquisition, canopy establishment and eventual yield among potato genotypes. It will report the development of a glasshouse assay for root system architecture (RSA) of juvenile potato plants, the correlations between root system architectures measured in the glasshouse and field, and the relationships between aspects of the juvenile root system and crop yields under drought conditions. It will then describe the development of HTP systems for assaying RSA of OSR seedlings, the identification of genetic loci affecting RSA in OSR, the development of mathematical models describing resource acquisition by OSR, and the correlations between root traits recorded in the HTP systems and yields of OSR in the field.

Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

PubMed

Heinig, Uwe; Scholz, Susanne; Dahm, Pia; Grabowy, Udo; Jennewein, Stefan

2010-08-01

Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures. Copyright 2010 Elsevier Inc. All rights reserved.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

High-Throughput Screening of Na(V)1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument.

PubMed

Chambers, Chris; Witton, Ian; Adams, Cathryn; Marrington, Luke; Kammonen, Juha

2016-03-01

Voltage-gated sodium (Na(V)) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na(V)1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na(V)1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube(®) is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na(V)1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC50 assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC50 potencies generated on the Qube correlate with data generated on the more established Sophion QPatch(®) APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na(V)1.7 in a robust and reliable APC assay for compound screening.

Survey of ecotoxicologically-relevant reproductive endpoint coverage within the ECOTOX database across ToxCast ER agonists (ASCCT)

EPA Science Inventory

The U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) has been charged with screening thousands of chemicals for their potential to affect the endocrine systems of humans and wildlife. In vitro high throughput screening (HTS) assays have been proposed as a way to prioritize...

A multi-analyte profile of serum proteins to screen for toxicological effects of anticholinesterase insecticides in the rat

EPA Science Inventory

The development of high throughput biochemical screens could be useful to assess the broad spectrum of physiological effects of environmental toxicants. To explore the prospect of using a screen in an in vivo exposure scenario, we applied a commercially available multianalyte pro...

Advantages of Crystallographic Fragment Screening: Functional and Mechanistic Insights from a Powerful Platform for Efficient Drug Discovery

PubMed Central

Patel, Disha; Bauman, Joseph D.; Arnold, Eddy

2015-01-01

X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this “chemical interrogation” of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility. PMID:25117499

TeraSCREEN: multi-frequency multi-mode Terahertz screening for border checks

NASA Astrophysics Data System (ADS)

Alexander, Naomi E.; Alderman, Byron; Allona, Fernando; Frijlink, Peter; Gonzalo, Ramón; Hägelen, Manfred; Ibáñez, Asier; Krozer, Viktor; Langford, Marian L.; Limiti, Ernesto; Platt, Duncan; Schikora, Marek; Wang, Hui; Weber, Marc Andree

2014-06-01

The challenge for any security screening system is to identify potentially harmful objects such as weapons and explosives concealed under clothing. Classical border and security checkpoints are no longer capable of fulfilling the demands of today's ever growing security requirements, especially with respect to the high throughput generally required which entails a high detection rate of threat material and a low false alarm rate. TeraSCREEN proposes to develop an innovative concept of multi-frequency multi-mode Terahertz and millimeter-wave detection with new automatic detection and classification functionalities. The system developed will demonstrate, at a live control point, the safe automatic detection and classification of objects concealed under clothing, whilst respecting privacy and increasing current throughput rates. This innovative screening system will combine multi-frequency, multi-mode images taken by passive and active subsystems which will scan the subjects and obtain complementary spatial and spectral information, thus allowing for automatic threat recognition. The TeraSCREEN project, which will run from 2013 to 2016, has received funding from the European Union's Seventh Framework Programme under the Security Call. This paper will describe the project objectives and approach.

Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole.

PubMed

Chia, Wan Ni; Lee, Yan Quan; Tan, Kevin Shyong-Wei

2017-01-01

Malaria, despite being one of the world's oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay. Copyright © 2016 Elsevier Inc. All rights reserved.

IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening

PubMed Central

Tidten-Luksch, Naomi; Grimaldi, Raffaella; Torrie, Leah S.; Frearson, Julie A.; Hunter, William N.; Brenk, Ruth

2012-01-01

CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens. PMID:22563402

Advantages of crystallographic fragment screening: functional and mechanistic insights from a powerful platform for efficient drug discovery.

PubMed

Patel, Disha; Bauman, Joseph D; Arnold, Eddy

2014-01-01

X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this "chemical interrogation" of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility. Copyright © 2014. Published by Elsevier Ltd.

Identifying Candidate Chemical-Disease Linkages ...

EPA Pesticide Factsheets

Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment. Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment.

Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

NASA Technical Reports Server (NTRS)

Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

2010-01-01

In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms.

PubMed

Ingham, Colin J; Sprenkels, Ad; Bomer, Johan; Molenaar, Douwe; van den Berg, Albert; van Hylckama Vlieg, Johan E T; de Vos, Willem M

2007-11-13

A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 x 7 mum, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolony in 10,000. (iii) In screening for an enzyme-based, nondominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without antibiotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of beta-galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

Parallel production and verification of protein products using a novel high-throughput screening method.

PubMed

Tegel, Hanna; Yderland, Louise; Boström, Tove; Eriksson, Cecilia; Ukkonen, Kaisa; Vasala, Antti; Neubauer, Peter; Ottosson, Jenny; Hober, Sophia

2011-08-01

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Precision and recall estimates for two-hybrid screens

PubMed Central

Huang, Hailiang; Bader, Joel S.

