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Sample records for kansas high-throughput screening

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

    Science.gov (United States)

    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.

  2. The University of Kansas High-Throughput Screening laboratory. Part I: meeting drug-discovery needs in the heartland of America with entrepreneurial flair.

    Science.gov (United States)

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

    2011-05-01

    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 applies pharmaceutical industry project-management principles in an academic setting by bringing together multidisciplinary teams to fill critical scientific and technology gaps, using an experienced team of industry-trained researchers and project managers. The KU HTS proactively engages in supporting grant applications for extramural funding, intellectual-property management and technology transfer. The KU HTS staff further provides educational opportunities for the KU faculty and students to learn cutting-edge technologies in drug-discovery platforms through seminars, workshops, internships and course teaching. This is the first instalment of a two-part contribution from the KU HTS laboratory.

  3. Application of ToxCast High-Throughput Screening and ...

    Science.gov (United States)

    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

  4. Applications of ambient mass spectrometry in high-throughput screening.

    Science.gov (United States)

    Li, Li-Ping; Feng, Bao-Sheng; Yang, Jian-Wang; Chang, Cui-Lan; Bai, Yu; Liu, Hu-Wei

    2013-06-07

    The development of rapid screening and identification techniques is of great importance for drug discovery, doping control, forensic identification, food safety and quality control. Ambient mass spectrometry (AMS) allows rapid and direct analysis of various samples in open air with little sample preparation. Recently, its applications in high-throughput screening have been in rapid progress. During the past decade, various ambient ionization techniques have been developed and applied in high-throughput screening. This review discusses typical applications of AMS, including DESI (desorption electrospray ionization), DART (direct analysis in real time), EESI (extractive electrospray ionization), etc., in high-throughput screening (HTS).

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  7. 20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

    Science.gov (United States)

    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...

  8. High throughput screening method for assessing heterogeneity of microorganisms

    NARCIS (Netherlands)

    Ingham, C.J.; Sprenkels, A.J.; van Hylckama Vlieg, J.E.T.; Bomer, Johan G.; de Vos, W.M.; van den Berg, Albert

    2006-01-01

    The invention relates to the field of microbiology. Provided is a method which is particularly powerful for High Throughput Screening (HTS) purposes. More specific a high throughput method for determining heterogeneity or interactions of microorganisms is provided.

  9. High-throughput fragment screening by affinity LC-MS.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  10. High-throughput screening to identify inhibitors of lysine demethylases.

    Science.gov (United States)

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

  11. High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale L

    2005-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  12. High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale

    2004-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  13. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  14. Optimization and high-throughput screening of antimicrobial peptides.

    Science.gov (United States)

    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.

  15. High-throughput screening with micro-x-ray fluorescence

    International Nuclear Information System (INIS)

    Havrilla, George J.; Miller, Thomasin C.

    2005-01-01

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity

  16. High-throughput screening of small molecule libraries using SAMDI mass spectrometry.

    Science.gov (United States)

    Gurard-Levin, Zachary A; Scholle, Michael D; Eisenberg, Adam H; Mrksich, Milan

    2011-07-11

    High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of "label-free" assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100,000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications.

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

    Science.gov (United States)

    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.

  18. High-throughput screening to enhance oncolytic virus immunotherapy

    Directory of Open Access Journals (Sweden)

    Allan KJ

    2016-04-01

    Full Text Available KJ Allan,1,2 David F Stojdl,1–3 SL Swift1 1Children’s Hospital of Eastern Ontario (CHEO Research Institute, 2Department of Biology, Microbiology and Immunology, 3Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Abstract: High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. Keywords: oncolytic, virus, screen, high-throughput, cancer, chemical, genomic, immunotherapy

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Creation of a small high-throughput screening facility.

    Science.gov (United States)

    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.

  2. High throughput screening of starch structures using carbohydrate microarrays

    DEFF Research Database (Denmark)

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated...

  3. Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.

    Science.gov (United States)

    Zhang, Haohai; Chan, Leo Li-Ying; Rice, William; Kassam, Nasim; Longhi, Maria Serena; Zhao, Haitao; Robson, Simon C; Gao, Wenda; Wu, Yan

    2017-08-01

    Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent K d binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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...

  5. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

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

    Science.gov (United States)

    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...

  7. Tiered High-Throughput Screening Approach to Identify ...

    Science.gov (United States)

    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 limited in the US EPA ToxCast screening assay portfolio. To fill one 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 1,074 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 two 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

  8. Development of automatic image analysis methods for high-throughput and high-content screening

    NARCIS (Netherlands)

    Di, Zi

    2013-01-01

    This thesis focuses on the development of image analysis methods for ultra-high content analysis of high-throughput screens where cellular phenotype responses to various genetic or chemical perturbations that are under investigation. Our primary goal is to deliver efficient and robust image analysis

  9. tcpl: The ToxCast Pipeline for High-Throughput Screening Data

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  13. Virtual high screening throughput and design of 14α-lanosterol ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Virtual high screening throughput and design of. 14α-lanosterol demethylase inhibitors against. Mycobacterium tuberculosis. Hildebert B. Maurice1*, Esther Tuarira1 and Kennedy Mwambete2. 1School of Pharmaceutical Sciences, Institute of Allied Health Sciences, Muhimbili University of Health and.

  14. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

    2015-01-01

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

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

    Czech Academy of Sciences Publication Activity Database

    Podolská, Kateřina; Sedlák, David; Bartůněk, Petr; Svoboda, Petr

    2014-01-01

    Roč. 19, č. 3 (2014), s. 417-426 ISSN 1087-0571 R&D Projects: GA ČR GA13-29531S; GA MŠk(CZ) LC06077; GA MŠk LM2011022 Grant - others:EMBO(DE) 1483 Institutional support: RVO:68378050 Keywords : Dicer * siRNA * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.423, year: 2014

  16. High-throughput screening of ionic conductivity in polymer membranes

    International Nuclear Information System (INIS)

    Zapata, Pedro; Basak, Pratyay; Carson Meredith, J.

    2009-01-01

    Combinatorial and high-throughput techniques have been successfully used for efficient and rapid property screening in multiple fields. The use of these techniques can be an advantageous new approach to assay ionic conductivity and accelerate the development of novel materials in research areas such as fuel cells. A high-throughput ionic conductivity (HTC) apparatus is described and applied to screening candidate polymer electrolyte membranes for fuel cell applications. The device uses a miniature four-point probe for rapid, automated point-to-point AC electrochemical impedance measurements in both liquid and humid air environments. The conductivity of Nafion 112 HTC validation standards was within 1.8% of the manufacturer's specification. HTC screening of 40 novel Kynar poly(vinylidene fluoride) (PVDF)/acrylic polyelectrolyte (PE) membranes focused on varying the Kynar type (5x) and PE composition (8x) using reduced sample sizes. Two factors were found to be significant in determining the proton conducting capacity: (1) Kynar PVDF series: membranes containing a particular Kynar PVDF type exhibited statistically identical mean conductivity as other membranes containing different Kynar PVDF types that belong to the same series or family. (2) Maximum effective amount of polyelectrolyte: increments in polyelectrolyte content from 55 wt% to 60 wt% showed no statistically significant effect in increasing conductivity. In fact, some membranes experienced a reduction in conductivity.

  17. Reverse Phase Protein Arrays for High-throughput Toxicity Screening

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    High-throughput screening is extensively applied for identification of drug targets and drug discovery and recently it found entry into toxicity testing. Reverse phase protein arrays (RPPAs) are used widespread for quantification of protein markers. We reasoned that RPPAs also can be utilized...... beneficially in automated high-throughput toxicity testing. An advantage of using RPPAs is that, in addition to the baseline toxicity readout, they allow testing of multiple markers of toxicity, such as inflammatory responses, which do not necessarily cumulate in cell death. We used transfection of si......RNAs with known killing effects as a model system to demonstrate that RPPA-based protein quantification can serve as substitute readout of cell viability, hereby reliably reflecting toxicity. In terms of automation, cell exposure, protein harvest, serial dilution and sample reformatting were performed using...

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

    Science.gov (United States)

    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.

  19. Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

    Science.gov (United States)

    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.

  20. Determining the optimal size of small molecule mixtures for high throughput NMR screening

    International Nuclear Information System (INIS)

    Mercier, Kelly A.; Powers, Robert

    2005-01-01

    High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a 'hit' for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library

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

    Science.gov (United States)

    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.

  2. Modular high-throughput test stand for versatile screening of thin-film materials libraries

    International Nuclear Information System (INIS)

    Thienhaus, Sigurd; Hamann, Sven; Ludwig, Alfred

    2011-01-01

    Versatile high-throughput characterization tools are required for the development of new materials using combinatorial techniques. Here, we describe a modular, high-throughput test stand for the screening of thin-film materials libraries, which can carry out automated electrical, magnetic and magnetoresistance measurements in the temperature range of −40 to 300 °C. As a proof of concept, we measured the temperature-dependent resistance of Fe–Pd–Mn ferromagnetic shape-memory alloy materials libraries, revealing reversible martensitic transformations and the associated transformation temperatures. Magneto-optical screening measurements of a materials library identify ferromagnetic samples, whereas resistivity maps support the discovery of new phases. A distance sensor in the same setup allows stress measurements in materials libraries deposited on cantilever arrays. A combination of these methods offers a fast and reliable high-throughput characterization technology for searching for new materials. Using this approach, a composition region has been identified in the Fe–Pd–Mn system that combines ferromagnetism and martensitic transformation.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 screened 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.

  4. High-Throughput Screening Using Fourier-Transform Infrared Imaging

    Directory of Open Access Journals (Sweden)

    Erdem Sasmaz

    2015-06-01

    Full Text Available Efficient parallel screening of combinatorial libraries is one of the most challenging aspects of the high-throughput (HT heterogeneous catalysis workflow. Today, a number of methods have been used in HT catalyst studies, including various optical, mass-spectrometry, and gas-chromatography techniques. Of these, rapid-scanning Fourier-transform infrared (FTIR imaging is one of the fastest and most versatile screening techniques. Here, the new design of the 16-channel HT reactor is presented and test results for its accuracy and reproducibility are shown. The performance of the system was evaluated through the oxidation of CO over commercial Pd/Al2O3 and cobalt oxide nanoparticles synthesized with different reducer-reductant molar ratios, surfactant types, metal and surfactant concentrations, synthesis temperatures, and ramp rates.

  5. A multilayer microdevice for cell-based high-throughput drug screening

    International Nuclear Information System (INIS)

    Liu, Chong; Wang, Lei; Li, Jingmin; Ding, Xiping; Chunyu, Li; Xu, Zheng; Wang, Qi

    2012-01-01

    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. (paper)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. High-throughput screening of chemicals as functional ...

    Science.gov (United States)

    Identifying chemicals that provide a specific function within a product, yet have minimal impact on the human body or environment, is the goal of most formulation chemists and engineers practicing green chemistry. We present a methodology to identify potential chemical functional substitutes from large libraries of chemicals using machine learning based models. We collect and analyze publicly available information on the function of chemicals in consumer products or industrial processes to identify a suite of harmonized function categories suitable for modeling. We use structural and physicochemical descriptors for these chemicals to build 41 quantitative structure–use relationship (QSUR) models for harmonized function categories using random forest classification. We apply these models to screen a library of nearly 6400 chemicals with available structure information for potential functional substitutes. Using our Functional Use database (FUse), we could identify uses for 3121 chemicals; 4412 predicted functional uses had a probability of 80% or greater. We demonstrate the potential application of the models to high-throughput (HT) screening for “candidate alternatives” by merging the valid functional substitute classifications with hazard metrics developed from HT screening assays for bioactivity. A descriptor set could be obtained for 6356 Tox21 chemicals that have undergone a battery of HT in vitro bioactivity screening assays. By applying QSURs, we wer

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

    Science.gov (United States)

    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.

  10. Correction of Microplate Data from High-Throughput Screening.

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan; Durmus, Naside Gozde

    2011-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan [Department of Medicine, Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Durmus, Naside Gozde, E-mail: udemirci@rics.bwh.harvard.edu [School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI (United States)

    2011-09-15

    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.

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

    Science.gov (United States)

    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.

  14. Statistical removal of background signals from high-throughput 1H NMR line-broadening ligand-affinity screens

    International Nuclear Information System (INIS)

    Worley, Bradley; Sisco, Nicholas J.; Powers, Robert

    2015-01-01

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1 H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1 H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hui [Iowa State Univ., Ames, IA (United States)

    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 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, 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/cm2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.

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

    International Nuclear Information System (INIS)

    Hui Su

    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 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, 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(sub 2) for 40-(micro)m wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection

  17. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  18. High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

    Directory of Open Access Journals (Sweden)

    Shen Pang

    Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

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

    Science.gov (United States)

    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...

  20. Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.

    Science.gov (United States)

    Sun, Shuwen; Kennedy, Robert T

    2014-09-16

    High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a "MS Plate Reader" that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high Z-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145,000 samples to be analyzed in 1 day.

  1. A high throughput mechanical screening device for cartilage tissue engineering.

    Science.gov (United States)

    Mohanraj, Bhavana; Hou, Chieh; Meloni, Gregory R; Cosgrove, Brian D; Dodge, George R; Mauck, Robert L

    2014-06-27

    Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying 'hits', or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput. © 2013 Published by Elsevier Ltd.

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

    Science.gov (United States)

    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.

  3. High-throughput screening for industrial enzyme production hosts by droplet microfluidics

    DEFF Research Database (Denmark)

    Sjostrom, Staffan L.; Bai, Yunpeng; Huang, Mingtao

    2014-01-01

    A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α......-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host...

  4. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

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

    Directory of Open Access Journals (Sweden)

    Boutros Michael

    2010-04-01

    Full Text Available Abstract Background 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. Results 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. Conclusions 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.

  6. Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible.

    Science.gov (United States)

    Theda, Christiane; Gibbons, Katy; Defor, Todd E; Donohue, Pamela K; Golden, W Christopher; Kline, Antonie D; Gulamali-Majid, Fizza; Panny, Susan R; Hubbard, Walter C; Jones, Richard O; Liu, Anita K; Moser, Ann B; Raymond, Gerald V

    2014-01-01

    X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. High-throughput screening of chemical effects on ...

    Science.gov (United States)

    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 on steroidogenesis via HPLC-MS/MS quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a three stage screening strategy. The first stage established the maximum tolerated concentration (MTC; >70% viability) per sample. The second stage quantified changes in hormone levels at the MTC while the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were pre-stimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2,060 chemical samples evaluated, 524 samples were selected for six-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into five distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A d

  8. Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti.

    Science.gov (United States)

    Cai, Yingying; Xia, Miaomiao; Dong, Huina; Qian, Yuan; Zhang, Tongcun; Zhu, Beiwei; Wu, Jinchuan; Zhang, Dawei

    2018-05-11

    As a very important coenzyme in the cell metabolism, Vitamin B 12 (cobalamin, VB 12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB 12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB 12 production. High-yield VB 12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. By the help of engineered strains with varied capacities of VB 12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 °C, the VB 12 titer of S. meliloti MC5-2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB 12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB 12 -yield strains.

  9. Uncertainty Quantification in High Throughput Screening ...

    Science.gov (United States)

    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 biochemical and cellular processes, including endocrine disruption, cytotoxicity, and zebrafish development. Over 2.6 million concentration response curves are fit to models to extract parameters related to potency and efficacy. Models built on ToxCast results are being used to rank and prioritize the toxicological risk of tested chemicals and to predict the toxicity of tens of thousands of chemicals not yet tested in vivo. However, the data size also presents challenges. When fitting the data, the choice of models, model selection strategy, and hit call criteria must reflect the need for computational efficiency and robustness, requiring hard and somewhat arbitrary cutoffs. When coupled with unavoidable noise in the experimental concentration response data, these hard cutoffs cause uncertainty in model parameters and the hit call itself. The uncertainty will then propagate through all of the models built on the data. Left unquantified, this uncertainty makes it difficult to fully interpret the data for risk assessment. We used bootstrap resampling methods to quantify the uncertainty in fitting models to the concentration response data. Bootstrap resampling determines confidence intervals for

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

    Science.gov (United States)

    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...

  11. DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

    KAUST Repository

    Soufan, Othman; Ba Alawi, Wail; Afeef, Moataz A.; Essack, Magbubah; Kalnis, Panos; Bajic, Vladimir B.

    2016-01-01

    Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods

  12. High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

    Science.gov (United States)

    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.

  13. New approach for high-throughput screening of drug activity on Plasmodium liver stages.

    NARCIS (Netherlands)

    Gego, A.; Silvie, O.; Franetich, J.F.; Farhati, K.; Hannoun, L.; Luty, A.J.F.; Sauerwein, R.W.; Boucheix, C.; Rubinstein, E.; Mazier, D.

    2006-01-01

    Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an

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

    Science.gov (United States)

    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.

  15. Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

    Science.gov (United States)

    Whitehurst, Charles E; Annis, D Allen

    2008-07-01

    Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

  16. Maximizing gain in high-throughput screening using conformal prediction.

    Science.gov (United States)

    Svensson, Fredrik; Afzal, Avid M; Norinder, Ulf; Bender, Andreas

    2018-02-21

    Iterative screening has emerged as a promising approach to increase the efficiency of screening campaigns compared to traditional high throughput approaches. By learning from a subset of the compound library, inferences on what compounds to screen next can be made by predictive models, resulting in more efficient screening. One way to evaluate screening is to consider the cost of screening compared to the gain associated with finding an active compound. In this work, we introduce a conformal predictor coupled with a gain-cost function with the aim to maximise gain in iterative screening. Using this setup we were able to show that by evaluating the predictions on the training data, very accurate predictions on what settings will produce the highest gain on the test data can be made. We evaluate the approach on 12 bioactivity datasets from PubChem training the models using 20% of the data. Depending on the settings of the gain-cost function, the settings generating the maximum gain were accurately identified in 8-10 out of the 12 datasets. Broadly, our approach can predict what strategy generates the highest gain based on the results of the cost-gain evaluation: to screen the compounds predicted to be active, to screen all the remaining data, or not to screen any additional compounds. When the algorithm indicates that the predicted active compounds should be screened, our approach also indicates what confidence level to apply in order to maximize gain. Hence, our approach facilitates decision-making and allocation of the resources where they deliver the most value by indicating in advance the likely outcome of a screening campaign.

  17. Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

    Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hui [Iowa State Univ., Ames, IA (United States)

    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 of 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/cm2 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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...

  5. Novel strategy for protein exploration: high-throughput screening assisted with fuzzy neural network.

    Science.gov (United States)

    Kato, Ryuji; Nakano, Hideo; Konishi, Hiroyuki; Kato, Katsuya; Koga, Yuchi; Yamane, Tsuneo; Kobayashi, Takeshi; Honda, Hiroyuki

    2005-08-19

    To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of

  6. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    DEFF Research Database (Denmark)

    Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon

    2015-01-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify....../oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models....

  7. High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model.

    Science.gov (United States)

    Conery, Annie L; Larkins-Ford, Jonah; Ausubel, Frederick M; Kirienko, Natalia V

    2014-03-14

    In recent history, the nematode Caenorhabditis elegans has provided a compelling platform for the discovery of novel antimicrobial drugs. In this protocol, we present an automated, high-throughput C. elegans pathogenesis assay, which can be used to screen for anti-infective compounds that prevent nematodes from dying due to Pseudomonas aeruginosa. New antibiotics identified from such screens would be promising candidates for treatment of human infections, and also can be used as probe compounds to identify novel targets in microbial pathogenesis or host immunity. Copyright © 2014 John Wiley & Sons, Inc.

  8. High-throughput screening of small-molecule adsorption in MOF-74

    Science.gov (United States)

    Thonhauser, T.; Canepa, P.