2009-01-01

Motivation: Yeast two-hybrid screens are an important method to map pairwise protein interactions. This method can generate spurious interactions (false discoveries), and true interactions can be missed (false negatives). Previously, we reported a capture–recapture estimator for bait-specific precision and recall. Here, we present an improved method that better accounts for heterogeneity in bait-specific error rates. Result: For yeast, worm and fly screens, we estimate the overall false discovery rates (FDRs) to be 9.9%, 13.2% and 17.0% and the false negative rates (FNRs) to be 51%, 42% and 28%. Bait-specific FDRs and the estimated protein degrees are then used to identify protein categories that yield more (or fewer) false positive interactions and more (or fewer) interaction partners. While membrane proteins have been suggested to have elevated FDRs, the current analysis suggests that intrinsic membrane proteins may actually have reduced FDRs. Hydrophobicity is positively correlated with decreased error rates and fewer interaction partners. These methods will be useful for future two-hybrid screens, which could use ultra-high-throughput sequencing for deeper sampling of interacting bait–prey pairs. Availability: All software (C source) and datasets are available as supplemental files and at http://www.baderzone.org under the Lesser GPL v. 3 license. Contact: joel.bader@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19091773

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

The High-Throughput Stochastic Human Exposure and Dose Simulation Model (SHEDS-HT) & The Chemical and Products Database (CPDat)

EPA Science Inventory

The Stochastic Human Exposure and Dose Simulation Model – High-Throughput (SHEDS-HT) is a U.S. Environmental Protection Agency research tool for predicting screening-level (low-tier) exposures to chemicals in consumer products. This course will present an overview of this m...

Forecasting Exposure in Order to Use High Throughput Hazard Data in a Risk-based Context (WC9)

EPA Science Inventory

The ToxCast program and Tox21 consortium have evaluated over 8000 chemicals using in vitro high-throughput screening (HTS) to identify potential hazards. Complementary exposure science needed to assess risk, and the U.S. Environmental Protection Agency (EPA)’s ExpoCast initiative...

Differentiating pathway-specific from non-specific effects in high-throughput toxicity data: A foundation for prioritizing adverse outcome pathway development

EPA Science Inventory

The U.S. Environmental Protection Agency’s ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with pote...

Differentiating pathway-based toxicity from non-specific effects in high throughput data: A foundation for prioritizing targets for AOP development.

EPA Science Inventory

The Environmental Protection Agency has implemented a high throughput screening program, ToxCast, to quickly evaluate large numbers of chemicals for their effects on hundreds of different biological targets. To understand how these measurements relate to adverse effects in an or...

Evaluating the Value of Augmenting In Vitro Hazard Assessment with Exposure and Pharmacokinetics Considerations for Chemical Prioritization

EPA Science Inventory

Over time, toxicity-testing paradigms have progressed from low-throughput in vivo animal studies for limited numbers of chemicals to high-throughput (HT) in vitro screening assays for thousands of chemicals. Such HT in vitro methods, along with HT in silico predictions of popula...

High Throughput PBTK: Evaluating EPA’s Open-Source Data and Tools for Dosimetry and Exposure Reconstruction

EPA Science Inventory

Thousands of chemicals have been profiled by high-throughput screening (HTS) programs such as ToxCast and Tox21; these chemicals are tested in part because most of them have limited or no data on hazard, exposure, or toxicokinetics (TK). While HTS generates in vitro bioactivity d...

High Throughput Prioritization for Integrated Toxicity Testing Based on ToxCast Chemical Profiling

EPA Science Inventory

The rational prioritization of chemicals for integrated toxicity testing is a central goal of the U.S. EPA’s ToxCast™ program (http://epa.gov/ncct/toxcast/). ToxCast includes a wide-ranging battery of over 500 in vitro high-throughput screening assays which in Phase I was used to...

Continuing Development of Alternative High-Throughput Screens to Determine Endocrine Disruption, Focusing on Androgen Receptor, Steroidogenesis, and Thyroid Pathways

EPA Science Inventory

The focus of this meeting is the SAP's review and comment on the Agency's proposed high-throughput computational model of androgen receptor pathway activity as an alternative to the current Tier 1 androgen receptor assay (OCSPP 890.1150: Androgen Receptor Binding Rat Prostate Cyt...

A Biologically Informed Framework for the Analysis of the PPAR Signaling Pathway using a Bayesian Network

EPA Science Inventory

The US EPA’s ToxCastTM program seeks to combine advances in high-throughput screening technology with methodologies from statistics and computer science to develop high-throughput decision support tools for assessing chemical hazard and risk. To develop new methods of analysis of...

ToxCast Assay Network (TCAN) Viewer: A Visualization Tool for High-throughput Assay Chemical Data (SOT)

EPA Science Inventory

USEPA’s ToxCast program has generated high-throughput bioactivity screening (HTS) data on thousands of chemicals. The ToxCast program has described and annotated the HTS assay battery with respect to assay design and target information (e.g., gene target). Recent stakeholder and ...

High-Throughput, Motility-Based Sorter for Microswimmers such as C. elegans

PubMed Central

Yuan, Jinzhou; Zhou, Jessie; Raizen, David M.; Bau, Haim H.