    2014-03-01

    Using high-throughput screening coupled with state-of-the-art van der Waals density functional theory, we investigate the adsorption properties of four important molecules, H2, CO2, CH4, and H2O in MOF-74-  with  = Be, Mg, Al, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Zr, Nb, Ru, Rh, Pd, La, W, Os, Ir, and Pt. We show that high-throughput techniques can aid in speeding up the development and refinement of effective materials for hydrogen storage, carbon capture, and gas separation. The exploration of the configurational adsorption space allows us to extract crucial information concerning, for example, the competition of water with CO2 for the adsorption binding sites. We find that only a few noble metals--Rh, Pd, Os, Ir, and Pt--favor the adsorption of CO2 and hence are potential candidates for effective carbon-capture materials. Our findings further reveal significant differences in the binding characteristics of H2, CO2, CH4, and H2O within the MOF structure, indicating that molecular blends can be successfully separated by these nano-porous materials. Supported by DOE DE-FG02-08ER46491.

  9. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening.

    Directory of Open Access Journals (Sweden)

    Michael P Friedmann

    Full Text Available Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.

  10. A simpler sampling interface of venturi easy ambient sonic-spray ionization mass spectrometry for high-throughput screening enzyme inhibitors.

    Science.gov (United States)

    Liu, Ning; Liu, Yang; Yang, YuHan; He, Lan; Ouyang, Jin

    2016-03-24

    High-throughput screening (HTS) is often required in enzyme inhibitor drugs screening. Mass spectrometry (MS) provides a powerful method for high-throughput screening enzyme inhibitors because its high speed, sensitivity and property of lable free. However, most of the MS methods need complicated sampling interface system. Overall throughput was limited by sample loading in these cases. In this study, we develop a simple interface which coupled droplet segmented system to a venturi easy ambient sonic-spray ionization mass spectrometer. It is fabricated by using a single capillary to act as both sampling probe and the emitter, which simplifies the construction, reduces the cost and shorten the sampling time. Samples sucked by venturi effect are segmented to nanoliter plugs by air, then the plugs can be detected by MS directly. This system eliminated the need for flow injection which was popular used in classic scheme. The new system is applied to screen angiotensin converting enzyme inhibitors. High-throughput was achieved in analyzing 96 samples at 1.6 s per sample. The plugs formation was at 0.5s per sample. Carry-over between samples was less than 5%, the peak height RSD was 2.92% (n = 15). Dose-response curves of 3 known inhibitors were also measured to validate its potential in drug discovery. The calculated IC50 agreed well with reported values. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. HIGH THROUGHPUT SCREENING METHOD AND APPARATUS FOR ANALYSING INTERACTIONS BETWEEN SURFACES WITH DIFFERENT TOPOGRAPHY AND THE ENVIRONMENT

    NARCIS (Netherlands)

    de Boer, Jan; van Blitterswijk, Clemens; Unadkat, H.V.; Stamatialis, Dimitrios; Papenburg, B.J.; Wessling, Matthias

    2009-01-01

    The invention is directed to a high throughput screening method for analysing and interaction between a surface of a material and an environment. The screening method of the invention comprises: providing a micro-array comprising said material and having a multitude of units at least part of which

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

    Science.gov (United States)

    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.

  13. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-01-01

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  14. poolHiTS: A Shifted Transversal Design based pooling strategy for high-throughput drug screening

    Directory of Open Access Journals (Sweden)

    Woolf Peter J

    2008-05-01

    Full Text Available Abstract Background A key goal of drug discovery is to increase the throughput of small molecule screens without sacrificing screening accuracy. High-throughput screening (HTS in drug discovery involves testing a large number of compounds in a biological assay to identify active compounds. Normally, molecules from a large compound library are tested individually to identify the activity of each molecule. Usually a small number of compounds are found to be active, however the presence of false positive and negative testing errors suggests that this one-drug one-assay screening strategy can be significantly improved. Pooling designs are testing schemes that test mixtures of compounds in each assay, thereby generating a screen of the whole compound library in fewer tests. By repeatedly testing compounds in different combinations, pooling designs also allow for error-correction. These pooled designs, for specific experiment parameters, can be simply and efficiently created using the Shifted Transversal Design (STD pooling algorithm. However, drug screening contains a number of key constraints that require specific modifications if this pooling approach is to be useful for practical screen designs. Results In this paper, we introduce a pooling strategy called poolHiTS (Pooled High-Throughput Screening which is based on the STD algorithm. In poolHiTS, we implement a limit on the number of compounds that can be mixed in a single assay. In addition, we show that the STD-based pooling strategy is limited in the error-correction that it can achieve. Due to the mixing constraint, we show that it is more efficient to split a large library into smaller blocks of compounds, which are then tested using an optimized strategy repeated for each block. We package the optimal block selection algorithm into poolHiTS. The MATLAB codes for the poolHiTS algorithm and the corresponding decoding strategy are also provided. Conclusion We have produced a practical version

  15. Use of a Fluorometric Imaging Plate Reader in high-throughput screening

    Science.gov (United States)

    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.

  16. A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

    Science.gov (United States)

    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

    2011-02-01

    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

  17. High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide.

    Science.gov (United States)

    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.

  18. High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

    Science.gov (United States)

    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.

  19. A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-β aggregation inhibitors.

    Directory of Open Access Journals (Sweden)

    Yukako Ishigaki

    Full Text Available The aggregation of amyloid β protein (Aβ is a key step in the pathogenesis of Alzheimer's disease (AD, and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50. We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology.

  20. Adaptation to high throughput batch chromatography enhances multivariate screening.

    Science.gov (United States)

    Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

    2015-09-01

    High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

    Directory of Open Access Journals (Sweden)

    Asli N Goktug

    Full Text Available High-throughput RNA interference (RNAi screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis

  2. High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth

    Directory of Open Access Journals (Sweden)

    Gregory D. Bowden

    2018-04-01

    Full Text Available The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM, a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60–70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83% have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18 of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ± 12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 μM [0.036–0.12; 95% CI] or 21.9 ng/ml [12.1–40.3; 95% CI]. These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection. Keywords: Drug repurposing, High-throughput screen, Sarcocystis neurona, Equine protozoal myeloencephalitis

  3. Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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...

  5. High-throughput screening and confirmation of 22 banned veterinary drugs in feedstuffs using LC-MS/MS and high-resolution Orbitrap mass spectrometry.

    Science.gov (United States)

    Wang, Xufeng; Liu, Yanghong; Su, Yijuan; Yang, Jianwen; Bian, Kui; Wang, Zongnan; He, Li-Min

    2014-01-15

    A new analytical strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with accurate mass high-resolution Orbitrap mass spectrometry (HR-Orbitrap MS) was performed for high-throughput screening, confirmation, and quantification of 22 banned or unauthorized veterinary drugs in feedstuffs according to Bulletin 235 of the Ministry of Agriculture, China. Feed samples were extracted with acidified acetonitrile, followed by cleanup using solid-phase extraction cartridge. The extracts were first screened by LC-MS/MS in a single selected reaction monitoring mode. The suspected positive samples were subjected to a specific pretreatment for confirmation and quantification of analyte of interest with LC-MS/MS and HR-Orbitrap MS. Mean recoveries for all target analytes (except for carbofuran and chlordimeform, which were about 35 and 45%, respectively) ranged from 52.2 to 90.4%, and the relative standard deviations were screening of real samples obtained from local feed markets and confirmation of the suspected target analytes. It provides a high-throughput, sensitive, and reliable screening, identification, and quantification of banned veterinary drugs in routine monitoring programs of feedstuffs.

  6. High throughput miniature drug-screening platform using bioprinting technology

    International Nuclear Information System (INIS)

    Rodríguez-Dévora, Jorge I; Reyna, Daniel; Xu Tao; Zhang Bimeng; Shi Zhidong

    2012-01-01

    In the pharmaceutical industry, new drugs are tested to find appropriate compounds for therapeutic purposes for contemporary diseases. Unfortunately, novel compounds emerge at expensive prices and current target evaluation processes have limited throughput, thus leading to an increase of cost and time for drug development. This work shows the development of the novel inkjet-based deposition method for assembling a miniature drug-screening platform, which can realistically and inexpensively evaluate biochemical reactions in a picoliter-scale volume at a high speed rate. As proof of concept, applying a modified Hewlett Packard model 5360 compact disc printer, green fluorescent protein expressing Escherichia coli cells along with alginate gel solution have been arrayed on a coverslip chip under a repeatable volume of 180% ± 26% picoliters per droplet; subsequently, different antibiotic droplets were patterned on the spots of cells to evaluate the inhibition of bacteria for antibiotic screening. The proposed platform was compared to the current screening process, validating its effectiveness. The viability and basic function of the printed cells were evaluated, resulting in cell viability above 98% and insignificant or no DNA damage to human kidney cells transfected. Based on the reduction of investment and compound volume used by this platform, this technique has the potential to improve the actual drug discovery process at its target evaluation stage. (paper)

  7. A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Ting Xiao

    2016-10-01

    Full Text Available Eukaryotic elongation factor 2 kinase (eEF2K inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

  8. High throughput screening of ligand binding to macromolecules using high resolution powder diffraction

    Science.gov (United States)

    Von Dreele, Robert B.; D'Amico, Kevin

    2006-10-31

    A process is provided for the high throughput screening of binding of ligands to macromolecules using high resolution powder diffraction data including producing a first sample slurry of a selected polycrystalline macromolecule material and a solvent, producing a second sample slurry of a selected polycrystalline macromolecule material, one or more ligands and the solvent, obtaining a high resolution powder diffraction pattern on each of said first sample slurry and the second sample slurry, and, comparing the high resolution powder diffraction pattern of the first sample slurry and the high resolution powder diffraction pattern of the second sample slurry whereby a difference in the high resolution powder diffraction patterns of the first sample slurry and the second sample slurry provides a positive indication for the formation of a complex between the selected polycrystalline macromolecule material and at least one of the one or more ligands.

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

    Science.gov (United States)

    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)

  10. Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

    NARCIS (Netherlands)

    Breier, J.M.; Gassmann, K.; Kayser, R.; Stegeman, H.; Groot, D.de; Fritsche, E.; Shafer, T.J.

    2010-01-01

    In vitro, high-throughput methods 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 ramifications for the accuracy,

  11. High-Throughput Screening Using Mass Spectrometry within Drug Discovery.

    Science.gov (United States)

    Rohman, Mattias; Wingfield, Jonathan

    2016-01-01

    In order to detect a biochemical analyte with a mass spectrometer (MS) it is necessary to ionize the analyte of interest. The analyte can be ionized by a number of different mechanisms, however, one common method is electrospray ionization (ESI). Droplets of analyte are sprayed through a highly charged field, the droplets pick up charge, and this is transferred to the analyte. High levels of salt in the assay buffer will potentially steal charge from the analyte and suppress the MS signal. In order to avoid this suppression of signal, salt is often removed from the sample prior to injection into the MS. Traditional ESI MS relies on liquid chromatography (LC) to remove the salt and reduce matrix effects, however, this is a lengthy process. Here we describe the use of RapidFire™ coupled to a triple-quadrupole MS for high-throughput screening. This system uses solid-phase extraction to de-salt samples prior to injection, reducing processing time such that a sample is injected into the MS ~every 10 s.

  12. Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Justesen, Sune Frederik Lamdahl; Lamberth, Kasper

    2009-01-01

    , better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput...... the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range...... screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009: 173-180)...

  13. Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

    Science.gov (United States)

    Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

    2017-02-01

    More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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%.

  17. High throughput protein production screening

    Science.gov (United States)

    Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

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

    Science.gov (United States)

    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.

  19. Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 ...

  1. Small-molecule fluorophores to detect cell-state switching in the context of high-throughput screening.

    Science.gov (United States)

    Wagner, Bridget K; Carrinski, Hyman A; Ahn, Young-Hoon; Kim, Yun Kyung; Gilbert, Tamara J; Fomina, Dina A; Schreiber, Stuart L; Chang, Young-Tae; Clemons, Paul A

    2008-04-02

    A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.

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

    Science.gov (United States)

    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.

  3. Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

    Full Text Available Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER. Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.

  4. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

    Science.gov (United States)

    Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

    2016-01-01

    Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

  5. HDAT: web-based high-throughput screening data analysis tools

    International Nuclear Information System (INIS)

    Liu, Rong; Hassan, Taimur; Rallo, Robert; Cohen, Yoram

    2013-01-01

    The increasing utilization of high-throughput screening (HTS) in toxicity studies of engineered nano-materials (ENMs) requires tools for rapid and reliable processing and analyses of large HTS datasets. In order to meet this need, a web-based platform for HTS data analyses tools (HDAT) was developed that provides statistical methods suitable for ENM toxicity data. As a publicly available computational nanoinformatics infrastructure, HDAT provides different plate normalization methods, various HTS summarization statistics, self-organizing map (SOM)-based clustering analysis, and visualization of raw and processed data using both heat map and SOM. HDAT has been successfully used in a number of HTS studies of ENM toxicity, thereby enabling analysis of toxicity mechanisms and development of structure–activity relationships for ENM toxicity. The online approach afforded by HDAT should encourage standardization of and future advances in HTS as well as facilitate convenient inter-laboratory comparisons of HTS datasets. (paper)

  6. Screening for Antifibrotic Compounds Using High Throughput System Based on Fluorescence Polarization

    Directory of Open Access Journals (Sweden)

    Branko Stefanovic

    2014-04-01

    Full Text Available Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I mRNA and α2(I mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL. Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis in vivo. Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.

  7. High Throughput Screen for Novel Antimicrobials using a Whole Animal Infection Model

    Science.gov (United States)

    Moy, Terence I.; Conery, Annie L.; Larkins-Ford, Jonah; Wu, Gang; Mazitschek, Ralph; Casadei, Gabriele; Lewis, Kim; Carpenter, Anne E.; Ausubel, Frederick M.

    2009-01-01

    The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen with a mortality rate of up to 37% that is increasingly acquiring resistance to antibiotics. Here, we describe an automated, high throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. The screen uses a robot to accurately dispense live, infected animals into 384-well plates, and automated microscopy and image analysis to generate quantitative, high content data. We identified 28 compounds and extracts that were not previously reported to have antimicrobial properties, including 6 structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use. Our versatile and robust screening system can be easily adapted for other whole animal assays to probe a broad range of biological processes. PMID:19572548

  8. High-Throughput Lipolysis in 96-Well Plates for Rapid Screening of Lipid-Based Drug Delivery Systems

    DEFF Research Database (Denmark)

    Mosgaard, Mette D; Sassene, Philip J; Mu, Huiling

    2017-01-01

    The high-throughput in vitro intestinal lipolysis model (HTP) applicable for rapid and low-scale screening of lipid-based drug delivery systems (LbDDSs) was optimized and adjusted as to be conducted in 96-well plates (HTP-96). Three different LbDDSs (I-III) loaded with danazol or cinnarizine were...

  9. Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1

    Science.gov (United States)

    Thompson, Scott; Messick, Troy; Schultz, David C.; Reichman, Melvin; Lieberman, Paul M.

    2012-01-01

    Latent infection with Epstein-Barr Virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA-binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. We demonstrate that EBNA1 binding to a fluorescent labeled DNA probe provides a robust assay with a Z-factor consistently greater than 0.6. A pilot screen of a small molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1, but not Zta. All three compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof-of-concept that small molecule inhibitors of EBNA1 can be identified by biochemical high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection. PMID:20930215

  10. Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Strange, Mette; Bräuner-Osborne, Hans; Jensen, Anders A.

    2006-01-01

    We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay...... assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors....

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

    Science.gov (United States)

    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.

  12. Chemiluminescence analyzer of NOx as a high-throughput screening tool in selective catalytic reduction of NO

    International Nuclear Information System (INIS)

    Oh, Kwang Seok; Woo, Seong Ihl

    2011-01-01

    A chemiluminescence-based analyzer of NO x gas species has been applied for high-throughput screening of a library of catalytic materials. The applicability of the commercial NO x 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 Al 2 O 3 substrate was tested for the efficiency of NO x removal using a home-built 64-channel parallel and sequential tubular reactor. The NO x concentrations measured by the NO x analyzer agreed well with the results obtained using micro gas chromatography for a reference catalyst consisting of 1 wt% Pt on γ-Al 2 O 3 . Most alloys showed high efficiency at 275 °C, which is typical of Pt-based catalysts for selective catalytic reduction of NO. The screening with NO x analyzer allowed to select Pt-Ce (X) (X=1–3) and Pt–Fe (2) as the optimal catalysts for NO x removal: 73% NO x 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

    Science.gov (United States)

    Fu, Wei; Wang, Huiyu; Wang, Chenguang; Mei, Lin; Lin, Xiangmei; Han, Xueqing; Zhu, Shuifang

    2016-03-15

    The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Assessing the utility and limitations of high throughput virtual screening

    Directory of Open Access Journals (Sweden)

    Paul Daniel Phillips

    2016-05-01

    Full Text Available Due to low cost, speed, and unmatched ability to explore large numbers of compounds, high throughput virtual screening and molecular docking engines have become widely utilized by computational scientists. It is generally accepted that docking engines, such as AutoDock, produce reliable qualitative results for ligand-macromolecular receptor binding, and molecular docking results are commonly reported in literature in the absence of complementary wet lab experimental data. In this investigation, three variants of the sixteen amino acid peptide, α-conotoxin MII, were docked to a homology model of the a3β2-nicotinic acetylcholine receptor. DockoMatic version 2.0 was used to perform a virtual screen of each peptide ligand to the receptor for ten docking trials consisting of 100 AutoDock cycles per trial. The results were analyzed for both variation in the calculated binding energy obtained from AutoDock, and the orientation of bound peptide within the receptor. The results show that, while no clear correlation exists between consistent ligand binding pose and the calculated binding energy, AutoDock is able to determine a consistent positioning of bound peptide in the majority of trials when at least ten trials were evaluated.

  17. High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

    Science.gov (United States)

    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...

  18. The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond.

    Science.gov (United States)

    Haslam, Carl; Hellicar, John; Dunn, Adrian; Fuetterer, Arne; Hardy, Neil; Marshall, Peter; Paape, Rainer; Pemberton, Michelle; Resemannand, Anja; Leveridge, Melanie

    2016-02-01

    Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays. © 2015 Society for Laboratory Automation and Screening.

  19. CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.

    Science.gov (United States)

    Klann, Tyler S; Black, Joshua B; Chellappan, Malathi; Safi, Alexias; Song, Lingyun; Hilton, Isaac B; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2017-06-01

    Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9 KRAB repressor and dCas9 p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

  20. Data-Driven Derivation of an "Informer Compound Set" for Improved Selection of Active Compounds in High-Throughput Screening.

    Science.gov (United States)

    Paricharak, Shardul; IJzerman, Adriaan P; Jenkins, Jeremy L; Bender, Andreas; Nigsch, Florian

    2016-09-26

    Despite the usefulness of high-throughput screening (HTS) in drug discovery, for some systems, low assay throughput or high screening cost can prohibit the screening of large numbers of compounds. In such cases, iterative cycles of screening involving active learning (AL) are employed, creating the need for smaller "informer sets" that can be routinely screened to build predictive models for selecting compounds from the screening collection for follow-up screens. Here, we present a data-driven derivation of an informer compound set with improved predictivity of active compounds in HTS, and we validate its benefit over randomly selected training sets on 46 PubChem assays comprising at least 300,000 compounds and covering a wide range of assay biology. The informer compound set showed improvement in BEDROC(α = 100), PRAUC, and ROCAUC values averaged over all assays of 0.024, 0.014, and 0.016, respectively, compared to randomly selected training sets, all with paired t-test p-values agnostic fashion. This approach led to a consistent improvement in hit rates in follow-up screens without compromising scaffold retrieval. The informer set is adjustable in size depending on the number of compounds one intends to screen, as performance gains are realized for sets with more than 3,000 compounds, and this set is therefore applicable to a variety of situations. Finally, our results indicate that random sampling may not adequately cover descriptor space, drawing attention to the importance of the composition of the training set for predicting actives.