2015-01-01

Animal motility varies with genotype, disease, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method is implemented in a simple microfluidic device capable of sorting thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriches for known C. elegans motility mutants. Furthermore, using this device, we isolate low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates C. elegans sleep. By performing genetic complementation tests, we demonstrate that our motility-based sorting device efficiently isolates mutants for the same gene identified by tedious visual inspection of behavior on an agar surface. Therefore, our motility-based sorter is capable of performing high throughput gene discovery approaches to investigate fundamental biological processes. PMID:26008643

Evolving the EPA Endocrine Disruptor Screening Program: The case for and against using high-throughput screening assays in EDSP Tier 1

EPA Science Inventory

Testing has begun as part of the EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 battery of 11 in vitro and in vivo tests. A recognized issue with the EDSP is that the current Tier 1 screening battery is highly resource intensive in terms of cost, time and animal usage fo...

Use of a Fluorometric Imaging Plate Reader in high-throughput screening

NASA Astrophysics Data System (ADS)

Groebe, Duncan R.; Gopalakrishnan, Sujatha; Hahn, Holly; Warrior, Usha; Traphagen, Linda; Burns, David J.

1999-04-01

High-throughput screening (HTS) efforts at Abbott Laboratories have been greatly facilitated by the use of a Fluorometric Imaging Plate Reader. The FLIPR consists of an incubated cabinet with integrated 96-channel pipettor and fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorescence is imaged by a cooled CCD camera. The image data is downloaded from the camera and processed to average the signal form each well of the microtiter pate for each time point. The data is presented in real time on the computer screen, facilitating interpretation and trouble-shooting. In addition to fluorescence, the camera can also detect luminescence form firefly luciferase.

UCLA's Molecular Screening Shared Resource: enhancing small molecule discovery with functional genomics and new technology.

PubMed

Damoiseaux, Robert

2014-05-01

The Molecular Screening Shared Resource (MSSR) offers a comprehensive range of leading-edge high throughput screening (HTS) services including drug discovery, chemical and functional genomics, and novel methods for nano and environmental toxicology. The MSSR is an open access environment with investigators from UCLA as well as from the entire globe. Industrial clients are equally welcome as are non-profit entities. The MSSR is a fee-for-service entity and does not retain intellectual property. In conjunction with the Center for Environmental Implications of Nanotechnology, the MSSR is unique in its dedicated and ongoing efforts towards high throughput toxicity testing of nanomaterials. In addition, the MSSR engages in technology development eliminating bottlenecks from the HTS workflow and enabling novel assays and readouts currently not available.

Rapid screening of illicit additives in weight loss dietary supplements with desorption corona beam ionisation (DCBI) mass spectrometry.

PubMed

Wang, H; Wu, Y; Zhao, Y; Sun, W; Ding, L; Guo, B; Chen, B

2012-08-01

Desorption corona beam ionisation (DCBI), the relatively novel ambient mass spectrometry (MS) technique, was utilised to screen for illicit additives in weight-loss food. The five usually abused chemicals - fenfluramine, N-di-desmethyl sibutramine, N-mono-desmethyl sibutramine, sibutramine and phenolphthalein - were detected with the proposed DCBI-MS method. Fast single-sample and high-throughput analysis was demonstrated. Semi-quantification was accomplished based on peak areas in the ion chromatograms. Four illicit additives were identified and semi-quantified in commercial samples. As there was no tedious sample pre-treatment compared with conventional HPLC methods, high-throughput analysis was achieved with DCBI. The results proved that DCBI-MS is a powerful tool for the rapid screening of illicit additives in weight-loss dietary supplements.

Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

PubMed Central

Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen TC

2008-01-01

Background The recent emergence of high-throughput automated image acquisition technologies has forever changed how cell biologists collect and analyze data. Historically, the interpretation of cellular phenotypes in different experimental conditions has been dependent upon the expert opinions of well-trained biologists. Such qualitative analysis is particularly effective in detecting subtle, but important, deviations in phenotypes. However, while the rapid and continuing development of automated microscope-based technologies now facilitates the acquisition of trillions of cells in thousands of diverse experimental conditions, such as in the context of RNA interference (RNAi) or small-molecule screens, the massive size of these datasets precludes human analysis. Thus, the development of automated methods which aim to identify novel and biological relevant phenotypes online is one of the major challenges in high-throughput image-based screening. Ideally, phenotype discovery methods should be designed to utilize prior/existing information and tackle three challenging tasks, i.e. restoring pre-defined biological meaningful phenotypes, differentiating novel phenotypes from known ones and clarifying novel phenotypes from each other. Arbitrarily extracted information causes biased analysis, while combining the complete existing datasets with each new image is intractable in high-throughput screens. Results Here we present the design and implementation of a novel and robust online phenotype discovery method with broad applicability that can be used in diverse experimental contexts, especially high-throughput RNAi screens. This method features phenotype modelling and iterative cluster merging using improved gap statistics. A Gaussian Mixture Model (GMM) is employed to estimate the distribution of each existing phenotype, and then used as reference distribution in gap statistics. This method is broadly applicable to a number of different types of image-based datasets derived from a wide spectrum of experimental conditions and is suitable to adaptively process new images which are continuously added to existing datasets. Validations were carried out on different dataset, including published RNAi screening using Drosophila embryos [Additional files 1, 2], dataset for cell cycle phase identification using HeLa cells [Additional files 1, 3, 4] and synthetic dataset using polygons, our methods tackled three aforementioned tasks effectively with an accuracy range of 85%–90%. When our method is implemented in the context of a Drosophila genome-scale RNAi image-based screening of cultured cells aimed to identifying the contribution of individual genes towards the regulation of cell-shape, it efficiently discovers meaningful new phenotypes and provides novel biological insight. We also propose a two-step procedure to modify the novelty detection method based on one-class SVM, so that it can be used to online phenotype discovery. In different conditions, we compared the SVM based method with our method using various datasets and our methods consistently outperformed SVM based method in at least two of three tasks by 2% to 5%. These results demonstrate that our methods can be used to better identify novel phenotypes in image-based datasets from a wide range of conditions and organisms. Conclusion We demonstrate that our method can detect various novel phenotypes effectively in complex datasets. Experiment results also validate that our method performs consistently under different order of image input, variation of starting conditions including the number and composition of existing phenotypes, and dataset from different screens. In our findings, the proposed method is suitable for online phenotype discovery in diverse high-throughput image-based genetic and chemical screens. PMID:18534020