  1. Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery

    DEFF Research Database (Denmark)

    Asmild, Margit; Oswald, Nicholas; Krzywkowski, Karen M

    2003-01-01

    by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation...... (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs....

  2. High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

    OpenAIRE

    Hyeonhu Bae; Minwoo Park; Byungryul Jang; Yura Kang; Jinwoo Park; Hosik Lee; Haegeun Chung; ChiHye Chung; Suklyun Hong; Yongkyung Kwon; Boris I. Yakobson; Hoonkyung Lee

    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...

  3. A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

    2015-03-01

    The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman; Ba Alawi, Wail; Afeef, Moataz A.; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  7. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman

    2015-12-14

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  8. Screensaver: an open source lab information management system (LIMS for high throughput screening facilities

    Directory of Open Access Journals (Sweden)

    Nale Jennifer

    2010-05-01

    Full Text Available Abstract 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.

  9. Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities.

    Science.gov (United States)

    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.

  10. Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

    Science.gov (United States)

    Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

    2017-12-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

  11. Automation in Cytomics: A Modern RDBMS Based Platform for Image Analysis and Management in High-Throughput Screening Experiments

    NARCIS (Netherlands)

    E. Larios (Enrique); Y. Zhang (Ying); K. Yan (Kuan); Z. Di; S. LeDévédec (Sylvia); F.E. Groffen (Fabian); F.J. Verbeek

    2012-01-01

    textabstractIn cytomics bookkeeping of the data generated during lab experiments is crucial. The current approach in cytomics is to conduct High-Throughput Screening (HTS) experiments so that cells can be tested under many different experimental conditions. Given the large amount of different

  12. AOPs and Biomarkers: Bridging High Throughput Screening ...

    Science.gov (United States)

    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 benefit from additional data sources that connect the magnitude of perturbation from the in vitro system to a level of concern at the organism or population level. The adverse outcome pathway (AOP) concept provides an ideal framework for combining these complementary data. Recent international efforts under the auspices of the Organization for Economic Co-operation and Development (OECD) have resulted in an AOP wiki designed to house formal descriptions of AOPs suitable for use in regulatory decision making. Recent efforts have built upon this to include an ontology describing the AOP with linkages to biological pathways, physiological terminology, and taxonomic applicability domains. Incorporation of an AOP network tool developed by the U.S. Army Corps of Engineers also allows consideration of cumulative risk from chemical and non-chemical stressors. Biomarkers are an important complement to formal AOP descriptions, particularly when dealing with susceptible subpopulations or lifestages in human health risk assessment. To address the issue of nonchemical stressors than may modify effects of criteria air pollutants, a novel method was used to integrate blood gene expression data with hema

  13. A novel screen-printed electrode array for rapid high-throughput detection.

    Science.gov (United States)

    Mu, Shuai; Wang, Xiao; Li, Yuan-Ting; Wang, Yang; Li, Da-Wei; Long, Yi-Tao

    2012-07-21

    A novel multi-channel electrode array sensing device was fabricated by screen-printing techniques using 96-well plate as the template. To confirm its practical value, we developed a one-step preparation of multi-walled carbon nanotubes (MWCNTs) doped electrode array by an ink containing MWCNTs, which was applied to the simultaneous detection of a variety of biological samples and environmental pollutants. Results demonstrated that the designed sensing device could carry out the multiple measurements of different analytes at the same time, while MWCNTs enhanced the electrocatalytic activity of electrodes toward electroactive molecules. The required amount of each sample was only ∼200 μL. Moreover, the excellent differential pulse voltammetric (DPV) response toward dopamine, hydroquinone and catechol was obtained and the detection limits was determined to be 0.337, 0.289 and 0.369 μM, respectively. Comparing it with the traditional screen-printed electrode (SPE), this sensing device possesses the advantages of high-throughput, fast electron transfer rate for electrodes, short-time analysis and low sample consumption.

  14. Towards high throughput screening of electrochemical stability of battery electrolytes

    International Nuclear Information System (INIS)

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-01-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5–2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi 0.5 Mn 1.5 O 4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen. (paper)

  15. An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

    Science.gov (United States)

    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.

  16. Fluorographene as a Mass Spectrometry Probe for High-Throughput Identification and Screening of Emerging Chemical Contaminants in Complex Samples.

    Science.gov (United States)

    Huang, Xiu; Liu, Qian; Huang, Xiaoyu; Nie, Zhou; Ruan, Ting; Du, Yuguo; Jiang, Guibin

    2017-01-17

    Mass spectrometry techniques for high-throughput analysis of complex samples are of profound importance in many areas such as food safety, omics studies, and environmental health science. Here we report the use of fluorographene (FG) as a new mass spectrometry probe for high-throughput identification and screening of emerging chemical contaminants in complex samples. FG was facilely synthesized by one-step exfoliation of fluorographite. With FG as a matrix or probe in matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI- or SELDI-TOF MS), higher sensitivity (detection limits at ppt or subppt levels), and better reproducibility were achieved than with other graphene-based materials due to the unique chemical structure and self-assembly properties of FG. The method was validated with different types of real complex samples. By using FG as a SELDI probe, we could easily detect trace amount of bisphenol S in paper products and high-fat canned food samples. Furthermore, we have successfully identified and screened as many as 28 quaternary ammonium halides in sewage sludge samples collected from municipal wastewater treatment plants. These results demonstrate that FG probe is a powerful tool for high-throughput analysis of complex samples by MS.

  17. Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

    Science.gov (United States)

    Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

    2014-03-20

    Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. High throughput label-free platform for statistical bio-molecular sensing

    DEFF Research Database (Denmark)

    Bosco, Filippo; Hwu, En-Te; Chen, Ching-Hsiu

    2011-01-01

    Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing...

  19. Optical tools for high-throughput screening of abrasion resistance of combinatorial libraries of organic coatings

    Science.gov (United States)

    Potyrailo, Radislav A.; Chisholm, Bret J.; Olson, Daniel R.; Brennan, Michael J.; Molaison, Chris A.

    2002-02-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8x6 arrays of coating elements that are deposited on a plastic substrate. Each coating element of the library is 10 mm in diameter and 2 to 5 micrometers thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the organic coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at single-or multiple-abrasion conditions, followed by high- throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0 to 30% haze. System precision of 0.1 to 2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. While the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied to a variety of other applications where materials ranking can be achieved using optical spectroscopic tools.

  20. Comparison of a rational vs. high throughput approach for rapid salt screening and selection.

    Science.gov (United States)

    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.

  1. Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

    Science.gov (United States)

    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.

  2. A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

    Science.gov (United States)

    Wigle, Tim J; Swinger, Kerren K; Campbell, John E; Scholle, Michael D; Sherrill, John; Admirand, Elizabeth A; Boriack-Sjodin, P Ann; Kuntz, Kevin W; Chesworth, Richard; Moyer, Mikel P; Scott, Margaret Porter; Copeland, Robert A

    2015-07-01

    Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work. © 2015 Society for Laboratory Automation and Screening.

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

    Science.gov (United States)

    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.

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

    Data.gov (United States)

    U.S. Environmental Protection Agency — High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations

    Czech Academy of Sciences Publication Activity Database

    Bruštíková, Kateřina; Sedlák, David; Kubíková, Jana; Škuta, Ctibor; Šolcová, Kateřina; Malík, Radek; Bartůněk, Petr; Svoboda, Petr

    2018-01-01

    Roč. 9 (2018), č. článku 45. ISSN 1664-8021 R&D Projects: GA ČR GA13-29531S; GA MŠk LO1220; GA MŠk LM2015063; GA MŠk LM2011022 Institutional support: RVO:68378050 Keywords : miRNA * high-throughput screening * miR-30 * let-7 * Argonaute Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.789, year: 2016

  8. Laterally orienting C. elegans using geometry at microscale for high-throughput visual screens in neurodegeneration and neuronal development studies.

    Directory of Open Access Journals (Sweden)

    Ivan de Carlos Cáceres

    Full Text Available C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens.

  9. IspE inhibitors identified by a combination of in silico and in vitro high-throughput screening.

    Directory of Open Access Journals (Sweden)

    Naomi Tidten-Luksch

    Full Text Available 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.

  10. High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

    Science.gov (United States)

    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 screening for inherited disorders by biochemical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

    Directory of Open Access Journals (Sweden)

    Heather L Martin

    Full Text Available Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

  12. High-throughput Screening for Protein-based Inheritance in S. cerevisiae.

    Science.gov (United States)

    Byers, James S; Jarosz, Daniel F

    2017-08-08

    The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrP Sc ) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that >1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Nicolas Pinto

    2009-11-01

    Full Text Available 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.

  15. High-Throughput Screening of the Asymmetric Decarboxylative Alkylation Reaction of Enolate-Stabilized Enol Carbonates

    KAUST Repository

    Stoltz, Brian

    2010-06-14

    The use of high-throughput screening allowed for the optimization of reaction conditions for the palladium-catalyzed asymmetric decarboxylative alkylation reaction of enolate-stabilized enol carbonates. Changing to a non-polar reaction solvent and to an electron-deficient PHOX derivative as ligand from our standard reaction conditions improved the enantioselectivity for the alkylation of a ketal-protected,1,3-diketone-derived enol carbonate from 28% ee to 84% ee. Similar improvements in enantioselectivity were seen for a β-keto-ester derived- and an α-phenyl cyclohexanone-derived enol carbonate.

  16. High-Throughput Screening of the Asymmetric Decarboxylative Alkylation Reaction of Enolate-Stabilized Enol Carbonates

    KAUST Repository

    Stoltz, Brian; McDougal, Nolan; Virgil, Scott

    2010-01-01

    The use of high-throughput screening allowed for the optimization of reaction conditions for the palladium-catalyzed asymmetric decarboxylative alkylation reaction of enolate-stabilized enol carbonates. Changing to a non-polar reaction solvent and to an electron-deficient PHOX derivative as ligand from our standard reaction conditions improved the enantioselectivity for the alkylation of a ketal-protected,1,3-diketone-derived enol carbonate from 28% ee to 84% ee. Similar improvements in enantioselectivity were seen for a β-keto-ester derived- and an α-phenyl cyclohexanone-derived enol carbonate.

  17. Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

    Science.gov (United States)

    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...

  18. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

    Science.gov (United States)

    Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

    2016-04-01

    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 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.

  19. High throughput reaction screening using desorption electrospray ionization mass spectrometry.

    Science.gov (United States)

    Wleklinski, Michael; Loren, Bradley P; Ferreira, Christina R; Jaman, Zinia; Avramova, Larisa; Sobreira, Tiago J P; Thompson, David H; Cooks, R Graham

    2018-02-14

    We report the high throughput analysis of reaction mixture arrays using methods and data handling routines that were originally developed for biological tissue imaging. Desorption electrospray ionization (DESI) mass spectrometry (MS) is applied in a continuous on-line process at rates that approach 10 4 reactions per h at area densities of up to 1 spot per mm 2 (6144 spots per standard microtiter plate) with the sprayer moving at ca. 10 4 microns per s. Data are analyzed automatically by MS using in-house software to create ion images of selected reagents and products as intensity plots in standard array format. Amine alkylation reactions were used to optimize the system performance on PTFE membrane substrates using methanol as the DESI spray/analysis solvent. Reaction times can be screening of processes like N -alkylation and Suzuki coupling reactions as reported herein. Products and by-products were confirmed by on-line MS/MS upon rescanning of the array.

  20. High-throughput bioaffinity mass spectrometry for screening and identification of designer anabolic steroids in dietary supplements.

    Science.gov (United States)

    Aqai, Payam; Cevik, Ebru; Gerssen, Arjen; Haasnoot, Willem; Nielen, Michel W F

    2013-03-19

    A generic high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of known and unknown recombinant human sex hormone-binding globulin (rhSHBG)-binding designer steroids in dietary supplements. For screening, a semi-automated competitive inhibition binding assay was combined with fast ultrahigh-performance-LC-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS). 17β-Testosterone-D3 was used as the stable isotope label of which the binding to rhSHBG-coated paramagnetic microbeads was inhibited by any other binding (designer) steroid. The assay was performed in a 96-well plate and combined with the fast LC-MS, 96 measurements could be performed within 4 h. The concentration-dependent inhibition of the label by steroids in buffer and dietary supplements was demonstrated. Following an adjusted bioaffinity isolation procedure, suspect extracts were injected into a chip-UPLC(NanoTile)-Q-time-of-flight-MS system for full-scan accurate mass identification. Next to known steroids, 1-testosterone was identified in three of the supplements studied and the designer steroid tetrahydrogestrinone was identified in a spiked supplement. The generic steroid-binding assay can be used for high-throughput screening of androgens, estrogens, and gestagens in dietary supplements to fight doping. When combined with chip-UPLC-MS, it is a powerful tool for early warning of unknown emerging rhSHBG bioactive designer steroids in dietary supplements.

  1. Combinatorial Strategies and High Throughput Screening in Drug Discovery Targeted to the Channel of Botulinum Neurotoxin

    National Research Council Canada - National Science Library

    Montal, Mauricio

    2006-01-01

    .... The major focus thus far has been the implementation of a reliable and robust high-throughput screen for blockers specific for BoNT using Neuro 2A cells in which BoNTA forms channels with similar properties to those previously characterized in lipid bilayers. The immediate task during the present reporting period involved the detailed characterization of the channel and chaperone activity of BoNTA on Neuro2A cells.

  2. High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

    Science.gov (United States)

    Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

    2017-12-01

    With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  4. High Throughput Screening of Valganciclovir in Acidic Microenvironments of Polyester Thin Films

    Directory of Open Access Journals (Sweden)

    Teilo Schaller

    2015-04-01

    Full Text Available Ganciclovir and valganciclor are antiviral agents used for the treatment of cytomegalovirus retinitis. The conventional method for administering ganciclovir in cytomegalovirus retinitis patients is repeated intravitreal injections. In order to obviate the possible detrimental effects of repeated intraocular injections, to improve compliance and to eliminate systemic side-effects, we investigated the tuning of the ganciclovir pro-drug valganciclovir and the release from thin films of poly(lactic-co-glycolic acid (PLGA, polycaprolactone (PCL, or mixtures of both, as a step towards prototyping periocular valganciclovir implants. To investigate the drug release, we established and evaluated a high throughput fluorescence-based quantification screening assay for the detection of valganciclovir. Our protocol allows quantifying as little as 20 ng of valganciclovir in 96-well polypropylene plates and a 50× faster analysis compared to traditional HPLC measurements. This improvement can hence be extrapolated to other polyester matrix thin film formulations using a high-throughput approach. The acidic microenvironment within the polyester matrix was found to protect valganciclovir from degradation with resultant increases in the half-life of the drug in the periocular implant to 100 days. Linear release profiles were obtained using the pure polyester polymers for 10 days and 60 days formulations; however, gross phase separations of PCL and acid-terminated PLGA prevented tuning within these timeframes due to the phase separation of the polymer, valganciclovir, or both.

  5. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    Directory of Open Access Journals (Sweden)

    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  6. High-throughput screening for compounds that modulate the cellular c-di-GMP level in bacteria

    DEFF Research Database (Denmark)

    Groizeleau, Julie; Andersen, Jens Bo; Givskov, Michael

    2017-01-01

    . The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. We describe a protocol for high......-throughput screening of chemical libraries for compounds that lower the c-di-GMP level in bacteria, and potentially can serve as lead compounds in the development of novel biofilm dismantling drugs....

  7. Using In Vitro High-Throughput Screening Data for Predicting ...

    Science.gov (United States)

    Today there are more than 80,000 chemicals in commerce and the environment. The potential human health risks are unknown for the vast majority of these chemicals as they lack human health risk assessments, toxicity reference values and risk screening values. We aim to use computational toxicology and quantitative high throughput screening (qHTS) technologies to fill these data gaps, and begin to prioritize these chemicals for additional assessment. By coupling qHTS data with adverse outcome pathways (AOPs) we can use ontologies to make predictions about potential hazards and to identify those assays which are sufficient to infer these same hazards. Once those assays are identified, we can use bootstrap natural spline-based metaregression to integrate the evidence across multiple replicates or assays (if a combination of assays are together necessary to be sufficient). In this pilot, we demonstrate how we were able to identify that benzo[k]fluoranthene (B[k]F) may induce DNA damage and steatosis using qHTS data and two separate AOPs. We also demonstrate how bootstrap natural spline-based metaregression can be used to integrate the data across multiple assay replicates to generate a concentration-response curve. We used this analysis to calculate an internal point of departure of 0.751µM and risk-specific concentrations of 0.378µM for both 1:1,000 and 1:10,000 additive risk for B[k]F induced DNA damage based on the p53 assay. Based on the available evidence, we

  8. Identifying genes that extend life span using a high-throughput screening system.

    Science.gov (United States)

    Chen, Cuiying; Contreras, Roland

    2007-01-01

    We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

  9. High-throughput screening of saliva for early detection of oral cancer: a pilot study.

    Science.gov (United States)

    Szanto, I; Mark, L; Bona, A; Maasz, G; Sandor, B; Gelencser, G; Turi, Z; Gallyas, F

    2012-04-01

    The success of tumour therapy depends considerably on early diagnosis. Therefore, we aimed to develop a widely available, cheap, non-invasive, high-throughput method suitable for screening high-risk populations, at least, for early signs of malignant transformation in the oral cavity. First, in order to identify suitable tumour marker candidates, we compared the protein patterns of five selected saliva samples obtained from healthy controls and tumour patients after electrophoretic separation, excised the bands that were consistently up-regulated in the tumour patients only, and performed matrix-assisted laser-desorption ionisation (MALDI)-time of flight (TOF) tandem mass spectrometry (MS/MS) analysis of the proteins in these bands after in-gel tryptic digestion. From the panel of proteins identified, we chose annexin 1 and peroxiredoxin 2 for further studies based on their presence in the saliva of all five oral cancer patients only. Then, we performed a homology search of protein databases using the primary sequence of each in silico tryptic fragment peptide of these two proteins as bait, and selected a unique peptide for each. Finally, we performed targeted MALDI-TOF MS peptide analysis in a blinded fashion on all samples obtained from 20 healthy controls and 22 tumour patients for the presence of these peptides. We found both peptides present in the saliva samples of all cancer patients only. Even though these tumour markers should be validated in a wider population, our results indicate that targeted MALDI-TOF MS analysis of unique peptides of putative saliva protein tumour biomarkers could be the method of choice for cost-efficient, high-throughput screening for the early detection of oral cancer.

  10. High-throughput screening of a diversity collection using biodefense category A and B priority pathogens.

    Science.gov (United States)

    Barrow, Esther W; Clinkenbeard, Patricia A; Duncan-Decocq, Rebecca A; Perteet, Rachel F; Hill, Kimberly D; Bourne, Philip C; Valderas, Michelle W; Bourne, Christina R; Clarkson, Nicole L; Clinkenbeard, Kenneth D; Barrow, William W

    2012-08-01

    One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.

  11. High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening.

    Science.gov (United States)

    Shcherbakova, Elena G; Brega, Valentina; Lynch, Vincent M; James, Tony D; Anzenbacher, Pavel

    2017-07-26

    A simple and efficient method for determination of the yield, enantiomeric/diasteriomeric excess (ee/de), and absolute configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach utilizes a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan methyl ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophysical properties and allow for fluorescence-based ee determination of molecules containing a 1,2- or 1,3-diol moiety. This method has been utilized for the screening of ee in a number of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asymmetric hydrogenation of benzil was performed in a highly parallel fashion with errors products from the parallel asymmetric synthesis in real time and in a high-throughput screening (HTS) fashion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. High-Throughput and Rapid Screening of Low-Mass Hazardous Compounds in Complex Samples.

    Science.gov (United States)

    Wang, Jing; Liu, Qian; Gao, Yan; Wang, Yawei; Guo, Liangqia; Jiang, Guibin

    2015-07-07

    Rapid screening and identification of hazardous chemicals in complex samples is of extreme importance for public safety and environmental health studies. In this work, we report a new method for high-throughput, sensitive, and rapid screening of low-mass hazardous compounds in complex media without complicated sample preparation procedures. This method is achieved based on size-selective enrichment on ordered mesoporous carbon followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis with graphene as a matrix. The ordered mesoporous carbon CMK-8 can exclude interferences from large molecules in complex samples (e.g., human serum, urine, and environmental water samples) and efficiently enrich a wide variety of low-mass hazardous compounds. The method can work at very low concentrations down to part per trillion (ppt) levels, and it is much faster and more facile than conventional methods. It was successfully applied to rapidly screen and identify unknown toxic substances such as perfluorochemicals in human serum samples from athletes and workers. Therefore, this method not only can sensitively detect target compounds but also can identify unknown hazardous compounds in complex media.

  13. A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors.

    Directory of Open Access Journals (Sweden)

    Andrew R Schwendeman

    Full Text Available Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery.

  14. High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kassir, Yona

    2017-01-01

    Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

  15. High Throughput Screening Method for Systematic Surveillance of Drugs of Abuse by Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry.

    Science.gov (United States)

    DiBattista, Alicia; Rampersaud, Dianne; Lee, Howard; Kim, Marcus; Britz-McKibbin, Philip

    2017-11-07

    New technologies are urgently required for reliable drug screening given a worldwide epidemic of prescription drug abuse and its devastating socioeconomic impacts on public health. Primary screening of drugs of abuse (DoA) currently relies on immunoassays that are prone to bias and are not applicable to detect an alarming array of psychoactive stimulants, tranquilizers, and synthetic opioids. These limitations impact patient safety when monitoring for medication compliance, drug substitution, or misuse/abuse and require follow-up confirmatory testing by more specific yet lower throughput instrumental methods. Herein, we introduce a high throughput platform for nontargeted screening of a broad spectrum of DoA and their metabolites based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). We demonstrate that MSI-CE-MS enables serial injections of 10 samples within a single run (high resolution MS with full-scan data acquisition. Unambiguous drug identification was achieved by four or more independent parameters, including comigration with a deuterated internal standard or in silico prediction of electromigration behavior together with accurate mass, most likely molecular formula, as well as MS/MS as required for confirmation testing. Acceptable precision was demonstrated for over 50 DoA at 3 concentration levels over 4 days (median coefficient of variance = 13%, n = 117) with minimal ion suppression, isobaric interferences, and sample carry-over (screening cutoff levels in human urine while allowing for systematic surveillance, specimen verification, and retrospective testing of designer drugs that elude conventional drug tests.

  16. Efficient high-throughput biological process characterization: Definitive screening design with the ambr250 bioreactor system.

    Science.gov (United States)

    Tai, Mitchell; Ly, Amanda; Leung, Inne; Nayar, Gautam

    2015-01-01

    The burgeoning pipeline for new biologic drugs has increased the need for high-throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250-mL automated mini bioreactor (ambr250™) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24-bioreactor ambr250™ system with 10-factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory-scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late-stage characterization. © 2015 American Institute of Chemical Engineers.

  17. A cell-based high-throughput protocol to screen entry inhibitors of highly pathogenic viruses with Traditional Chinese Medicines.

    Science.gov (United States)

    Yang, Yong; Cheng, Han; Yan, Hui; Wang, Peng-Zhan; Rong, Rong; Zhang, Ying-Ying; Zhang, Cheng-Bo; Du, Rui-Kun; Rong, Li-Jun

    2017-05-01

    Emerging viruses such as Ebola virus (EBOV), Lassa virus (LASV), and avian influenza virus H5N1 (AIV) are global health concerns. Since there is very limited options (either vaccine or specific therapy) approved for humans against these viruses, there is an urgent need to develop prophylactic and therapeutic treatments. Previously we reported a high-throughput screening (HTS) protocol to identify entry inhibitors for three highly pathogenic viruses (EBOV, LASV, and AIV) using a human immunodeficiency virus-based pseudotyping platform which allows us to perform the screening in a BSL-2 facility. In this report, we have adopted this screening protocol to evaluate traditional Chinese Medicines (TCMs) in an effort to discover entry inhibitors against these viruses. Here we show that extracts of the following Chinese medicinal herbs exhibit potent anti-Ebola viral activities: Gardenia jasminoides Ellis, Citrus aurantium L., Viola yedoensis Makino, Prunella vulgaris L., Coix lacryma-jobi L. var. mayuen (Roman.) Stapf, Pinellia ternata (Thunb.) Breit., and Morus alba L. This study represents a proof-of-principle investigation supporting the suitability of this assay for rapid screening TCMs and identifying putative entry inhibitors for these viruses. J. Med. Virol. 89:908-916, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits.

    Science.gov (United States)

    Choi, Su-Lim; Rha, Eugene; Lee, Sang Jun; Kim, Haseong; Kwon, Kilkoang; Jeong, Young-Su; Rhee, Young Ha; Song, Jae Jun; Kim, Hak-Sung; Lee, Seung-Goo

    2014-03-21

    Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

  19. High-throughput screen for novel antimicrobials using a whole animal infection model.

    Science.gov (United States)

    Moy, Terence I; Conery, Annie L; Larkins-Ford, Jonah; Wu, Gang; Mazitschek, Ralph; Casadei, Gabriele; Lewis, Kim; Carpenter, Anne E; Ausubel, Frederick M

    2009-07-17

    The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen. Here, we describe an automated, high-throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. Using a robot to dispense live, infected animals into 384-well plates and automated microscopy and image analysis, we identified 28 compounds and extracts not previously reported to have antimicrobial properties, including six structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use.

  20. A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs.

    Science.gov (United States)

    Ihlow, Alexander; Schweizer, Patrick; Seiffert, Udo

    2008-01-23

    To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps. We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects. Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.

  1. Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening.

    Science.gov (United States)

    Lee, Moo-Yeal; Dordick, Jonathan S; Clark, Douglas S

    2010-01-01

    Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To accelerate the identification of safer drug candidates and improve the clinical progression of drug candidates to pharmaceuticals, it is important to develop high-throughput tools that can provide early-stage predictive toxicology data. In particular, in vitro cell-based systems that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process. The in vitro techniques that provide a high degree of human toxicity prediction will be perhaps more important in cosmetic and chemical industries in Europe, as animal toxicity testing is being phased out entirely in the immediate future.We have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized three-dimensional (3D) cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products. The human or rat MetaChip contains an array of encapsulated metabolic enzymes that is designed to emulate the metabolic reactions in the human or rat liver. The human or rat DataChip contains an array of 3D human or rat cells encapsulated in alginate gels for cell-based toxicity screening. By combining the DataChip with the complementary MetaChip, in vitro toxicity results are obtained that correlate well with in vivo rat data.

  2. DockoMatic: automated peptide analog creation for high throughput virtual screening.

    Science.gov (United States)

    Jacob, Reed B; Bullock, Casey W; Andersen, Tim; McDougal, Owen M

    2011-10-01

    The purpose of this manuscript is threefold: (1) to describe an update to DockoMatic that allows the user to generate cyclic peptide analog structure files based on protein database (pdb) files, (2) to test the accuracy of the peptide analog structure generation utility, and (3) to evaluate the high throughput capacity of DockoMatic. The DockoMatic graphical user interface interfaces with the software program Treepack to create user defined peptide analogs. To validate this approach, DockoMatic produced cyclic peptide analogs were tested for three-dimensional structure consistency and binding affinity against four experimentally determined peptide structure files available in the Research Collaboratory for Structural Bioinformatics database. The peptides used to evaluate this new functionality were alpha-conotoxins ImI, PnIA, and their published analogs. Peptide analogs were generated by DockoMatic and tested for their ability to bind to X-ray crystal structure models of the acetylcholine binding protein originating from Aplysia californica. The results, consisting of more than 300 simulations, demonstrate that DockoMatic predicts the binding energy of peptide structures to within 3.5 kcal mol(-1), and the orientation of bound ligand compares to within 1.8 Å root mean square deviation for ligand structures as compared to experimental data. Evaluation of high throughput virtual screening capacity demonstrated that Dockomatic can collect, evaluate, and summarize the output of 10,000 AutoDock jobs in less than 2 hours of computational time, while 100,000 jobs requires approximately 15 hours and 1,000,000 jobs is estimated to take up to a week. Copyright © 2011 Wiley Periodicals, Inc.

  3. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening

    Directory of Open Access Journals (Sweden)

    Pothineni VR

    2016-04-01

    Full Text Available Venkata Raveendra Pothineni,1 Dhananjay Wagh,1 Mustafeez Mujtaba Babar,1 Mohammed Inayathullah,1 David Solow-Cordero,2 Kwang-Min Kim,1 Aneesh V Samineni,1 Mansi B Parekh,1 Lobat Tayebi,3 Jayakumar Rajadas1 1Biomaterials and Advanced Drug Delivery Laboratory, Stanford Cardiovascular Pharmacology Division, Cardiovascular Institute, Stanford University School of Medicine, Palo Alto, 2Chemical & Systems Biology, Stanford University School of Medicine, Stanford, CA, 3Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI, USA Abstract: Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved that were screened are Library of Pharmacologically Active Compounds (LOPAC1280, the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150

  4. Development of a high throughput single-particle screening for inorganic semiconductor nanorods as neural voltage sensor

    Science.gov (United States)

    Kuo, Yung; Park, Kyoungwon; Li, Jack; Ingargiola, Antonino; Park, Joonhyuck; Shvadchak, Volodymyr; Weiss, Shimon

    2017-08-01

    Monitoring membrane potential in neurons requires sensors with minimal invasiveness, high spatial and temporal (sub-ms) resolution, and large sensitivity for enabling detection of sub-threshold activities. While organic dyes and fluorescent proteins have been developed to possess voltage-sensing properties, photobleaching, cytotoxicity, low sensitivity, and low spatial resolution have obstructed further studies. Semiconductor nanoparticles (NPs), as prospective voltage sensors, have shown excellent sensitivity based on Quantum confined Stark effect (QCSE) at room temperature and at single particle level. Both theory and experiment have shown their voltage sensitivity can be increased significantly via material, bandgap, and structural engineering. Based on theoretical calculations, we synthesized one of the optimal candidates for voltage sensors: 12 nm type-II ZnSe/CdS nanorods (NRs), with an asymmetrically located seed. The voltage sensitivity and spectral shift were characterized in vitro using spectrally-resolved microscopy using electrodes grown by thin film deposition, which "sandwich" the NRs. We characterized multiple batches of such NRs and iteratively modified the synthesis to achieve higher voltage sensitivity (ΔF/F> 10%), larger spectral shift (>5 nm), better homogeneity, and better colloidal stability. Using a high throughput screening method, we were able to compare the voltage sensitivity of our NRs with commercial spherical quantum dots (QDs) with single particle statistics. Our method of high throughput screening with spectrally-resolved microscope also provides a versatile tool for studying single particles spectroscopy under field modulation.

  5. Time of flight secondary ion mass spectrometry: A powerful high throughput screening tool

    International Nuclear Information System (INIS)

    Smentkowski, Vincent S.; Ostrowski, Sara G.

    2007-01-01

    Combinatorial materials libraries are becoming more complicated; successful screening of these libraries requires the development of new high throughput screening methodologies. Time of flight secondary ion mass spectrometry (ToF-SIMS) is a surface analytical technique that is able to detect and image all elements (including hydrogen which is problematic for many other analysis instruments) and molecular fragments, with high mass resolution, during a single measurement. Commercial ToF-SIMS instruments can image 500 μm areas by rastering the primary ion beam over the region of interest. In this work, we will show that large area analysis can be performed, in one single measurement, by rastering the sample under the ion beam. We show that an entire 70 mm diameter wafer can be imaged in less than 90 min using ToF-SIMS stage (macro)rastering techniques. ToF-SIMS data sets contain a wealth of information since an entire high mass resolution mass spectrum is saved at each pixel in an ion image. Multivariate statistical analysis (MVSA) tools are being used in the ToF-SIMS community to assist with data interpretation; we will demonstrate that MVSA tools provide details that were not obtained using manual (univariate) analysis

  6. Benchmarking Ligand-Based Virtual High-Throughput Screening with the PubChem Database

    Directory of Open Access Journals (Sweden)

    Mariusz Butkiewicz

    2013-01-01

    Full Text Available With the rapidly increasing availability of High-Throughput Screening (HTS data in the public domain, such as the PubChem database, methods for ligand-based computer-aided drug discovery (LB-CADD have the potential to accelerate and reduce the cost of probe development and drug discovery efforts in academia. We assemble nine data sets from realistic HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods. Each data set is public domain through PubChem and carefully collated through confirmation screens validating active compounds. These data sets provide the foundation for benchmarking a new cheminformatics framework BCL::ChemInfo, which is freely available for non-commercial use. Quantitative structure activity relationship (QSAR models are built using Artificial Neural Networks (ANNs, Support Vector Machines (SVMs, Decision Trees (DTs, and Kohonen networks (KNs. Problem-specific descriptor optimization protocols are assessed including Sequential Feature Forward Selection (SFFS and various information content measures. Measures of predictive power and confidence are evaluated through cross-validation, and a consensus prediction scheme is tested that combines orthogonal machine learning algorithms into a single predictor. Enrichments ranging from 15 to 101 for a TPR cutoff of 25% are observed.

  7. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  8. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    International Nuclear Information System (INIS)

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s −1 ) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening

  9. Development of droplets‐based microfluidic systems for single­‐cell high‐throughput screening

    DEFF Research Database (Denmark)

    Chen, Jun; Jensen, Thomas Glasdam; Godina, Alexei

    2014-01-01

    High-throughput screening (HTS) plays an important role in the development of microbial cell factories. One of the most popular approaches is to use microplates combined with the application of robotics, liquid handling and sophisticated detection methods. However, these workstations require large...... investment, and a logarithmic increase to screen large combinatorial libraries over the decades also makes it gradually out of depth. Here, we are trying to develop a feasible high‐throughput system that uses microfluidics to compartmentalize a single cell for propagation and analysis in monodisperse...... picoliter aqueous droplets surround by an immiscible fluorinated oil phase. Our aim is to use this system to facilitate the screening process for both the biotechnology and food industry....

  10. High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production.

    Science.gov (United States)

    Cheng, Xiaoliang; Hiras, Jennifer; Deng, Kai; Bowen, Benjamin; Simmons, Blake A; Adams, Paul D; Singer, Steven W; Northen, Trent R

    2013-01-01

    Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS)-based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC), Medium 84 + rolled oats, and M9TE + MCC at 45°C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45°C than at all other temperatures. While T. bispora is reported to grow optimally at 60°C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45°C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

  11. High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production

    Directory of Open Access Journals (Sweden)

    Xiaoliang eCheng

    2013-12-01

    Full Text Available Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC, Medium 84 + rolled oats, and M9TE + MCC at 45 °C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45 °C than at all other temperatures. While T. bispora is reported to grow optimally at 60 °C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45 °C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

  12. Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

    Directory of Open Access Journals (Sweden)

    Sun Youxian

    2008-06-01

    Full Text Available Abstract 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

  13. HTTK: R Package for High-Throughput Toxicokinetics

    Science.gov (United States)

    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...

  14. High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.

    Science.gov (United States)

    Czerniecki, Stefan M; Cruz, Nelly M; Harder, Jennifer L; Menon, Rajasree; Annis, James; Otto, Edgar A; Gulieva, Ramila E; Islas, Laura V; Kim, Yong Kyun; Tran, Linh M; Martins, Timothy J; Pippin, Jeffrey W; Fu, Hongxia; Kretzler, Matthias; Shankland, Stuart J; Himmelfarb, Jonathan; Moon, Randall T; Paragas, Neal; Freedman, Benjamin S

    2018-05-15

    Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

    Science.gov (United States)

    Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

    2017-11-17

    Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

  16. Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  19. MIPHENO: Data normalization for high throughput metabolic analysis.

    Science.gov (United States)

    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...

  20. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer?s-Associated A? Oligomers

    OpenAIRE

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein int...

  1. Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory.

    Science.gov (United States)

    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

  2. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    Directory of Open Access Journals (Sweden)

    Michael S Rogers

    Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

  3. Advances in High Throughput Screening of Biomass Recalcitrance (Poster)

    Energy Technology Data Exchange (ETDEWEB)

    Turner, G. B.; Decker, S. R.; Tucker, M. P.; Law, C.; Doeppke, C.; Sykes, R. W.; Davis, M. F.; Ziebell, A.

    2012-06-01

    This was a poster displayed at the Symposium. Advances on previous high throughput screening of biomass recalcitrance methods have resulted in improved conversion and replicate precision. Changes in plate reactor metallurgy, improved preparation of control biomass, species-specific pretreatment conditions, and enzymatic hydrolysis parameters have reduced overall coefficients of variation to an average of 6% for sample replicates. These method changes have improved plate-to-plate variation of control biomass recalcitrance and improved confidence in sugar release differences between samples. With smaller errors plant researchers can have a higher degree of assurance more low recalcitrance candidates can be identified. Significant changes in plate reactor, control biomass preparation, pretreatment conditions and enzyme have significantly reduced sample and control replicate variability. Reactor plate metallurgy significantly impacts sugar release aluminum leaching into reaction during pretreatment degrades sugars and inhibits enzyme activity. Removal of starch and extractives significantly decreases control biomass variability. New enzyme formulations give more consistent and higher conversion levels, however required re-optimization for switchgrass. Pretreatment time and temperature (severity) should be adjusted to specific biomass types i.e. woody vs. herbaceous. Desalting of enzyme preps to remove low molecular weight stabilizers and improved conversion levels likely due to water activity impacts on enzyme structure and substrate interactions not attempted here due to need to continually desalt and validate precise enzyme concentration and activity.

  4. Use of High Throughput Screening Data in IARC Monograph ...

    Science.gov (United States)

    Purpose: Evaluation of carcinogenic mechanisms serves a critical role in IARC monograph evaluations, and can lead to “upgrade” or “downgrade” of the carcinogenicity conclusions based on human and animal evidence alone. Three recent IARC monograph Working Groups (110, 112, and 113) pioneered analysis of high throughput in vitro screening data from the U.S. Environmental Protection Agency’s ToxCast program in evaluations of carcinogenic mechanisms. Methods: For monograph 110, ToxCast assay data across multiple nuclear receptors were used to test the hypothesis that PFOA acts exclusively through the PPAR family of receptors, with activity profiles compared to several prototypical nuclear receptor-activating compounds. For monographs 112 and 113, ToxCast assays were systematically evaluated and used as an additional data stream in the overall evaluation of the mechanistic evidence. Specifically, ToxCast assays were mapped to 10 “key characteristics of carcinogens” recently identified by an IARC expert group, and chemicals’ bioactivity profiles were evaluated both in absolute terms (number of relevant assays positive for bioactivity) and relative terms (ranking with respect to other compounds evaluated by IARC, using the ToxPi methodology). Results: PFOA activates multiple nuclear receptors in addition to the PPAR family in the ToxCast assays. ToxCast assays offered substantial coverage for 5 of the 10 “key characteristics,” with the greates

  5. ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    McGown, Alexander; Shaw, Dame Pamela J; Ramesh, Tennore

    2016-07-26

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2-3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well suited for the study of diseasepathophysiology and also for high throughput (HT) drug screening. The mutant sod1 zebrafish model of ALS mimics the hallmark features of ALS. Using a fluorescence based readout of neuronal stress, we developed a high throughput (HT) screen to identify neuroprotective compounds. Here we show that the zebrafish screen is a robust system that can be used to rapidly screen thousands ofcompounds and also demonstrate that riluzole is capable of reducing neuronal stress in this model system. The screen shows optimal quality control, maintaining a high sensitivity and specificity withoutcompromising throughput. Most importantly, we demonstrate that many compounds previously failed in human clinical trials, showed no stress reducing activity in the zebrafish assay. We conclude that HT drug screening using a mutant sod1 zebrafish is a reliable model system which supplemented with secondary assays would be useful in identifying drugs with potential for neuroprotective efficacy in ALS.