High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries.

PubMed

Pérez Del Palacio, José; Díaz, Caridad; de la Cruz, Mercedes; Annang, Frederick; Martín, Jesús; Pérez-Victoria, Ignacio; González-Menéndez, Víctor; de Pedro, Nuria; Tormo, José R; Algieri, Francesca; Rodriguez-Nogales, Alba; Rodríguez-Cabezas, M Elena; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; Gálvez, Julio

2016-07-01

It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and a high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. In this work, we present a validated high-throughput screening approach in 96-well plate format for the discovery of new molecules with anti-inflammatory/immunomodulatory activity. The in vitro models are based on the quantitation of nitrite levels in RAW264.7 murine macrophages and interleukin-8 in Caco-2 cells. We have used this platform in a pilot project to screen a subset of 5976 noncytotoxic crude microbial extracts from the MEDINA microbial natural product collection. To our knowledge, this is the first report on an high-throughput screening of microbial natural product extracts for the discovery of immunomodulators. © 2016 Society for Laboratory Automation and Screening.

RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

PubMed

Fu, Jiaqi; Fernandez, Daniel; Ferrer, Marc; Titus, Steven A; Buehler, Eugen; Lal-Nag, Madhu A

2017-06-01

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.

Comparison of a rational vs. high throughput approach for rapid salt screening and selection.

PubMed

Collman, Benjamin M; Miller, Jonathan M; Seadeek, Christopher; Stambek, Julie A; Blackburn, Anthony C

2013-01-01

In recent years, high throughput (HT) screening has become the most widely used approach for early phase salt screening and selection in a drug discovery/development setting. The purpose of this study was to compare a rational approach for salt screening and selection to those results previously generated using a HT approach. The rational approach involved a much smaller number of initial trials (one salt synthesis attempt per counterion) that were selected based on a few strategic solubility determinations of the free form combined with a theoretical analysis of the ideal solvent solubility conditions for salt formation. Salt screening results for sertraline, tamoxifen, and trazodone using the rational approach were compared to those previously generated by HT screening. The rational approach produced similar results to HT screening, including identification of the commercially chosen salt forms, but with a fraction of the crystallization attempts. Moreover, the rational approach provided enough solid from the very initial crystallization of a salt for more thorough and reliable solid-state characterization and thus rapid decision-making. The crystallization techniques used in the rational approach mimic larger-scale process crystallization, allowing smoother technical transfer of the selected salt to the process chemist.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

PubMed

Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C; Johnson, Michael E

2014-01-01

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development. Copyright © 2013. Published by Elsevier Ltd.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

PubMed

Qin, Lina; Jiang, Xianzhang; Dong, Zhiyang; Huang, Jianzhong; Chen, Xiuzhen

2018-01-01

The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei . Currently, only the locus of exocellobiohydrolases I encoding gene ( cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei . Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Screening and identification of host factors interacting with UL14 of herpes simplex virus 1.

PubMed

Wu, Fuqing; Xing, Junji; Wang, Shuai; Li, Meili; Zheng, Chunfu

2011-08-01

The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

Combinatorial and high-throughput screening of materials libraries: review of state of the art.

PubMed

Potyrailo, Radislav; Rajan, Krishna; Stoewe, Klaus; Takeuchi, Ichiro; Chisholm, Bret; Lam, Hubert

2011-11-14

Rational materials design based on prior knowledge is attractive because it promises to avoid time-consuming synthesis and testing of numerous materials candidates. However with the increase of complexity of materials, the scientific ability for the rational materials design becomes progressively limited. As a result of this complexity, combinatorial and high-throughput (CHT) experimentation in materials science has been recognized as a new scientific approach to generate new knowledge. This review demonstrates the broad applicability of CHT experimentation technologies in discovery and optimization of new materials. We discuss general principles of CHT materials screening, followed by the detailed discussion of high-throughput materials characterization approaches, advances in data analysis/mining, and new materials developments facilitated by CHT experimentation. We critically analyze results of materials development in the areas most impacted by the CHT approaches, such as catalysis, electronic and functional materials, polymer-based industrial coatings, sensing materials, and biomaterials.

Life in the fast lane: high-throughput chemistry for lead generation and optimisation.