  6. RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

    Science.gov (United States)

    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.

  7. High Throughout Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale

    2003-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  8. Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

  9. High-Throughput Screening and Quantitation of Target Compounds in Biofluids by Coated Blade Spray-Mass Spectrometry.

    Science.gov (United States)

    Tascon, Marcos; Gómez-Ríos, Germán Augusto; Reyes-Garcés, Nathaly; Poole, Justen; Boyacı, Ezel; Pawliszyn, Janusz

    2017-08-15

    Most contemporary methods of screening and quantitating controlled substances and therapeutic drugs in biofluids typically require laborious, time-consuming, and expensive analytical workflows. In recent years, our group has worked toward developing microextraction (μe)-mass spectrometry (MS) technologies that merge all of the tedious steps of the classical methods into a simple, efficient, and low-cost methodology. Unquestionably, the automation of these technologies allows for faster sample throughput, greater reproducibility, and radically reduced analysis times. Coated blade spray (CBS) is a μe technology engineered for extracting/enriching analytes of interest in complex matrices, and it can be directly coupled with MS instruments to achieve efficient screening and quantitative analysis. In this study, we introduced CBS as a technology that can be arranged to perform either rapid diagnostics (single vial) or the high-throughput (96-well plate) analysis of biofluids. Furthermore, we demonstrate that performing 96-CBS extractions at the same time allows the total analysis time to be reduced to less than 55 s per sample. Aiming to validate the versatility of CBS, substances comprising a broad range of molecular weights, moieties, protein binding, and polarities were selected. Thus, the high-throughput (HT)-CBS technology was used for the concomitant quantitation of 18 compounds (mixture of anabolics, β-2 agonists, diuretics, stimulants, narcotics, and β-blockers) spiked in human urine and plasma samples. Excellent precision (∼2.5%), accuracy (≥90%), and linearity (R 2 ≥ 0.99) were attained for all the studied compounds, and the limits of quantitation (LOQs) were within the range of 0.1 to 10 ng·mL -1 for plasma and 0.25 to 10 ng·mL -1 for urine. The results reported in this paper confirm CBS's great potential for achieving subsixty-second analyses of target compounds in a broad range of fields such as those related to clinical diagnosis, food, the

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

    Science.gov (United States)

    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.

  11. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Directory of Open Access Journals (Sweden)

    Kyle C Wilcox

    Full Text Available Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs. AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs. This method gives a soluble membrane protein library (SMPL--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can

  12. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Science.gov (United States)

    Wilcox, Kyle C; Marunde, Matthew R; Das, Aditi; Velasco, Pauline T; Kuhns, Benjamin D; Marty, Michael T; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G; Klein, William L

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery

  13. Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase.

    Science.gov (United States)

    Lin, Zhi-Jian; Zheng, Ren-Chao; Lei, Li-Hua; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-06-01

    Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

    Directory of Open Access Journals (Sweden)

    Elizabeth R Sharlow

    2010-04-01

    Full Text Available 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 gamma-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.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 approximately 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

  15. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  16. High throughput screening and profiling of high-value carotenoids from a wide diversity of bacteria in surface seawater.

    Science.gov (United States)

    Asker, Dalal

    2018-09-30

    Carotenoids are valuable natural colorants that exhibit numerous health promoting properties, and thus are widely used in food, feeds, pharmaceutical and nutraceuticals industries. In this study, we isolated and identified novel microbial sources that produced high-value carotenoids using high throughput screening (HTS). A total of 701 pigmented microbial strains library including marine bacteria and red yeast was constructed. Carotenoids profiling using HPLC-DAD-MS methods showed 88 marine bacterial strains with potential for the production of high-value carotenoids including astaxanthin (28 strains), zeaxanthin (21 strains), lutein (1 strains) and canthaxanthin (2 strains). A comprehensive 16S rRNA gene based phylogenetic analysis revealed that these strains can be classified into 30 species belonging to five bacterial classes (Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli). Importantly, we discovered novel producers of zeaxanthin and lutein, and a high diversity in both carotenoids and producing microbial strains, which are promising and highly selective biotechnological sources for high-value carotenoids. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Fun with High Throughput Toxicokinetics (CalEPA webinar)

    Science.gov (United States)

    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 ...

  18. High throughput experimentation for the discovery of new catalysts

    International Nuclear Information System (INIS)

    Thomson, S.; Hoffmann, C.; Johann, T.; Wolf, A.; Schmidt, H.-W.; Farrusseng, D.; Schueth, F.

    2002-01-01

    Full text: The use of combinatorial chemistry to obtain new materials has been developed extensively by the pharmaceutical and biochemical industries, but such approaches have been slow to impact on the field of heterogeneous catalysis. The reasons for this lie in with difficulties associated in the synthesis, characterisation and determination of catalytic properties of such materials. In many synthetic and catalytic reactions, the conditions used are difficult to emulate using High Throughput Experimentation (HTE). Furthermore, the ability to screen these catalysts simultaneously in real time, requires the development and/or modification of characterisation methods. Clearly, there is a need for both high throughput synthesis and screening of new and novel reactions, and we describe several new concepts that help to achieve these goals. Although such problems have impeded the development of combinatorial catalysis, the fact remains that many highly attractive processes still exist for which no suitable catalysts have been developed. The ability to decrease the tiFme needed to evaluate catalyst is therefore essential and this makes the use of high throughput techniques highly desirable. In this presentation we will describe the synthesis, catalytic testing, and novel screening methods developed at the Max Planck Institute. Automated synthesis procedures, performed by the use of a modified Gilson pipette robot, will be described, as will the development of two 16 and 49 sample fixed bed reactors and two 25 and 29 sample three phase reactors for catalytic testing. We will also present new techniques for the characterisation of catalysts and catalytic products using standard IR microscopy and infrared focal plane array detection, respectively

  19. High-Throughput Screening by Nuclear Magnetic Resonance (HTS by NMR) for the Identification of PPIs Antagonists.

    Science.gov (United States)

    Wu, Bainan; Barile, Elisa; De, Surya K; Wei, Jun; Purves, Angela; Pellecchia, Maurizio

    2015-01-01

    In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.

  20. A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer.

    Science.gov (United States)

    Cribbes, Scott; Kessel, Sarah; McMenemy, Scott; Qiu, Jean; Chan, Leo Li-Ying

    2017-06-01

    Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

  1. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  2. High-throughput theoretical design of lithium battery materials

    International Nuclear Information System (INIS)

    Ling Shi-Gang; Gao Jian; Xiao Rui-Juan; Chen Li-Quan

    2016-01-01

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

  3. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

    Science.gov (United States)

    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 (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

  4. Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

    Science.gov (United States)

    Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

    2015-01-01

    Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

  5. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang Leyu [Research Technology Center, Pfizer Global Research and Development, Cambridge, MA (United States); Moore, Jennifer; Kuo, Ming-Shang T. [Pfizer Global Research and Development, San Diego, CA (United States); LaMarr, William A.; Ozbal, Can C. [Biotrove, Inc., Woburn, MA (United States); Bhat, B. Ganesh [Pfizer Global Research and Development, San Diego, CA (United States)], E-mail: gbhat@gnf.org

    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{sub m} = 10.5 {mu}M). The assay was highly reproducible with an average Z' value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC{sub 50} values of 0.88 and 0.12 {mu}M, 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 {mu}M - 14% conformation rate). Of the confirmed hits 172 had IC{sub 50} values of <10 {mu}M, including 111 <1 {mu}M 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

  6. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    International Nuclear Information System (INIS)

    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

    2008-01-01

    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 μM). 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 μM, 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 μM - 14% conformation rate). Of the confirmed hits 172 had IC 50 values of <10 μM, including 111 <1 μM 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

  7. Blood group genotyping: from patient to high-throughput donor screening.

    Science.gov (United States)

    Veldhuisen, B; van der Schoot, C E; de Haas, M

    2009-10-01

    Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

  8. A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening.

    Science.gov (United States)

    An, Steven S; Askovich, Peter S; Zarembinski, Thomas I; Ahn, Kwangmi; Peltier, John M; von Rechenberg, Moritz; Sahasrabudhe, Sudhir; Fredberg, Jeffrey J

    2011-01-13

    A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

  9. A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

    Directory of Open Access Journals (Sweden)

    von Rechenberg Moritz

    2011-01-01

    Full Text Available Abstract Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20 and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM. Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Results Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. Conclusions This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

  10. A high throughput mass spectrometry screening analysis based on two-dimensional carbon microfiber fractionation system.

    Science.gov (United States)

    Ma, Biao; Zou, Yilin; Xie, Xuan; Zhao, Jinhua; Piao, Xiangfan; Piao, Jingyi; Yao, Zhongping; Quinto, Maurizio; Wang, Gang; Li, Donghao

    2017-06-09

    A novel high-throughput, solvent saving and versatile integrated two-dimensional microscale carbon fiber/active carbon fiber system (2DμCFs) that allows a simply and rapid separation of compounds in low-polar, medium-polar and high-polar fractions, has been coupled with ambient ionization-mass spectrometry (ESI-Q-TOF-MS and ESI-QqQ-MS) for screening and quantitative analyses of real samples. 2DμCFs led to a substantial interference reduction and minimization of ionization suppression effects, thus increasing the sensitivity and the screening capabilities of the subsequent MS analysis. The method has been applied to the analysis of Schisandra Chinensis extracts, obtaining with a single injection a simultaneous determination of 33 compounds presenting different polarities, such as organic acids, lignans, and flavonoids in less than 7min, at low pressures and using small solvent amounts. The method was also validated using 10 model compounds, giving limit of detections (LODs) ranging from 0.3 to 30ngmL -1 , satisfactory recoveries (from 75.8 to 93.2%) and reproducibilities (relative standard deviations, RSDs, from 1.40 to 8.06%). Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

    Science.gov (United States)

    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.

  12. Database for High Throughput Screening Hits (dHITS): a simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.

    Science.gov (United States)

    Chuartzman, Silvia G; Schuldiner, Maya

    2018-03-25

    In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice ('hits') are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen - all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community. © 2018 The Authors Yeast Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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.

  14. Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

    2018-05-03

    L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

  15. On-chip polarimetry for high-throughput screening of nanoliter and smaller sample volumes

    Science.gov (United States)

    Bachmann, Brian O. (Inventor); Bornhop, Darryl J. (Inventor); Dotson, Stephen (Inventor)

    2012-01-01

    A polarimetry technique for measuring optical activity that is particularly suited for high throughput screening employs a chip or substrate (22) having one or more microfluidic channels (26) formed therein. A polarized laser beam (14) is directed onto optically active samples that are disposed in the channels. The incident laser beam interacts with the optically active molecules in the sample, which slightly alter the polarization of the laser beam as it passes multiple times through the sample. Interference fringe patterns (28) are generated by the interaction of the laser beam with the sample and the channel walls. A photodetector (34) is positioned to receive the interference fringe patterns and generate an output signal that is input to a computer or other analyzer (38) for analyzing the signal and determining the rotation of plane polarized light by optically active material in the channel from polarization rotation calculations.

  16. Surface Plasmon Resonance: New Biointerface Designs and High-Throughput Affinity Screening

    Science.gov (United States)

    Linman, Matthew J.; Cheng, Quan Jason

    Surface plasmon resonance (SPR) is a surface optical technique that measures minute changes in refractive index at a metal-coated surface. It has become increasingly popular in the study of biological and chemical analytes because of its label-free measurement feature. In addition, SPR allows for both quantitative and qualitative assessment of binding interactions in real time, making it ideally suited for probing weak interactions that are often difficult to study with other methods. This chapter presents the biosensor development in the last 3 years or so utilizing SPR as the principal analytical technique, along with a concise background of the technique itself. While SPR has demonstrated many advantages, it is a nonselective method and so, building reproducible and functional interfaces is vital to sensing applications. This chapter, therefore, focuses mainly on unique surface chemistries and assay approaches to examine biological interactions with SPR. In addition, SPR imaging for high-throughput screening based on microarrays and novel hyphenated techniques involving the coupling of SPR to other analytical methods is discussed. The chapter concludes with a commentary on the current state of SPR biosensing technology and the general direction of future biosensor research.

  17. Using information from historical high-throughput screens to predict active compounds.

    Science.gov (United States)

    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

  18. A high-throughput multiplex method adapted for GMO detection.

    Science.gov (United States)

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  19. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  20. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

    Science.gov (United States)

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

  1. High-throughput and automatic typing via human papillomavirus identification map for cervical cancer screening and prognosis.

    Science.gov (United States)

    Yi, Linglu; Xu, Xueqin; Lin, Xuexia; Li, Haifang; Ma, Yuan; Lin, Jin-Ming

    2014-07-07

    A novel human papillomavirus (HPV) typing assay for cervical cancer screening and prognosis was developed by the combination of restriction fragment length polymorphism (RFLP) and microchip electrophoresis (MCE) to achieve higher levels of sensitivity and throughput. The detection limit of 2 × 10(2) copies, high sensitivity and typing accuracy on the account of PCR-RFLP-MCE method guarantee the successful diagnosis results of 4-fold higher infection rate over cytologic tests. From clinical samples, eleven kinds of HPV types were identified with a good compatibility degree of over 90%. The described method showed good reliability in clinical samples and provided a promising alternative for pathological studies at the molecular level.

  2. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  3. Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    2008-01-01

    Full Text Available Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR and peroxiredoxin (Prx and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50s ranging from micromolar to the assay response limit ( approximately 25 nM. This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump

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

    International Nuclear Information System (INIS)

    Druwe, Ingrid; Freudenrich, Theresa M.; Wallace, Kathleen; Shafer, Timothy J.; Mundy, William R.

    2015-01-01

    High-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 be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery

  5. Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.

    Directory of Open Access Journals (Sweden)

    Bo Zhu

    Full Text Available The C1a isoenzyme of horseradish peroxidase (HRP is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT and inactive mutant (MUT genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

  6. A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

    OpenAIRE

    von Rechenberg Moritz; Peltier John M; Ahn Kwangmi; Zarembinski Thomas I; Askovich Peter S; An Steven S; Sahasrabudhe Sudhir; Fredberg Jeffrey J

    2011-01-01

    Abstract Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two qua...

  7. Chiral Amine Synthesis Using ω-Transaminases: An Amine Donor that Displaces Equilibria and Enables High-Throughput Screening**

    Science.gov (United States)

    Green, Anthony P; Turner, Nicholas J; O'Reilly, Elaine

    2014-01-01

    The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity. PMID:25138082

  8. Structural, dielectric and ferroelectric properties of (Bi,Na)TiO3–BaTiO3 system studied by high throughput screening

    International Nuclear Information System (INIS)

    Hayden, Brian E.; Yakovlev, Sergey

    2016-01-01

    Thin-film materials libraries of the Bi 2 O 3 –Na 2 O–TiO 2 –BaO system in a broad composition range have been deposited in ultra-high vacuum from elemental evaporation sources and an oxygen plasma source. A high throughput approach was used for systematic compositional and structural characterization and the screening of the dielectric and ferroelectric properties. The perovskite (Bi,Na)TiO 3 –BaTiO 3 phase with a Ba concentration near the morphotropic phase boundary (ca. 6 at.%) exhibited a relative dielectric permittivity of 180, a loss tangent of 0.04 and remnant polarization of 19 μC/cm 2 . Compared to published data, observed remnant polarization is close to that known for epitaxially grown films but higher than the values reported for polycrystalline films. The high throughput methodology and systematic nature of the study allowed us to establish the composition boundaries of the phase with optimal dielectric and ferroelectric characteristics. - Highlights: • Bi 2 O 3 –Na 2 O–TiO 2 –BaO high throughput materials library was deposited using PVD method. • Materials were processed from individual molecular beam epitaxy sources of elements. • High throughput approach was used for structural, dielectric and ferroelectric study. • Composition boundaries of perovskite compounds with optimum properties are reported.

  9. High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth.

    Science.gov (United States)

    Bowden, Gregory D; Land, Kirkwood M; O'Connor, Roberta M; Fritz, Heather M

    2018-04-01

    The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM), a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60-70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83%) have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18) of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ± 12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 μM [0.036-0.12; 95% CI] or 21.9 ng/ml [12.1-40.3; 95% CI]). These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection. Copyright © 2018. Published by Elsevier Ltd.

  10. Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

    Science.gov (United States)

    Yong, K J; Scott, D J

    2015-03-01

    Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

  11. Predictive models for anti-tubercular molecules using machine learning on high-throughput biological screening datasets.

    Science.gov (United States)

    Periwal, Vinita; Rajappan, Jinuraj K; Jaleel, Abdul Uc; Scaria, Vinod

    2011-11-18

    Tuberculosis is a contagious disease caused by Mycobacterium tuberculosis (Mtb), affecting more than two billion people around the globe and is one of the major causes of morbidity and mortality in the developing world. Recent reports suggest that Mtb has been developing resistance to the widely used anti-tubercular drugs resulting in the emergence and spread of multi drug-resistant (MDR) and extensively drug-resistant (XDR) strains throughout the world. In view of this global epidemic, there is an urgent need to facilitate fast and efficient lead identification methodologies. Target based screening of large compound libraries has been widely used as a fast and efficient approach for lead identification, but is restricted by the knowledge about the target structure. Whole organism screens on the other hand are target-agnostic and have been now widely employed as an alternative for lead identification but they are limited by the time and cost involved in running the screens for large compound libraries. This could be possibly be circumvented by using computational approaches to prioritize molecules for screening programmes. We utilized physicochemical properties of compounds to train four supervised classifiers (Naïve Bayes, Random Forest, J48 and SMO) on three publicly available bioassay screens of Mtb inhibitors and validated the robustness of the predictive models using various statistical measures. This study is a comprehensive analysis of high-throughput bioassay data for anti-tubercular activity and the application of machine learning approaches to create target-agnostic predictive models for anti-tubercular agents.

  12. Predictive models for anti-tubercular molecules using machine learning on high-throughput biological screening datasets

    Directory of Open Access Journals (Sweden)

    Periwal Vinita

    2011-11-01

    Full Text Available Abstract Background Tuberculosis is a contagious disease caused by Mycobacterium tuberculosis (Mtb, affecting more than two billion people around the globe and is one of the major causes of morbidity and mortality in the developing world. Recent reports suggest that Mtb has been developing resistance to the widely used anti-tubercular drugs resulting in the emergence and spread of multi drug-resistant (MDR and extensively drug-resistant (XDR strains throughout the world. In view of this global epidemic, there is an urgent need to facilitate fast and efficient lead identification methodologies. Target based screening of large compound libraries has been widely used as a fast and efficient approach for lead identification, but is restricted by the knowledge about the target structure. Whole organism screens on the other hand are target-agnostic and have been now widely employed as an alternative for lead identification but they are limited by the time and cost involved in running the screens for large compound libraries. This could be possibly be circumvented by using computational approaches to prioritize molecules for screening programmes. Results We utilized physicochemical properties of compounds to train four supervised classifiers (Naïve Bayes, Random Forest, J48 and SMO on three publicly available bioassay screens of Mtb inhibitors and validated the robustness of the predictive models using various statistical measures. Conclusions This study is a comprehensive analysis of high-throughput bioassay data for anti-tubercular activity and the application of machine learning approaches to create target-agnostic predictive models for anti-tubercular agents.

  13. High-throughput screening of tick-borne pathogens in Europe

    DEFF Research Database (Denmark)

    Michelet, Lorraine; Delannoy, Sabine; Devillers, Elodie

    2014-01-01

    was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia......, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia...

  14. High-Throughput Screening of Heterogeneous Catalysts for the Conversion of Furfural to Bio-Based Fuel Components

    Directory of Open Access Journals (Sweden)

    Roberto Pizzi

    2015-12-01

    Full Text Available The one-pot catalytic reductive etherification of furfural to 2-methoxymethylfuran (furfuryl methyl ether, FME, a valuable bio-based chemical or fuel, is reported. A large number of commercially available hydrogenation heterogeneous catalysts based on nickel, copper, cobalt, iridium, palladium and platinum catalysts on various support were evaluated by a high-throughput screening approach. The reaction was carried out in liquid phase with a 10% w/w furfural in methanol solution at 50 bar of hydrogen. Among all the samples tested, carbon-supported noble metal catalysts were found to be the most promising in terms of productivity and selectivity. In particular, palladium on charcoal catalysts show high selectivity (up to 77% to FME. Significant amounts of furfuryl alcohol (FA and 2-methylfuran (2-MF are observed as the major by-products.

  15. High-throughput technology for novel SO2 oxidation catalysts

    International Nuclear Information System (INIS)

    Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F

    2011-01-01

    We review the state of the art and explain the need for better SO 2 oxidation catalysts for the production of sulfuric acid. A high-throughput technology has been developed for the study of potential catalysts in the oxidation of SO 2 to SO 3 . High-throughput methods are reviewed and the problems encountered with their adaptation to the corrosive conditions of SO 2 oxidation are described. We show that while emissivity-corrected infrared thermography (ecIRT) can be used for primary screening, it is prone to errors because of the large variations in the emissivity of the catalyst surface. UV-visible (UV-Vis) spectrometry was selected instead as a reliable analysis method of monitoring the SO 2 conversion. Installing plain sugar absorbents at reactor outlets proved valuable for the detection and quantitative removal of SO 3 from the product gas before the UV-Vis analysis. We also overview some elements used for prescreening and those remaining after the screening of the first catalyst generations. (topical review)

  16. High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Mazzio, E; Deiab, S; Park, K; Soliman, KFA

    2012-01-01

    Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

  17. High-throughput preparation and testing of ion-exchanged zeolites

    International Nuclear Information System (INIS)

    Janssen, K.P.F.; Paul, J.S.; Sels, B.F.; Jacobs, P.A.

    2007-01-01

    A high-throughput research platform was developed for the preparation and subsequent catalytic liquid-phase screening of ion-exchanged zeolites, for instance with regard to their use as heterogeneous catalysts. In this system aqueous solutions and other liquid as well as solid reagents are employed as starting materials and 24 samples are prepared on a library plate with a 4 x 6 layout. Volumetric dispensing of metal precursor solutions, weighing of zeolite and subsequent mixing/washing cycles of the starting materials and distributing reaction mixtures to the library plate are automatically performed by liquid and solid handlers controlled by a single common and easy-to-use programming software interface. The thus prepared materials are automatically contacted with reagent solutions, heated, stirred and sampled continuously using a modified liquid handling. The high-throughput platform is highly promising in enhancing synthesis of catalysts and their screening. In this paper the preparation of lanthanum-exchanged NaY zeolites (LaNaY) on the platform is reported, along with their use as catalyst for the conversion of renewables

  18. An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization.

    Directory of Open Access Journals (Sweden)

    Fuqiang Ma

    Full Text Available High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >10⁸ clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k(cat/K(M values approaching the diffusion-limited efficiency of ∼10⁸ M⁻¹s⁻¹.

  19. A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Qin, Chong-Zhen; Ren, Xian; Tan, Zhi-Rong; Chen, Yao; Yin, Ji-Ye; Yu, Jing; Qu, Jian; Zhou, Hong-Hao; Liu, Zhao-Qian

    2014-02-01

    A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.

  20. En Garde: Fencing at Kansas City's Central Computers Unlimited/Classical Greek Magnet High School, 1991-1995

    Science.gov (United States)

    Poos, Bradley W.

    2015-01-01

    Central High School in Kansas City, Missouri is one of the oldest schools west of the Mississippi and the first public high school built in Kansas City. Kansas City's magnet plan resulted in Central High School being rebuilt as the Central Computers Unlimited/Classical Greek Magnet High School, a school that was designed to offer students an…

  1. High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

    Directory of Open Access Journals (Sweden)

    Thomas J.J. Gintjee

    2014-11-01

    Full Text Available Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD, the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD.

  2. High throughput screening in duchenne muscular dystrophy: from drug discovery to functional genomics.

    Science.gov (United States)

    Gintjee, Thomas J J; Magh, Alvin S H; Bertoni, Carmen

    2014-11-14

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD.

  3. Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

    Directory of Open Access Journals (Sweden)

    Oliver N F King

    2010-11-01

    Full Text Available Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors.High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4 family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II and to modulate demethylation at the H3K9 locus in a cell-based assay.These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

  4. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

    Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

  5. Adherence to cancer screening guidelines and predictors of improvement among participants in the Kansas State Employee Wellness Program.

    Science.gov (United States)

    Hui, Siu-kuen Azor; Engelman, Kimberly K; Shireman, Theresa I; Ellerbeck, Edward F

    2013-07-11

    Employee wellness programs (EWPs) have been used to implement worksite-based cancer prevention and control interventions. However, little is known about whether these programs result in improved adherence to cancer screening guidelines or how participants' characteristics affect subsequent screening. This study was conducted to describe cancer screening behaviors among participants in a state EWP and identify factors associated with screening adherence among those who were initially nonadherent. We identified employees and their dependents who completed health risk assessments (HRAs) as part of the Kansas state EWP in both 2008 and 2009. We examined baseline rates of adherence to cancer screening guidelines in 2008 and factors associated with adherence in 2009 among participants who were initially nonadherent. Of 53,095 eligible participants, 13,222 (25%) participated in the EWP in 2008 and 6,205 (12%) participated in both years. Among the multiyear participants, adherence was high at baseline to screening for breast (92.5%), cervical (91.8%), and colorectal cancer (72.7%). Of participants who were initially nonadherent in 2008, 52.4%, 41.3%, and 33.5%, respectively, became adherent in the following year to breast, cervical, and colorectal cancer screening. Suburban/urban residence and more frequent doctor visits predicted adherence to breast and colorectal cancer screening guidelines. The effectiveness of EWPs for increasing cancer screening is limited by low HRA participation rates, high rates of adherence to screening at baseline, and failure of nonadherent participants to get screening. Improving overall adherence to cancer screening guidelines among employees will require efforts to increase HRA participation, stronger interventions for nonadherent participants, and better access to screening for rural employees.

  6. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    International Nuclear Information System (INIS)

    Shin, Hyeong-Moo; Ernstoff, Alexi; Csiszar, Susan A.

    2015-01-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models

  7. High-throughput gene expression profiling of memory differentiation in primary human T cells

    Directory of Open Access Journals (Sweden)

    Russell Kate

    2008-08-01

    Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

  8. A CRISPR CASe for High-Throughput Silencing

    Directory of Open Access Journals (Sweden)

    Jacob eHeintze

    2013-10-01

    Full Text Available Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

  9. Toward a Molecular Understanding of the Interaction of Dual Specificity Phosphatases with Substrates: Insights from Structure-Based Modeling and High Throughput Screening

    OpenAIRE

    Bakan, Ahmet; Lazo, John S; Wipf, Peter; Brummond, Kay M; Bahar, Ivet

    2008-01-01

    Dual-specificity phosphatases (DSPs) are important, but poorly understood, cell signaling enzymes that remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Deregulation of DSPs has been implicated in cancer, obesity, diabetes, inflammation, and Alzheimer’s disease. Due to their biological and biomedical significance, DSPs have increasingly become the subject of drug discovery high-throughput screening (HTS) and focused compound library development efforts. P...

  10. Analytical Validation of a Portable Mass Spectrometer Featuring Interchangeable, Ambient Ionization Sources for High Throughput Forensic Evidence Screening.

    Science.gov (United States)

    Lawton, Zachary E; Traub, Angelica; Fatigante, William L; Mancias, Jose; O'Leary, Adam E; Hall, Seth E; Wieland, Jamie R; Oberacher, Herbert; Gizzi, Michael C; Mulligan, Christopher C

    2017-06-01

    Forensic evidentiary backlogs are indicative of the growing need for cost-effective, high-throughput instrumental methods. One such emerging technology that shows high promise in meeting this demand while also allowing on-site forensic investigation is portable mass spectrometric (MS) instrumentation, particularly that which enables the coupling to ambient ionization techniques. While the benefits of rapid, on-site screening of contraband can be anticipated, the inherent legal implications of field-collected data necessitates that the analytical performance of technology employed be commensurate with accepted techniques. To this end, comprehensive analytical validation studies are required before broad incorporation by forensic practitioners can be considered, and are the focus of this work. Pertinent performance characteristics such as throughput, selectivity, accuracy/precision, method robustness, and ruggedness have been investigated. Reliability in the form of false positive/negative response rates is also assessed, examining the effect of variables such as user training and experience level. To provide flexibility toward broad chemical evidence analysis, a suite of rapidly-interchangeable ion sources has been developed and characterized through the analysis of common illicit chemicals and emerging threats like substituted phenethylamines. Graphical Abstract ᅟ.

  11. Analytical Validation of a Portable Mass Spectrometer Featuring Interchangeable, Ambient Ionization Sources for High Throughput Forensic Evidence Screening

    Science.gov (United States)

    Lawton, Zachary E.; Traub, Angelica; Fatigante, William L.; Mancias, Jose; O'Leary, Adam E.; Hall, Seth E.; Wieland, Jamie R.; Oberacher, Herbert; Gizzi, Michael C.; Mulligan, Christopher C.

    2017-06-01

    Forensic evidentiary backlogs are indicative of the growing need for cost-effective, high-throughput instrumental methods. One such emerging technology that shows high promise in meeting this demand while also allowing on-site forensic investigation is portable mass spectrometric (MS) instrumentation, particularly that which enables the coupling to ambient ionization techniques. While the benefits of rapid, on-site screening of contraband can be anticipated, the inherent legal implications of field-collected data necessitates that the analytical performance of technology employed be commensurate with accepted techniques. To this end, comprehensive analytical validation studies are required before broad incorporation by forensic practitioners can be considered, and are the focus of this work. Pertinent performance characteristics such as throughput, selectivity, accuracy/precision, method robustness, and ruggedness have been investigated. Reliability in the form of false positive/negative response rates is also assessed, examining the effect of variables such as user training and experience level. To provide flexibility toward broad chemical evidence analysis, a suite of rapidly-interchangeable ion sources has been developed and characterized through the analysis of common illicit chemicals and emerging threats like substituted phenethylamines. [Figure not available: see fulltext.

  12. High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

    Directory of Open Access Journals (Sweden)

    Baoshan Guo

    Full Text Available The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary and nitrogen-deficient (lipid-accumulated E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

  13. A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT

    DEFF Research Database (Denmark)

    Simmons, Katie J; Gotfryd, Kamil; Billesbølle, Christian B

    2013-01-01

    Abstract Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the e...... this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family....

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

    Czech Academy of Sciences Publication Activity Database

    De Diego, N.; Fürst, T.; Humplík, Jan; Ugena, L.; Podlešáková, K.; Spíchal, L.

    2017-01-01

    Roč. 8, OCT 4 (2017), č. článku 1702. ISSN 1664-462X R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : salt stress * chlorophyll fluorescence * salinity tolerance * plant-responses * cold-tolerance * water-deficit * thaliana * selection * platform * reveals * high-throughput screening assay * Arabidopsis * multi-well plates * rosette growth * stress conditions Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 4.298, year: 2016

  15. Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

    Science.gov (United States)

    Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

    2016-10-01

    The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

  16. High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

    Directory of Open Access Journals (Sweden)

    Ehud Goldin

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase. Previously, wildtype GCase was used for high throughput screening (HTS of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

  17. Residential radon in Kansas City-black shales aren't the prime suspect

    International Nuclear Information System (INIS)

    Spencer, C.G.

    1993-01-01

    The US EPA preliminary assessment of potential radon risk (EPA, 1986) depicted a large area of the mid-continent in which radon levels might be elevated due to the presence of uranium-rich black shales. A preliminary study (Hilpman, Coveney ampersand Spencer, 1988) indicated that a significant percentage of homes in the greater Kansas City area had radon screening levels above 4 pCi/L. However, their lab tests with crushed black shale, and radon tests in limestone mines with black shale floors showed that the shale did not yield extremely high radon levels. This expanded study presents additional results of screening tests in homes, and correlates those results to bedrock geology and soil type. High radon levels in the Kansas city area are not due primarily to black shale sources. The highest readings are associated with limestone and non-organic shale. Mean radon level is higher in younger cyclothemic deposits, and a loessial soil. The EPA initial assessment overstated the radon risk attributable to black uraniferous shale sources. Assessment of the overall potential risk for the greater Kansas City area requires further evaluation of other sources

  18. Novel heparan sulfate assay by using automated high-throughput mass spectrometry: Application to monitoring and screening for mucopolysaccharidoses.

    Science.gov (United States)

    Shimada, Tsutomu; Kelly, Joan; LaMarr, William A; van Vlies, Naomi; Yasuda, Eriko; Mason, Robert W; Mackenzie, William; Kubaski, Francyne; Giugliani, Roberto; Chinen, Yasutsugu; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji E; Fukao, Toshiyuki; Orii, Tadao; Tomatsu, Shunji

    2014-01-01

    Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity

  19. High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH).

    Science.gov (United States)

    Hollox, E J; Atia, T; Cross, G; Parkin, T; Armour, J A L

    2002-11-01

    Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone.

  20. High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

    DEFF Research Database (Denmark)

    Adler, Andrew F; Speidel, Alessondra T; Christoforou, Nicolas

    2011-01-01

    of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM....... Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library...... and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems....

  1. Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

    Science.gov (United States)

    Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

    2017-12-01

    Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of

  2. Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control.

    Science.gov (United States)

    Musenga, Alessandro; Cowan, David A

    2013-05-03

    We describe a sensitive, comprehensive and fast screening method based on liquid chromatography-high resolution mass spectrometry for the detection of a large number of analytes in sports samples. UHPLC coupled to high resolution mass spectrometry with polarity switching capability is applied for the rapid screening of a large number of analytes in human urine samples. Full scan data are acquired alternating both positive and negative ionisation. Collision-induced dissociation with positive ionisation is also performed to produce fragment ions to improve selectivity for some analytes. Data are reviewed as extracted ion chromatograms based on narrow mass/charge windows (±5ppm). A simple sample preparation method was developed, using direct enzymatic hydrolysis of glucuronide conjugates, followed by solid phase extraction with mixed mode ion-exchange cartridges. Within a 10min run time (including re-equilibration) the method presented allows for the analysis of a large number of analytes from most of the classes in the World Anti-Doping Agency (WADA) Prohibited List, including anabolic agents, β2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids and β-blockers, and does so while meeting the WADA sensitivity requirements. The high throughput of the method and the fast sample pre-treatment reduces analysis cost and increases productivity. The method presented has been used for the analysis of over 5000 samples in about one month and proved to be reliable. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. A High Throughput, 384-Well, Semi-Automated, Hepatocyte Intrinsic Clearance Assay for Screening New Molecular Entities in Drug Discovery.

    Science.gov (United States)

    Heinle, Lance; Peterkin, Vincent; de Morais, Sonia M; Jenkins, Gary J; Badagnani, Ilaria

    2015-01-01

    A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.

  4. Predictive Power of Machine Learning for Optimizing Solar Water Heater Performance: The Potential Application of High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Hao Li

    2017-01-01

    Full Text Available Predicting the performance of solar water heater (SWH is challenging due to the complexity of the system. Fortunately, knowledge-based machine learning can provide a fast and precise prediction method for SWH performance. With the predictive power of machine learning models, we can further solve a more challenging question: how to cost-effectively design a high-performance SWH? Here, we summarize our recent studies and propose a general framework of SWH design using a machine learning-based high-throughput screening (HTS method. Design of water-in-glass evacuated tube solar water heater (WGET-SWH is selected as a case study to show the potential application of machine learning-based HTS to the design and optimization of solar energy systems.

  5. Comparison of Points of Departure for Health Risk Assessment Based on High-Throughput Screening Data

    Science.gov (United States)

    Sand, Salomon; Parham, Fred; Portier, Christopher J.; Tice, Raymond R.; Krewski, Daniel

    2016-01-01

    Background: The National Research Council’s vision for toxicity testing in the 21st century anticipates that points of departure (PODs) for establishing human exposure guidelines in future risk assessments will increasingly be based on in vitro high-throughput screening (HTS) data. Objectives: The aim of this study was to compare different PODs for HTS data. Specifically, benchmark doses (BMDs) were compared to the signal-to-noise crossover dose (SNCD), which has been suggested as the lowest dose applicable as a POD. Methods: Hill models were fit to > 10,000 in vitro concentration–response curves, obtained for > 1,400 chemicals tested as part of the U.S. Tox21 Phase I effort. BMDs and lower confidence limits on the BMDs (BMDLs) corresponding to extra effects (i.e., changes in response relative to the maximum response) of 5%, 10%, 20%, 30%, and 40% were estimated for > 8,000 curves, along with BMDs and BMDLs corresponding to additional effects (i.e., absolute changes in response) of 5%, 10%, 15%, 20%, and 25%. The SNCD, defined as the dose where the ratio between the additional effect and the difference between the upper and lower bounds of the two-sided 90% confidence interval on absolute effect was 1, 0.67, and 0.5, respectively, was also calculated and compared with the BMDLs. Results: The BMDL40, BMDL25, and BMDL18, defined in terms of extra effect, corresponded to the SNCD1.0, SNCD0.67, and SNCD0.5, respectively, at the median. Similarly, the BMDL25, BMDL17, and BMDL13, defined in terms of additional effect, corresponded to the SNCD1.0, SNCD0.67, and SNCD0.5, respectively, at the median. Conclusions: The SNCD may serve as a reference level that guides the determination of standardized BMDs for risk assessment based on HTS concentration–response data. The SNCD may also have application as a POD for low-dose extrapolation. Citation: Sand S, Parham F, Portier CJ, Tice RR, Krewski D. 2017. Comparison of points of departure for health risk assessment based on

  6. Generating information-rich high-throughput experimental materials genomes using functional clustering via multitree genetic programming and information theory.