PubMed

Hunter, D

2001-01-01

The pharmaceutical industry has come under increasing pressure due to regulatory restrictions on the marketing and pricing of drugs, competition, and the escalating costs of developing new drugs. These forces can be addressed by the identification of novel targets, reductions in the development time of new drugs, and increased productivity. Emphasis has been placed on identifying and validating new targets and on lead generation: the response from industry has been very evident in genomics and high throughput screening, where new technologies have been applied, usually coupled with a high degree of automation. The combination of numerous new potential biological targets and the ability to screen large numbers of compounds against many of these targets has generated the need for large diverse compound collections. To address this requirement, high-throughput chemistry has become an integral part of the drug discovery process. Copyright 2002 Wiley-Liss, Inc.

A high-throughput colorimetric assay for glucose detection based on glucose oxidase-catalyzed enlargement of gold nanoparticles

NASA Astrophysics Data System (ADS)

Xiong, Yanmei; Zhang, Yuyan; Rong, Pengfei; Yang, Jie; Wang, Wei; Liu, Dingbin

2015-09-01

We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose.We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose. Electronic supplementary information (ESI) available: Experimental section and additional figures. See DOI: 10.1039/c5nr03758a

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

PubMed

Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

2017-06-27

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

High-throughput cultivation and screening platform for unicellular phototrophs.

PubMed

Tillich, Ulrich M; Wolter, Nick; Schulze, Katja; Kramer, Dan; Brödel, Oliver; Frohme, Marcus

2014-09-16

High-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria. In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PCC6803. The required technical automation for these processes was achieved with a Tecan Freedom Evo 200 pipetting robot. The cultivation was performed in 2.2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. Each microtiter-well within the chamber functions as a separate cultivation vessel with reproducible conditions. The automated measurement of various parameters such as growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have already been established and can be monitored during cultivation. Measurement of growth parameters can be used as inputs for the system to allow for periodic automatic dilutions and therefore a semi-continuous cultivation of hundreds of cultures in parallel. The system also allows the automatic generation of mid and long term backups of cultures to repeat experiments or to retrieve strains of interest. The presented platform allows for high-throughput cultivation and screening of Synechocystis sp. PCC6803. The platform should be usable for many phototrophic microorganisms as is, and be adaptable for even more. A variety of analyses are already established and the platform is easily expandable both in quality, i.e. with further parameters to screen for additional targets and in quantity, i.e. size or number of processed samples.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

PubMed

Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

2015-03-01

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Gas pressure assisted microliquid-liquid extraction coupled online to direct infusion mass spectrometry: a new automated screening platform for bioanalysis.

PubMed

Raterink, Robert-Jan; Witkam, Yoeri; Vreeken, Rob J; Ramautar, Rawi; Hankemeier, Thomas

2014-10-21

In the field of bioanalysis, there is an increasing demand for miniaturized, automated, robust sample pretreatment procedures that can be easily connected to direct-infusion mass spectrometry (DI-MS) in order to allow the high-throughput screening of drugs and/or their metabolites in complex body fluids like plasma. Liquid-Liquid extraction (LLE) is a common sample pretreatment technique often used for complex aqueous samples in bioanalysis. Despite significant developments that have been made in automated and miniaturized LLE procedures, fully automated LLE techniques allowing high-throughput bioanalytical studies on small-volume samples using direct infusion mass spectrometry, have not been matured yet. Here, we introduce a new fully automated micro-LLE technique based on gas-pressure assisted mixing followed by passive phase separation, coupled online to nanoelectrospray-DI-MS. Our method was characterized by varying the gas flow and its duration through the solvent mixture. For evaluation of the analytical performance, four drugs were spiked to human plasma, resulting in highly acceptable precision (RSD down to 9%) and linearity (R(2) ranging from 0.990 to 0.998). We demonstrate that our new method does not only allow the reliable extraction of analytes from small sample volumes of a few microliters in an automated and high-throughput manner, but also performs comparable or better than conventional offline LLE, in which the handling of small volumes remains challenging. Finally, we demonstrate the applicability of our method for drug screening on dried blood spots showing excellent linearity (R(2) of 0.998) and precision (RSD of 9%). In conclusion, we present the proof of principe of a new high-throughput screening platform for bioanalysis based on a new automated microLLE method, coupled online to a commercially available nano-ESI-DI-MS.

Cheminformatic Analysis of the US EPA ToxCast Chemical Library

EPA Science Inventory

The ToxCast project is employing high throughput screening (HTS) technologies, along with chemical descriptors and computational models, to develop approaches for screening and prioritizing environmental chemicals for further toxicity testing. ToxCast Phase I generated HTS data f...

Response to “Accurate Risk-Based Chemical Screening Relies on Robust Exposure Estimates”

EPA Science Inventory

This is a correspondence (letter to the editor) with reference to comments by Rudel and Perovich on the article "Integration of Dosimetry, Exposure, and High-Throughput Screening Data in Chemical Toxicity Assessment". Article Reference: SI # 238882

High-throughput spectrometer designs in a compact form-factor: principles and applications

NASA Astrophysics Data System (ADS)

Norton, S. M.

2013-05-01

Many compact, portable Raman spectrometers have entered the market in the past few years with applications in narcotics and hazardous material identification, as well as verification applications in pharmaceuticals and security screening. Often, the required compact form-factor has forced designers to sacrifice throughput and sensitivity for portability and low-cost. We will show that a volume phase holographic (VPH)-based spectrometer design can achieve superior throughput and thus sensitivity over conventional Czerny-Turner reflective designs. We will look in depth at the factors influencing throughput and sensitivity and illustrate specific VPH-based spectrometer examples that highlight these design principles.

Field-based high throughput phenotyping rapidly identifies genomic regions controlling yield components in rice

PubMed Central

Tanger, Paul; Klassen, Stephen; Mojica, Julius P.; Lovell, John T.; Moyers, Brook T.; Baraoidan, Marietta; Naredo, Maria Elizabeth B.; McNally, Kenneth L.; Poland, Jesse; Bush, Daniel R.; Leung, Hei; Leach, Jan E.; McKay, John K.

2017-01-01

To ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. Here we demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor-intensive measures of flowering time, height, biomass, grain yield, and harvest index. Genetic mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution. PMID:28220807

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate.

PubMed

Kojima, Takashi; Tsutsumi, Shunichirou; Yamamoto, Katsuhiko; Ikeda, Yukihiro; Moriwaki, Toshiya

2010-10-31

Cocrystal has attracted much attention in order to improve poor physicochemical properties, since cocrystal former crystallize with the ionic drugs as well as nonionic drugs. Cocrystal screening was usually conducted by crystallization, slurry and co-grinding techniques, however sensitivity, cost and time for screening were limited because of issues such as dissociation of cocrystal during crystallization and cost and time required for slurry and co-grinding methods. To overcome these issues, novel high-throughput cocrystal slurry screening was developed by using in situ Raman microscope and a multi-well plate. Cocrystal screening of indomethacin was conducted with 46 cocrystal formers and potential cocrystals were prepared on a large scale for the characterization with powder X-ray diffractometry, thermal analysis, and Raman microscopy and (1)H NMR spectroscopy. Compared with the characterization of scale-up cocrystals, the cocrystal screening indicated that indomethacin structured novel cocrystals with D/L-mandelic acid, nicotinamide, lactamide and benzamide which was not obtained in the screening with crystallization technique previously reported. In addition, the screening provided not only information of cocrystal formation within a day but also information of equilibrium of cocrystal formation and polymorphic transformation in one screening. Information obtained in this screening allows effective solid form selection by saving cost and time for the development. Copyright © 2010 Elsevier B.V. All rights reserved.

The multidimensional perturbation value: a single metric to measure similarity and activity of treatments in high-throughput multidimensional screens.

PubMed

Hutz, Janna E; Nelson, Thomas; Wu, Hua; McAllister, Gregory; Moutsatsos, Ioannis; Jaeger, Savina A; Bandyopadhyay, Somnath; Nigsch, Florian; Cornett, Ben; Jenkins, Jeremy L; Selinger, Douglas W

2013-04-01

Screens using high-throughput, information-rich technologies such as microarrays, high-content screening (HCS), and next-generation sequencing (NGS) have become increasingly widespread. Compared with single-readout assays, these methods produce a more comprehensive picture of the effects of screened treatments. However, interpreting such multidimensional readouts is challenging. Univariate statistics such as t-tests and Z-factors cannot easily be applied to multidimensional profiles, leaving no obvious way to answer common screening questions such as "Is treatment X active in this assay?" and "Is treatment X different from (or equivalent to) treatment Y?" We have developed a simple, straightforward metric, the multidimensional perturbation value (mp-value), which can be used to answer these questions. Here, we demonstrate application of the mp-value to three data sets: a multiplexed gene expression screen of compounds and genomic reagents, a microarray-based gene expression screen of compounds, and an HCS compound screen. In all data sets, active treatments were successfully identified using the mp-value, and simulations and follow-up analyses supported the mp-value's statistical and biological validity. We believe the mp-value represents a promising way to simplify the analysis of multidimensional data while taking full advantage of its richness.

A workflow to investigate exposure and pharmacokinetic influences on high-throughput in vitro chemical screening based on adverse outcome pathways, OpenTox USA 2015 Poster

EPA Science Inventory

Adverse outcome pathways (AOP) link known population outcomes to a molecular initiating event (MIE) that can be quantified using high-throughput in vitro methods. Practical application of AOPs in chemical-specific risk assessment requires consideration of exposure and absorption,...

Use of Threshold of Toxicological Concern (TTC) with High Throughput Exposure Predictions as a Risk-Based Screening Approach to Prioritize More Than Seven Thousand Chemicals (ASCCT)

EPA Science Inventory

Here, we present results of an approach for risk-based prioritization using the Threshold of Toxicological Concern (TTC) combined with high-throughput exposure (HTE) modelling. We started with 7968 chemicals with calculated population median oral daily intakes characterized by an...

*Biomarkers of acute respiratory allergen exposure: Screening for sensitization potential

EPA Science Inventory

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens fol...

SCREENING CHEMICALS FOR ESTROGEN RECEPTOR BIOACTIVITY USING A COMPUTATIONAL MODEL

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) is considering the use high-throughput and computational methods for regulatory applications in the Endocrine Disruptor Screening Program (EDSP). To use these new tools for regulatory decision making, computational methods must be a...

Using Neural Progenitor Cells in High-Throughput Screens for Developmental Neurotoxicants: Triumphs and Tragedies

EPA Science Inventory

Current protocols for developmental neurotoxicity testing are insufficient to test thousands of commercial chemicals. Thus, development of highthroughput screens (HTS) to detect and prioritize chemicals that may cause developmental neurotoxicity is needed to improve protection of...

Pathway Profiling and Tissue Modeling Using ToxCast HTS Data

EPA Science Inventory

High-throughput screening (HTS) and high-content screening (HCS) assays are providing data-rich studies to probe and profile the direct cellular effects of thousands of chemical compounds in commerce or potentially entering the environment. In vitro profiling may compare unknown ...