    Science.gov (United States)

    Suram, Santosh K; Haber, Joel A; Jin, Jian; Gregoire, John M

    2015-04-13

    High-throughput experimental methodologies are capable of synthesizing, screening and characterizing vast arrays of combinatorial material libraries at a very rapid rate. These methodologies strategically employ tiered screening wherein the number of compositions screened decreases as the complexity, and very often the scientific information obtained from a screening experiment, increases. The algorithm used for down-selection of samples from higher throughput screening experiment to a lower throughput screening experiment is vital in achieving information-rich experimental materials genomes. The fundamental science of material discovery lies in the establishment of composition-structure-property relationships, motivating the development of advanced down-selection algorithms which consider the information value of the selected compositions, as opposed to simply selecting the best performing compositions from a high throughput experiment. Identification of property fields (composition regions with distinct composition-property relationships) in high throughput data enables down-selection algorithms to employ advanced selection strategies, such as the selection of representative compositions from each field or selection of compositions that span the composition space of the highest performing field. Such strategies would greatly enhance the generation of data-driven discoveries. We introduce an informatics-based clustering of composition-property functional relationships using a combination of information theory and multitree genetic programming concepts for identification of property fields in a composition library. We demonstrate our approach using a complex synthetic composition-property map for a 5 at. % step ternary library consisting of four distinct property fields and finally explore the application of this methodology for capturing relationships between composition and catalytic activity for the oxygen evolution reaction for 5429 catalyst compositions in a

  7. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

    Science.gov (United States)

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-09-02

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

  8. High-throughput selection for cellulase catalysts using chemical complementation.

    Science.gov (United States)

    Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

    2008-12-24

    Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

  9. Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and a trial of oral S-adenosyl-l-methionine as a treatment method.

    Science.gov (United States)

    van Werkhoven, Michiel A; Duley, John A; McGown, Ivan; Munce, Teresa; Freeman, Jeremy L; Pitt, James J

    2013-11-01

    The aim of this study was to develop a high-throughput urine screening technique for adenylosuccinate lyase (ADSL) deficiency and to evaluate S-adenosyl-l-methionine (SAMe) as a potential treatment for this disorder. Testing for succinyladenosine (S-Ado), a marker of ADSL deficiency, was incorporated into a screening panel for urine biomarkers for inborn errors of metabolism using electrospray tandem mass spectrometry. Liquid chromatography-mass spectrometry and high-performance liquid chromatography were used to confirm and monitor the response of metabolites to oral SAMe treatment. Increased levels of S-Ado were detected in a 3-month-old male infant with hypotonia and seizures. ADSL gene sequencing revealed a previously described c.-49T>C mutation and a novel c.889_891dupAAT mutation, which was likely to disrupt enzyme function. After 9 months of SAMe treatment, there was no clear response evidenced in urine metabolite levels or clinical parameters. These results demonstrate proof of the principle for the high-throughput urine screening technique, allowing earlier diagnosis of patients with ADSL deficiency. However, early treatment with SAMe does not appear to be effective in ADSL deficiency. It is suggested that although SAMe treatment may ameliorate purine nucleotide deficiency, it cannot correct metabolic syndromes in which a toxic nucleotide is present, in this case presumed to be succinylaminoimidazole carboxamide ribotide. © 2013 Mac Keith Press.

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

    Science.gov (United States)

    Wang, Jun; Hallinger, Daniel R; Murr, Ashley S; Buckalew, Angela R; Simmons, Steven O; Laws, Susan C; Stoker, Tammy E

    2018-05-01

    Thyroid uptake of iodide via the sodium-iodide symporter (NIS) is the first step in the biosynthesis of thyroid hormones that are critical for health and development in humans and wildlife. Despite having long been a known target of endocrine disrupting chemicals such as perchlorate, information regarding NIS inhibition activity is still unavailable for the vast majority of environmental chemicals. This study applied a previously validated high-throughput approach to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important environmental chemicals. Here 310 blinded samples were screened in a tiered-approach using an initial single-concentration (100 μM) radioactive-iodide uptake (RAIU) assay, followed by 169 samples further evaluated in multi-concentration (0.001 μM-100 μM) testing in parallel RAIU and cell viability assays. A novel chemical ranking system that incorporates multi-concentration RAIU and cytotoxicity responses was also developed as a standardized method for chemical prioritization in current and future screenings. Representative chemical responses and thyroid effects of high-ranking chemicals are further discussed. This study significantly expands current knowledge of NIS inhibition potential in environmental chemicals and provides critical support to U.S. EPA's Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).

  11. High-Throughput Behavioral Screens: the First Step towards Finding Genes Involved in Vertebrate Brain Function Using Zebrafish

    Directory of Open Access Journals (Sweden)

    Robert Gerlai

    2010-04-01

    Full Text Available The zebrafish has been in the forefront of developmental biology for three decades and has become a favorite of geneticists. Due to the accumulated genetic knowledge and tools developed for the zebrafish it is gaining popularity in other disciplines, including neuroscience. The zebrafish offers a compromise between system complexity (it is a vertebrate similar in many ways to our own species and practical simplicity (it is small, easy to keep, and prolific. Such features make zebrafish an excellent choice for high throughput mutation and drug screening. For the identification of mutation or drug induced alteration of brain function arguably the best methods are behavioral test paradigms. This review does not present experimental examples for the identification of particular genes or drugs. Instead it describes how behavioral screening methods may enable one to find functional alterations in the vertebrate brain. Furthermore, the review is not comprehensive. The behavioral test examples presented are biased according to the personal interests of the author. They will cover research areas including learning and memory, fear and anxiety, and social behavior. Nevertheless, the general principles will apply to other functional domains and should represent a snapshot of the rapidly evolving behavioral screening field with zebrafish.

  12. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

    Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

  13. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter

    2003-01-01

    . A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct....... The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery....

  14. Structural, dielectric and ferroelectric properties of (Bi,Na)TiO{sub 3}–BaTiO{sub 3} system studied by high throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Hayden, Brian E. [Ilika Technologies Plc., Kenneth Dibben House, Enterprise Road, University of Southampton Science Park, Chilworth, Southampton SO16 7NS (United Kingdom); Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ (United Kingdom); Yakovlev, Sergey, E-mail: sergey.yakovlev@ilika.com [Ilika Technologies Plc., Kenneth Dibben House, Enterprise Road, University of Southampton Science Park, Chilworth, Southampton SO16 7NS (United Kingdom)

    2016-03-31

    Thin-film materials libraries of the Bi{sub 2}O{sub 3}–Na{sub 2}O–TiO{sub 2}–BaO system in a broad composition range have been deposited in ultra-high vacuum from elemental evaporation sources and an oxygen plasma source. A high throughput approach was used for systematic compositional and structural characterization and the screening of the dielectric and ferroelectric properties. The perovskite (Bi,Na)TiO{sub 3}–BaTiO{sub 3} phase with a Ba concentration near the morphotropic phase boundary (ca. 6 at.%) exhibited a relative dielectric permittivity of 180, a loss tangent of 0.04 and remnant polarization of 19 μC/cm{sup 2}. Compared to published data, observed remnant polarization is close to that known for epitaxially grown films but higher than the values reported for polycrystalline films. The high throughput methodology and systematic nature of the study allowed us to establish the composition boundaries of the phase with optimal dielectric and ferroelectric characteristics. - Highlights: • Bi{sub 2}O{sub 3}–Na{sub 2}O–TiO{sub 2}–BaO high throughput materials library was deposited using PVD method. • Materials were processed from individual molecular beam epitaxy sources of elements. • High throughput approach was used for structural, dielectric and ferroelectric study. • Composition boundaries of perovskite compounds with optimum properties are reported.

  15. Genetic high throughput screening in Retinitis Pigmentosa based on high resolution melting (HRM) analysis.

    Science.gov (United States)

    Anasagasti, Ander; Barandika, Olatz; Irigoyen, Cristina; Benitez, Bruno A; Cooper, Breanna; Cruchaga, Carlos; López de Munain, Adolfo; Ruiz-Ederra, Javier

    2013-11-01

    Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n = 96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4% of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for

  16. High Throughput In Situ XAFS Screening of Catalysts

    International Nuclear Information System (INIS)

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Schroeder, Sven L. M.; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu

    2007-01-01

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on γ-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2

  17. Meeting Report: High-Throughput Technologies for In Vivo Imaging Agents

    Directory of Open Access Journals (Sweden)

    Robert J. Gillies

    2005-04-01

    Full Text Available Combinatorial chemistry and high-throughput screening have become standard tools for discovering new drug candidates with suitable pharmacological properties. Now, those same technologies are starting to be applied to the problem of discovering novel in vivo imaging agents. Important differences in the biological and pharmacological properties needed for imaging agents, compared to those for a therapeutic agent, require new screening methods that emphasize those characteristics, such as optimized residence time and tissue specificity, that make for a good imaging agent candidate.

  18. A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53.

    Science.gov (United States)

    Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena; Spiotto, Michael T

    2016-01-01

    p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways.

  19. Fully-Automated High-Throughput NMR System for Screening of Haploid Kernels of Maize (Corn by Measurement of Oil Content.

    Directory of Open Access Journals (Sweden)

    Hongzhi Wang

    Full Text Available One of the modern crop breeding techniques uses doubled haploid plants that contain an identical pair of chromosomes in order to accelerate the breeding process. Rapid haploid identification method is critical for large-scale selections of double haploids. The conventional methods based on the color of the endosperm and embryo seeds are slow, manual and prone to error. On the other hand, there exists a significant difference between diploid and haploid seeds generated by high oil inducer, which makes it possible to use oil content to identify the haploid. This paper describes a fully-automated high-throughput NMR screening system for maize haploid kernel identification. The system is comprised of a sampler unit to select a single kernel to feed for measurement of NMR and weight, and a kernel sorter to distribute the kernel according to the measurement result. Tests of the system show a consistent accuracy of 94% with an average screening time of 4 seconds per kernel. Field test result is described and the directions for future improvement are discussed.

  20. Fully-Automated High-Throughput NMR System for Screening of Haploid Kernels of Maize (Corn) by Measurement of Oil Content

    Science.gov (United States)

    Xu, Xiaoping; Huang, Qingming; Chen, Shanshan; Yang, Peiqiang; Chen, Shaojiang; Song, Yiqiao

    2016-01-01

    One of the modern crop breeding techniques uses doubled haploid plants that contain an identical pair of chromosomes in order to accelerate the breeding process. Rapid haploid identification method is critical for large-scale selections of double haploids. The conventional methods based on the color of the endosperm and embryo seeds are slow, manual and prone to error. On the other hand, there exists a significant difference between diploid and haploid seeds generated by high oil inducer, which makes it possible to use oil content to identify the haploid. This paper describes a fully-automated high-throughput NMR screening system for maize haploid kernel identification. The system is comprised of a sampler unit to select a single kernel to feed for measurement of NMR and weight, and a kernel sorter to distribute the kernel according to the measurement result. Tests of the system show a consistent accuracy of 94% with an average screening time of 4 seconds per kernel. Field test result is described and the directions for future improvement are discussed. PMID:27454427

  1. Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

    Directory of Open Access Journals (Sweden)

    Arnaud Carpentier

    2016-05-01

    Full Text Available The establishment of protocols to differentiate human pluripotent stem cells (hPSCs including embryonic (ESC and induced pluripotent (iPSC stem cells into functional hepatocyte-like cells (HLCs creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

  2. Intersection of toxicogenomics and high throughput screening in the Tox21 program: an NIEHS perspective.

    Science.gov (United States)

    Merrick, B Alex; Paules, Richard S; Tice, Raymond R

    Humans are exposed to thousands of chemicals with inadequate toxicological data. Advances in computational toxicology, robotic high throughput screening (HTS), and genome-wide expression have been integrated into the Tox21 program to better predict the toxicological effects of chemicals. Tox21 is a collaboration among US government agencies initiated in 2008 that aims to shift chemical hazard assessment from traditional animal toxicology to target-specific, mechanism-based, biological observations using in vitro assays and lower organism models. HTS uses biocomputational methods for probing thousands of chemicals in in vitro assays for gene-pathway response patterns predictive of adverse human health outcomes. In 1999, NIEHS began exploring the application of toxicogenomics to toxicology and recent advances in NextGen sequencing should greatly enhance the biological content obtained from HTS platforms. We foresee an intersection of new technologies in toxicogenomics and HTS as an innovative development in Tox21. Tox21 goals, priorities, progress, and challenges will be reviewed.

  3. High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

    Directory of Open Access Journals (Sweden)

    Brandon J Walters

    Full Text Available p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique compounds, of which 830 are Food and Drug Administration (FDA approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

  4. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter

    2003-01-01

    Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels....... A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct...... characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion...

  5. High-content screening of yeast mutant libraries by shotgun lipidomics

    DEFF Research Database (Denmark)

    Tarasov, Kirill; Stefanko, Adam; Casanovas, Albert

    2014-01-01

    To identify proteins with a functional role in lipid metabolism and homeostasis we designed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries. To this end, we combined culturing and lipid extraction in 96-well format, automated direct infusion...... factor KAR4 precipitated distinct lipid metabolic phenotypes. These results demonstrate that the high-throughput shotgun lipidomics platform is a valid and complementary proxy for high-content screening of yeast mutant libraries....... nanoelectrospray ionization, high-resolution Orbitrap mass spectrometry, and a dedicated data processing framework to support lipid phenotyping across hundreds of Saccharomyces cerevisiae mutants. Our novel approach revealed that the absence of genes with unknown function YBR141C and YJR015W, and the transcription...

  6. High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

    Science.gov (United States)

    Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

    2012-07-01

    Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    Science.gov (United States)

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. © 2015 Society for Laboratory Automation and Screening.

  8. Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

    Science.gov (United States)

    Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J Stephen

    2012-09-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

  9. Life in the fast lane: high-throughput chemistry for lead generation and optimisation.

    Science.gov (United States)

    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.

  10. Miniaturized microscope for high throughput screening of tumor spheroids in microfluidic devices

    Science.gov (United States)

    Uranga, Javier; Rodríguez-Pena, Alejandro; Gahigiro, Desiré; Ortiz-de-Solorzano, Carlos

    2018-02-01

    High-throughput in vitro screening of highly physiological three-dimensional cell cultures (3D-HTS) is rapidly gaining importance in preclinical studies, to study the effect of the microenvironment in tumor development, and to evaluate the efficacy of new anticancer drugs. Furthermore, it could also be envisioned the use of 3D-HTS systems in personalized anti-cancer treatment planning, based on tumor organoids or spheroids grown from tumor biopsies or isolated tumor circulating cells. Most commercial, multi-well plate based 3D-HTS systems are large, expensive, and are based on the use of multi-well plates that hardly provide a physiological environment and require the use of large amounts of biological material and reagents. In this paper we present a novel, miniaturized inverted microscope (hereinafter miniscospe), made up of low-cost, mass producible parts, that can be used to monitor the growth of living tumor cell spheroids within customized three-dimensional microfluidic platforms. Our 3D-HTS miniscope combines phase contrast imaging based on oblique back illumination technique with traditional widefield epi-fluorescence imaging, implemented using miniaturized electro-optical parts and gradient-index refraction lenses. This small (3x6x2cm), lightweight device can effectively image overtime the growth of (>200) tumor spheroids in a controlled and reproducible environment. Our miniscope can be used to acquire time-lapse images of cellular living spheroids over the course of several hours and captures their growth before and after drug treatment, to evaluate the effectiveness of the drug.

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

    Science.gov (United States)

    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...

  12. A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

    Science.gov (United States)

    Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

    2010-01-01

    Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

  13. High Throughput Facility

    Data.gov (United States)

    Federal Laboratory Consortium — Argonne?s high throughput facility provides highly automated and parallel approaches to material and materials chemistry development. The facility allows scientists...

  14. Rapid evaporative ionisation mass spectrometry and chemometrics for high-throughput screening of growth promoters in meat producing animals.

    Science.gov (United States)

    Guitton, Yann; Dervilly-Pinel, Gaud; Jandova, Renata; Stead, Sara; Takats, Zoltan; Le Bizec, Bruno

    2018-01-17

    In a proof of concept perspective, Rapid Evaporative Ionisation Mass Spectrometry (REIMS) was explored for the direct analysis of meat samples from β-agonist treated livestock. In this context, the combination of REIMS with untargeted metabolomics was investigated to identify carcasses from treated animals on the basis of a modification of indirect metabolites profile. The REIMS analysis generated specific lipid profiles which enabled the differentiation of meat samples collected from pigs treated with ractopamine via their feeding regime. Furthermore, the strategy was found successful when tested on different muscle types (loin, shoulder and thigh), which further expands its applicability. Classification performances were greater than 95% accurate which fully answers requirements of a screening strategy. This research indicates that REIMS implemented in an untargeted-metabolomics workflow can be considered as a high-throughput and accurate strategy for real-time meat classification in relation to ractopamine (and wider β-agonists) treatment in pig production. This approach may subsequently be implemented as a rapid screening test, at the slaughterhouse or at border inspection points, to detect such practice.

  15. Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening.

    Science.gov (United States)

    Green, Anthony P; Turner, Nicholas J; O'Reilly, Elaine

    2014-09-26

    The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  16. Label-free detection of cellular drug responses by high-throughput bright-field imaging and machine learning.

    Science.gov (United States)

    Kobayashi, Hirofumi; Lei, Cheng; Wu, Yi; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

    2017-09-29

    In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological change from different experiments can be inferred from the classification accuracy of a single classification model. Our work lays the groundwork for label-free drug screening in pharmaceutical science and industry.

  17. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

    Directory of Open Access Journals (Sweden)

    Michael J Smout

    Full Text Available BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

  18. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    Science.gov (United States)

    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

  19. Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Cristiana Lalli

    2015-01-01

    Full Text Available Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

  20. High Throughput Single-cell and Multiple-cell Micro-encapsulation

    OpenAIRE

    Lagus, Todd P.; Edd, Jon F.

    2012-01-01

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of ...

  1. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    Science.gov (United States)

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

  2. Understanding the stable boron clusters: A bond model and first-principles calculations based on high-throughput screening

    International Nuclear Information System (INIS)

    Xu, Shao-Gang; Liao, Ji-Hai; Zhao, Yu-Jun; Yang, Xiao-Bao

    2015-01-01

    The unique electronic property induced diversified structure of boron (B) cluster has attracted much interest from experimentalists and theorists. B 30–40 were reported to be planar fragments of triangular lattice with proper concentrations of vacancies recently. Here, we have performed high-throughput screening for possible B clusters through the first-principles calculations, including various shapes and distributions of vacancies. As a result, we have determined the structures of B n clusters with n = 30–51 and found a stable planar cluster of B 49 with a double-hexagon vacancy. Considering the 8-electron rule and the electron delocalization, a concise model for the distribution of the 2c–2e and 3c–2e bonds has been proposed to explain the stability of B planar clusters, as well as the reported B cages

  3. High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology.

    Science.gov (United States)

    Watson, Christa; Ge, Jing; Cohen, Joel; Pyrgiotakis, Georgios; Engelward, Bevin P; Demokritou, Philip

    2014-03-25

    The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO>Ag>Fe2O3>CeO2>SiO2 in TK6 cells at 4 h and Ag>Fe2O3>ZnO>CeO2>SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.

  4. HTS-DB: an online resource to publish and query data from functional genomics high-throughput siRNA screening projects.

    Science.gov (United States)

    Saunders, Rebecca E; Instrell, Rachael; Rispoli, Rossella; Jiang, Ming; Howell, Michael

    2013-01-01

    High-throughput screening (HTS) uses technologies such as RNA interference to generate loss-of-function phenotypes on a genomic scale. As these technologies become more popular, many research institutes have established core facilities of expertise to deal with the challenges of large-scale HTS experiments. As the efforts of core facility screening projects come to fruition, focus has shifted towards managing the results of these experiments and making them available in a useful format that can be further mined for phenotypic discovery. The HTS-DB database provides a public view of data from screening projects undertaken by the HTS core facility at the CRUK London Research Institute. All projects and screens are described with comprehensive assay protocols, and datasets are provided with complete descriptions of analysis techniques. This format allows users to browse and search data from large-scale studies in an informative and intuitive way. It also provides a repository for additional measurements obtained from screens that were not the focus of the project, such as cell viability, and groups these data so that it can provide a gene-centric summary across several different cell lines and conditions. All datasets from our screens that can be made available can be viewed interactively and mined for further hit lists. We believe that in this format, the database provides researchers with rapid access to results of large-scale experiments that might facilitate their understanding of genes/compounds identified in their own research. DATABASE URL: http://hts.cancerresearchuk.org/db/public.