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

PubMed

Kelley, Brian D; Tobler, Scott A; Brown, Paul; Coffman, Jonathan L; Godavarti, Ranga; Iskra, Timothy; Switzer, Mary; Vunnum, Suresh

2008-10-15

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved. (c) 2008 Wiley Periodicals, Inc.

Automated chest-radiography as a triage for Xpert testing in resource-constrained settings: a prospective study of diagnostic accuracy and costs

NASA Astrophysics Data System (ADS)

Philipsen, R. H. H. M.; Sánchez, C. I.; Maduskar, P.; Melendez, J.; Peters-Bax, L.; Peter, J. G.; Dawson, R.; Theron, G.; Dheda, K.; van Ginneken, B.

2015-07-01

Molecular tests hold great potential for tuberculosis (TB) diagnosis, but are costly, time consuming, and HIV-infected patients are often sputum scarce. Therefore, alternative approaches are needed. We evaluated automated digital chest radiography (ACR) as a rapid and cheap pre-screen test prior to Xpert MTB/RIF (Xpert). 388 suspected TB subjects underwent chest radiography, Xpert and sputum culture testing. Radiographs were analysed by computer software (CAD4TB) and specialist readers, and abnormality scores were allocated. A triage algorithm was simulated in which subjects with a score above a threshold underwent Xpert. We computed sensitivity, specificity, cost per screened subject (CSS), cost per notified TB case (CNTBC) and throughput for different diagnostic thresholds. 18.3% of subjects had culture positive TB. For Xpert alone, sensitivity was 78.9%, specificity 98.1%, CSS $13.09 and CNTBC $90.70. In a pre-screening setting where 40% of subjects would undergo Xpert, CSS decreased to $6.72 and CNTBC to $54.34, with eight TB cases missed and throughput increased from 45 to 113 patients/day. Specialists, on average, read 57% of radiographs as abnormal, reducing CSS ($8.95) and CNTBC ($64.84). ACR pre-screening could substantially reduce costs, and increase daily throughput with few TB cases missed. These data inform public health policy in resource-constrained settings.

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

NASA Astrophysics Data System (ADS)

Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

2013-11-01

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

High throughput screening technologies for ion channels

PubMed Central

Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

2016-01-01

Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

Quantitative high throughput screening identifies inhibitors of anthrax-induced cell death

PubMed Central

Zhu, Ping Jun; Hobson, Peyton; Southall, Noel; Qiu, Cunping; Thomas, Craig J.; Lu, Jiamo; Inglese, James; Zheng, Wei; Leppla, Stephen H.; Bugge, Thomas H.; Austin, Christopher P.; Liu, Shihui

2009-01-01

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization. PMID:19540764

A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

PubMed Central

2016-01-01

Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

Convenient, Sensitive and High-Throughput Method for Screening Botanic Origin

NASA Astrophysics Data System (ADS)

Yuan, Yuan; Jiang, Chao; Liu, Libing; Yu, Shulin; Cui, Zhanhu; Chen, Min; Lin, Shufang; Wang, Shu; Huang, Luqi

2014-06-01

In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

PubMed

Shaneyfelt, Mark E; Burke, Anna D; Graff, Joel W; Jutila, Mark A; Hardy, Michele E

2006-09-01

There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell–like diffuse large B-cell lymphoma cells

PubMed Central

Mathews Griner, Lesley A.; Guha, Rajarshi; Shinn, Paul; Young, Ryan M.; Keller, Jonathan M.; Liu, Dongbo; Goldlust, Ian S.; Yasgar, Adam; McKnight, Crystal; Boxer, Matthew B.; Duveau, Damien Y.; Jiang, Jian-Kang; Michael, Sam; Mierzwa, Tim; Huang, Wenwei; Walsh, Martin J.; Mott, Bryan T.; Patel, Paresma; Leister, William; Maloney, David J.; Leclair, Christopher A.; Rai, Ganesha; Jadhav, Ajit; Peyser, Brian D.; Austin, Christopher P.; Martin, Scott E.; Simeonov, Anton; Ferrer, Marc; Staudt, Louis M.; Thomas, Craig J.

2014-01-01

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug–drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell–like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton’s tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL. PMID:24469833

High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

PubMed

Hu, Junqiang; Gondarenko, Alexander A; Dang, Alex P; Bashour, Keenan T; O'Connor, Roddy S; Lee, Sunwoo; Liapis, Anastasia; Ghassemi, Saba; Milone, Michael C; Sheetz, Michael P; Dustin, Michael L; Kam, Lance C; Hone, James C

2016-04-13

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 μm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 μm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

High-throughput screening of PLGA thin films utilizing hydrophobic fluorescent dyes for hydrophobic drug compounds.

PubMed

Steele, Terry W J; Huang, Charlotte L; Kumar, Saranya; Widjaja, Effendi; Chiang Boey, Freddy Yin; Loo, Joachim S C; Venkatraman, Subbu S

2011-10-01

Hydrophobic, antirestenotic drugs such as paclitaxel (PCTX) and rapamycin are often incorporated into thin film coatings for local delivery using implantable medical devices and polymers such as drug-eluting stents and balloons. Selecting the optimum coating formulation through screening the release profile of these drugs in thin films is time consuming and labor intensive. We describe here a high-throughput assay utilizing three model hydrophobic fluorescent compounds: fluorescein diacetate (FDAc), coumarin-6, and rhodamine 6G that were incorporated into poly(d,l-lactide-co-glycolide) (PLGA) and PLGA-polyethylene glycol films. Raman microscopy determined the hydrophobic fluorescent dye distribution within the PLGA thin films in comparison with that of PCTX. Their subsequent release was screened in a high-throughput assay and directly compared with HPLC quantification of PCTX release. It was observed that PCTX controlled-release kinetics could be mimicked by a hydrophobic dye that had similar octanol-water partition coefficient values and homogeneous dissolution in a PLGA matrix as the drug. In particular, FDAc was found to be the optimal hydrophobic dye at modeling the burst release as well as the total amount of PCTX released over a period of 30 days. Copyright © 2011 Wiley-Liss, Inc.

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Automatic Segmentation of High-Throughput RNAi Fluorescent Cellular Images

PubMed Central

Yan, Pingkum; Zhou, Xiaobo; Shah, Mubarak; Wong, Stephen T. C.

2010-01-01

High-throughput genome-wide RNA interference (RNAi) screening is emerging as an essential tool to assist biologists in understanding complex cellular processes. The large number of images produced in each study make manual analysis intractable; hence, automatic cellular image analysis becomes an urgent need, where segmentation is the first and one of the most important steps. In this paper, a fully automatic method for segmentation of cells from genome-wide RNAi screening images is proposed. Nuclei are first extracted from the DNA channel by using a modified watershed algorithm. Cells are then extracted by modeling the interaction between them as well as combining both gradient and region information in the Actin and Rac channels. A new energy functional is formulated based on a novel interaction model for segmenting tightly clustered cells with significant intensity variance and specific phenotypes. The energy functional is minimized by using a multiphase level set method, which leads to a highly effective cell segmentation method. Promising experimental results demonstrate that automatic segmentation of high-throughput genome-wide multichannel screening can be achieved by using the proposed method, which may also be extended to other multichannel image segmentation problems. PMID:18270043

[HTRF-based high-throughput PGE2 release prohibition model and application in discovering traditional Chinese medicine active ingredients].

PubMed

Bai, Zhi-Ru; Fei, Hong-Qiang; Li, Na; Cao, Liang; Zhang, Chen-Feng; Wang, Tuan-Jie; Ding, Gang; Wang, Zhen-Zhong; Xiao, Wei

2016-02-01

Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC₅₀ value for positive compounds was (7.3±0.1) μmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage. Copyright© by the Chinese Pharmaceutical Association.

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.

PubMed

Fiene, Amelie; Baqi, Younis; Lecka, Joanna; Sévigny, Jean; Müller, Christa E

2015-01-07

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

PubMed

Damnjanović, Jasmina; Takahashi, Rie; Suzuki, Atsuo; Nakano, Hideo; Iwasaki, Yugo

2012-08-01

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

High-throughput screening technologies for botulinum neurotoxins.

PubMed

Bompiani, Kristin M; Dickerson, Tobin J

2014-01-01

Botulinum neurotoxins (BoNTs) are a class of bacterial neurotoxins that are the most potent toxic compounds reported to date. Exposure to relatively low concentrations of the toxin protein can result in major muscle paralysis, which may result in death in severe cases. In addition to their role in natural human disease, BoNTs are currently under close scrutiny because of their potential to be used as biowarfare agents. Clinical treatment options for botulism are currently limited, and finite stockpiles of antitoxin exist. In light of current bioterrorist threats, researchers have focused on identifying new molecules that can be applied to either sensitive toxin detection or improved clinical treatment. High-throughput screening (HTS) is a laboratory technique commonly employed to screen large libraries of diverse compounds based on specific compound binding capabilities or function. Here we review existing HTS platforms that have been applied to identify novel BoNT diagnostic or therapeutic agents. HTS platforms for screening antibodies, peptides, small molecules, and aptamers are described, as well as the screening results and current progress of the identified compounds.

High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress.

PubMed

Thorne, Natasha; Malik, Nasir; Shah, Sonia; Zhao, Jean; Class, Bradley; Aguisanda, Francis; Southall, Noel; Xia, Menghang; McKew, John C; Rao, Mahendra; Zheng, Wei

2016-05-01

Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. ©AlphaMed Press.

Dr. Janie Merkel is interviewed by Ryan Blum and Janice Friend.

PubMed

Merkel, Janie

2007-12-01

Dr. Janie Merkel is the director of Yale's Chemical Genomics Screening Facility, a high-throughput screening laboratory that is part of the Yale University Center for Genomics and Proteomics. The Screening Facility connects Yale researchers with industry-quality robotic machinery and a diverse group of compound libraries, which have been used successfully to link therapeutic targets with potential therapies.

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening.

PubMed

Nebane, N Miranda; Coric, Tatjana; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E Lucile

2016-02-01

The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte's Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research's HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens. © 2015 Society for Laboratory Automation and Screening.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

PubMed Central

Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

2008-01-01

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses.

PubMed

Shoshan, Maria C; Havelka, Associate Professor Principal Investigator Aleksandra Mandic; Neumann, Frank; Linder, Stig

2006-11-01

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.

High Throughput Exposure Prioritization of Chemicals Using a Screening-Level Probabilistic SHEDS-Lite Exposure Model

EPA Science Inventory

These novel modeling approaches for screening, evaluating and classifying chemicals based on the potential for biologically-relevant human exposures will inform toxicity testing and prioritization for chemical risk assessment. The new modeling approach is derived from the Stocha...