  5. DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

    KAUST Repository

    Soufan, Othman

    2016-11-10

    Background Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemann–Pick type C disease. Conclusion We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between existing

  6. High-Throughput Computational Screening of the Metal Organic Framework Database for CH4/H2 Separations.

    Science.gov (United States)

    Altintas, Cigdem; Erucar, Ilknur; Keskin, Seda

    2018-01-31

    Metal organic frameworks (MOFs) have been considered as one of the most exciting porous materials discovered in the last decade. Large surface areas, high pore volumes, and tailorable pore sizes make MOFs highly promising in a variety of applications, mainly in gas separations. The number of MOFs has been increasing very rapidly, and experimental identification of materials exhibiting high gas separation potential is simply impractical. High-throughput computational screening studies in which thousands of MOFs are evaluated to identify the best candidates for target gas separation is crucial in directing experimental efforts to the most useful materials. In this work, we used molecular simulations to screen the most complete and recent collection of MOFs from the Cambridge Structural Database to unlock their CH 4 /H 2 separation performances. This is the first study in the literature, which examines the potential of all existing MOFs for adsorption-based CH 4 /H 2 separation. MOFs (4350) were ranked based on several adsorbent evaluation metrics including selectivity, working capacity, adsorbent performance score, sorbent selection parameter, and regenerability. A large number of MOFs were identified to have extraordinarily large CH 4 /H 2 selectivities compared to traditional adsorbents such as zeolites and activated carbons. We examined the relations between structural properties of MOFs such as pore sizes, porosities, and surface areas and their selectivities. Correlations between the heat of adsorption, adsorbility, metal type of MOFs, and selectivities were also studied. On the basis of these relations, a simple mathematical model that can predict the CH 4 /H 2 selectivity of MOFs was suggested, which will be very useful in guiding the design and development of new MOFs with extraordinarily high CH 4 /H 2 separation performances.

  7. High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

    Science.gov (United States)

    Hop, Cornelis E C A; Cole, Mark J; Davidson, Ralph E; Duignan, David B; Federico, James; Janiszewski, John S; Jenkins, Kelly; Krueger, Suzanne; Lebowitz, Rebecca; Liston, Theodore E; Mitchell, Walter; Snyder, Mark; Steyn, Stefan J; Soglia, John R; Taylor, Christine; Troutman, Matt D; Umland, John; West, Michael; Whalen, Kevin M; Zelesky, Veronica; Zhao, Sabrina X

    2008-11-01

    Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

  8. A new pooling strategy for high-throughput screening: the Shifted Transversal Design

    Directory of Open Access Journals (Sweden)

    Thierry-Mieg Nicolas

    2006-01-01

    Full Text Available Abstract Background In binary high-throughput screening projects where the goal is the identification of low-frequency events, beyond the obvious issue of efficiency, false positives and false negatives are a major concern. Pooling constitutes a natural solution: it reduces the number of tests, while providing critical duplication of the individual experiments, thereby correcting for experimental noise. The main difficulty consists in designing the pools in a manner that is both efficient and robust: few pools should be necessary to correct the errors and identify the positives, yet the experiment should not be too vulnerable to biological shakiness. For example, some information should still be obtained even if there are slightly more positives or errors than expected. This is known as the group testing problem, or pooling problem. Results In this paper, we present a new non-adaptive combinatorial pooling design: the "shifted transversal design" (STD. It relies on arithmetics, and rests on two intuitive ideas: minimizing the co-occurrence of objects, and constructing pools of constant-sized intersections. We prove that it allows unambiguous decoding of noisy experimental observations. This design is highly flexible, and can be tailored to function robustly in a wide range of experimental settings (i.e., numbers of objects, fractions of positives, and expected error-rates. Furthermore, we show that our design compares favorably, in terms of efficiency, to the previously described non-adaptive combinatorial pooling designs. Conclusion This method is currently being validated by field-testing in the context of yeast-two-hybrid interactome mapping, in collaboration with Marc Vidal's lab at the Dana Farber Cancer Institute. Many similar projects could benefit from using the Shifted Transversal Design.

  9. High-Throughput Screening as a Supplemental Tool for the Development of Advanced Emission Control Catalysts: Methodological Approaches and Data Processing

    Directory of Open Access Journals (Sweden)

    Andreas Sundermann

    2016-01-01

    Full Text Available A high-throughput (HT screening platform developed at hte with the application focus on automotive catalysis is described. hte HT units are configured for performing steady-state testing, as well as dynamic tests with fast feed switches, such as lean/rich excursions for the evaluation of NOx storage capacity and efficiency of lean NOx traps (LNT, ammonia storage capacity for selective catalytic reduction (SCR, evaluation of oxygen storage capacity (OSC, as well as lambda sweep tests for screening of three-way catalysts (TWC. Even though catalysts are screened on a rather small scale (~100 mg powder, experience showed that dosing rather complex gas mixtures in concentrations close to that found in real exhaust for the given application is mandatory to generate relevant data. The objective of this work is to give additional insight into HT technology. In the industrial research laboratory, HT screening has matured to become a reliable approach for rapid screening of both reaction parameter spaces, as well as material properties relevant for exhaust gas catalyst development. Due to the speed of optimized screening involving 48 parallel reactors, automated handling of primary data is an imported requirement. Software for data reduction, like estimation of light-off temperature, needs to be robust and handle results for diverse sample libraries in an unattended fashion. In combination with the statistical design of experiment and multivariate data analysis, HT testing has become a valuable enhancement to automotive catalyst development.

  10. Ultra-high frequency ultrasound biomicroscopy and high throughput cardiovascular phenotyping in a large scale mouse mutagenesis screen

    Science.gov (United States)

    Liu, Xiaoqin; Francis, Richard; Tobita, Kimimasa; Kim, Andy; Leatherbury, Linda; Lo, Cecilia W.

    2013-02-01

    Ultrasound biomicroscopy (UBM) is ideally suited for phenotyping fetal mice for congenital heart disease (CHD), as imaging can be carried out noninvasively to provide both hemodynamic and structural information essential for CHD diagnosis. Using the UBM (Vevo 2100; 40Hz) in conjunction with the clinical ultrasound system (Acuson Sequioa C512; 15Hz), we developed a two-step screening protocol to scan thousands fetuses derived from ENU mutagenized pedigrees. A wide spectrum of CHD was detected by the UBM, which were subsequently confirmed with follow-up necropsy and histopathology examination with episcopic fluorescence image capture. CHD observed included outflow anomalies, left/right heart obstructive lesions, septal/valvular defects and cardiac situs anomalies. Meanwhile, various extracardiac defects were found, such as polydactyly, craniofacial defects, exencephaly, omphalocele-cleft palate, most of which were associated with cardiac defects. Our analyses showed the UBM was better at assessing cardiac structure and blood flow profiles, while conventional ultrasound allowed higher throughput low-resolution screening. Our study showed the integration of conventional clinical ultrasound imaging with the UBM for fetal mouse cardiovascular phenotyping can maximize the detection and recovery of CHD mutants.

  11. Combined whole-cell high-throughput functional screening for identification of new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries

    Directory of Open Access Journals (Sweden)

    Rubén Zapata-Pérez

    2016-11-01

    Full Text Available Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide to produce ammonia and nicotinic acid. These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or nicotinic acid to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside. After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the sodium nitroprusside method allowed the finding of the first nicotinamidase with balanced catalytic efficiency towards nicotinamide (nicotinamidase activity and pyrazinamide (pyrazinamidase activity. Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

  12. Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries.

    Science.gov (United States)

    Zapata-Pérez, Rubén; García-Saura, Antonio G; Jebbar, Mohamed; Golyshin, Peter N; Sánchez-Ferrer, Álvaro

    2016-01-01

    Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD + salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

  13. High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

    Science.gov (United States)

    Annang, F; Pérez-Moreno, G; García-Hernández, R; Cordon-Obras, C; Martín, J; Tormo, J R; Rodríguez, L; de Pedro, N; Gómez-Pérez, V; Valente, M; Reyes, F; Genilloud, O; Vicente, F; Castanys, S; Ruiz-Pérez, L M; Navarro, M; Gamarro, F; González-Pacanowska, D

    2015-01-01

    African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites. © 2014 Society for Laboratory Automation and Screening.

  14. Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

    Science.gov (United States)

    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.

  15. Combining thermodynamic modeling and 3D printing of elemental powder blends for high-throughput investigation of high-entropy alloys – Towards rapid alloy screening and design

    International Nuclear Information System (INIS)

    Haase, Christian; Tang, Florian; Wilms, Markus B.; Weisheit, Andreas; Hallstedt, Bengt

    2017-01-01

    High-entropy alloys have gained high interest of both academia and industry in recent years due to their excellent properties and large variety of possible alloy systems. However, so far prediction of phase constitution and stability is based on empirical rules that can only be applied to selected alloy systems. In the current study, we introduce a methodology that enables high-throughput theoretical and experimental alloy screening and design. As a basis for thorough thermodynamic calculations, a new database was compiled for the Co–Cr–Fe–Mn–Ni system and used for Calphad and Scheil simulations. For bulk sample production, laser metal deposition (LMD) of an elemental powder blend was applied to build up the equiatomic CoCrFeMnNi Cantor alloy as a first demonstrator. This production approach allows high flexibility in varying the chemical composition and, thus, renders itself suitable for high-throughput alloy production. The microstructure, texture, and mechanical properties of the material processed were characterized using optical microscopy, EBSD, EDX, XRD, hardness and compression testing. The LMD-produced alloy revealed full density, strongly reduced segregation compared to conventionally cast material, pronounced texture, and excellent mechanical properties. Phase constitution and elemental distribution were correctly predicted by simulations. The applicability of the introduced methodology to high-entropy alloys and extension to compositionally complex alloys is discussed.

  16. Combining thermodynamic modeling and 3D printing of elemental powder blends for high-throughput investigation of high-entropy alloys – Towards rapid alloy screening and design

    Energy Technology Data Exchange (ETDEWEB)

    Haase, Christian, E-mail: christian.haase@iehk.rwth-aachen.de [Department of Ferrous Metallurgy, RWTH Aachen University, 52072 Aachen (Germany); Tang, Florian [Institute for Materials Applications in Mechanical Engineering, RWTH Aachen University, 52062 Aachen (Germany); Wilms, Markus B.; Weisheit, Andreas [Fraunhofer Institute for Laser Technology ILT, 52074 Aachen (Germany); Hallstedt, Bengt [Institute for Materials Applications in Mechanical Engineering, RWTH Aachen University, 52062 Aachen (Germany)

    2017-03-14

    High-entropy alloys have gained high interest of both academia and industry in recent years due to their excellent properties and large variety of possible alloy systems. However, so far prediction of phase constitution and stability is based on empirical rules that can only be applied to selected alloy systems. In the current study, we introduce a methodology that enables high-throughput theoretical and experimental alloy screening and design. As a basis for thorough thermodynamic calculations, a new database was compiled for the Co–Cr–Fe–Mn–Ni system and used for Calphad and Scheil simulations. For bulk sample production, laser metal deposition (LMD) of an elemental powder blend was applied to build up the equiatomic CoCrFeMnNi Cantor alloy as a first demonstrator. This production approach allows high flexibility in varying the chemical composition and, thus, renders itself suitable for high-throughput alloy production. The microstructure, texture, and mechanical properties of the material processed were characterized using optical microscopy, EBSD, EDX, XRD, hardness and compression testing. The LMD-produced alloy revealed full density, strongly reduced segregation compared to conventionally cast material, pronounced texture, and excellent mechanical properties. Phase constitution and elemental distribution were correctly predicted by simulations. The applicability of the introduced methodology to high-entropy alloys and extension to compositionally complex alloys is discussed.

  17. High-throughput characterization for solar fuels materials discovery

    Science.gov (United States)

    Mitrovic, Slobodan; Becerra, Natalie; Cornell, Earl; Guevarra, Dan; Haber, Joel; Jin, Jian; Jones, Ryan; Kan, Kevin; Marcin, Martin; Newhouse, Paul; Soedarmadji, Edwin; Suram, Santosh; Xiang, Chengxiang; Gregoire, John; High-Throughput Experimentation Team

    2014-03-01

    In this talk I will present the status of the High-Throughput Experimentation (HTE) project of the Joint Center for Artificial Photosynthesis (JCAP). JCAP is an Energy Innovation Hub of the U.S. Department of Energy with a mandate to deliver a solar fuel generator based on an integrated photoelectrochemical cell (PEC). However, efficient and commercially viable catalysts or light absorbers for the PEC do not exist. The mission of HTE is to provide the accelerated discovery through combinatorial synthesis and rapid screening of material properties. The HTE pipeline also features high-throughput material characterization using x-ray diffraction and x-ray photoemission spectroscopy (XPS). In this talk I present the currently operating pipeline and focus on our combinatorial XPS efforts to build the largest free database of spectra from mixed-metal oxides, nitrides, sulfides and alloys. This work was performed at Joint Center for Artificial Photosynthesis, a DOE Energy Innovation Hub, supported through the Office of Science of the U.S. Department of Energy under Award No. DE-SC0004993.

  18. Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Rasmussen, Michael; Røder, Gustav Andreas

    2011-01-01

    and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.......Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely...... timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal...

  19. NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening.

    Science.gov (United States)

    Thornburg, Christopher C; Britt, John R; Evans, Jason R; Akee, Rhone K; Whitt, James A; Trinh, Spencer K; Harris, Matthew J; Thompson, Jerell R; Ewing, Teresa L; Shipley, Suzanne M; Grothaus, Paul G; Newman, David J; Schneider, Joel P; Grkovic, Tanja; O'Keefe, Barry R

    2018-06-13

    The US National Cancer Institute's (NCI) Natural Product Repository is one of the world's largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions. This library, representing perhaps the largest accumulation of natural-product based fractions in the world, will be made available free of charge in 384-well plates for screening against all disease states in an effort to reinvigorate natural product-based drug discovery.

  20. High-throughput continuous cryopump

    International Nuclear Information System (INIS)

    Foster, C.A.

    1986-01-01

    A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible

  1. Fast Gradient Elution Reversed-Phase HPLC with Diode-Array Detection as a High Throughput Screening Method for Drugs of Abuse

    Energy Technology Data Exchange (ETDEWEB)

    Peter W. Carr; K.M. Fuller; D.R. Stoll; L.D. Steinkraus; M.S. Pasha; Glenn G. Hardin

    2005-12-30

    A new approach has been developed by modifying a conventional gradient elution liquid chromatograph for the high throughput screening of biological samples to detect the presence of regulated intoxicants. The goal of this work was to improve the speed of a gradient elution screening method over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which are the basis for the identification. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between gradient runs, thereby reducing the total time for each analysis. The total analysis time for each gradient elution run is only 2.8 minutes, including 0.3 minutes for column reequilibration between analyses. Retention times standard calibration solutes are reproducible to better than 0.002 minutes in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. The discriminating power and mean list length were calculated for a library of 47 intoxicants and compared with previous work from other laboratories to evaluate fast gradient elution HPLC as a screening tool.

  2. Quantitative in vitro-to-in vivo extrapolation in a high-throughput environment

    International Nuclear Information System (INIS)

    Wetmore, Barbara A.

    2015-01-01

    High-throughput in vitro toxicity screening provides an efficient way to identify potential biological targets for environmental and industrial chemicals while conserving limited testing resources. However, reliance on the nominal chemical concentrations in these in vitro assays as an indicator of bioactivity may misrepresent potential in vivo effects of these chemicals due to differences in clearance, protein binding, bioavailability, and other pharmacokinetic factors. Development of high-throughput in vitro hepatic clearance and protein binding assays and refinement of quantitative in vitro-to-in vivo extrapolation (QIVIVE) methods have provided key tools to predict xenobiotic steady state pharmacokinetics. Using a process known as reverse dosimetry, knowledge of the chemical steady state behavior can be incorporated with HTS data to determine the external in vivo oral exposure needed to achieve internal blood concentrations equivalent to those eliciting bioactivity in the assays. These daily oral doses, known as oral equivalents, can be compared to chronic human exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. This review will describe the use of QIVIVE methods in a high-throughput environment and the promise they hold in shaping chemical testing priorities and, potentially, high-throughput risk assessment strategies

  3. Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension.

    Science.gov (United States)

    Su, Hao; Yan, Ji; Xu, Jian; Fan, Xi-Zhen; Sun, Xian-Lin; Chen, Kang-Yu

    2015-08-01

    Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH. In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products. In the pipeline, the stepwise high-throughput virtual screening, quantitative structure-activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol. With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 μM, respectively. Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.

  4. In-situ nanoelectrospray for high-throughput screening of enzymes and real-time monitoring of reactions.

    Science.gov (United States)

    Yang, Yuhan; Han, Feifei; Ouyang, Jin; Zhao, Yunling; Han, Juan; Na, Na

    2016-01-01

    The in-situ and high-throughput evaluation of enzymes and real-time monitoring of enzyme catalyzed reactions in liquid phase is quite significant in the catalysis industry. In-situ nanoelectrospray, the direct sampling and ionization method for mass spectrometry, has been applied for high-throughput evaluation of enzymes, as well as the on-line monitoring of reactions. Simply inserting a capillary into a liquid system with high-voltage applied, analytes in liquid reaction system can be directly ionized at the capillary tip with small volume consumption. With no sample pre-treatment or injection procedure, different analytes such as saccharides, amino acids, alkaloids, peptides and proteins can be rapidly and directly extracted from liquid phase and ionized at the capillary tip. Taking irreversible transesterification reaction of vinyl acetate and ethanol as an example, this technique has been used for the high-throughput evaluation of enzymes, fast optimizations, as well as real-time monitoring of reaction catalyzed by different enzymes. In addition, it is even softer than traditional electrospray ionization. The present method can also be used for the monitoring of other homogenous and heterogeneous reactions in liquid phases, which will show potentials in the catalysis industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Selective anti-proliferation of HER2-positive breast cancer cells by anthocyanins identified by high-throughput screening.

    Directory of Open Access Journals (Sweden)

    Weihua Liu

    Full Text Available Overexpressed Human epidermal growth factor receptor 2 (HER2 drives the biology of 20% breast cancer and is a prediction of a poor prognosis for patients. HER2-targeted therapies significantly improve outcomes for HER2-positive patients. Traditional Chinese herbs/medicines have been used to treat breast cancer patients including HER2-positive patients in Asia for decades. Although the traditional medicines demonstrate efficacy in clinics for HER2-positive patients, the mechanism is largely unknown. In this article, we screened a 10,000 natural product library in 6 different cell lines representing breast cancer, and assessed the ability of each drug to cause cytotoxicity through a high-throughput screening approach. We have identified eight natural compounds that selectively inhibit the proliferation of HER2-positive cells. Two of the hit compounds, peonidin-3-glucoside and cyaniding-3-glucoside, are both extracts from black rice. They inhibit the phospho-HER2 and phospho-AKT and were confirmed to induce HER2-psotive breast cancer cells apoptosis both in vitro and in vivo. Peonidin-3-glucoside and cyaniding-3-glucoside treatments significantly reduced the tumor size and volume in vivo compared to the control group. There is no significant difference of antitumorgenic effects between peonidin-3-glucoside and cyaniding-3-glucoside treatments.

  6. “httk”: EPA’s Tool for High Throughput Toxicokinetics (CompTox CoP)

    Science.gov (United States)

    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...

  7. An automatic on-line 2,2-diphenyl-1-picrylhydrazyl-high performance liquid chromatography method for high-throughput screening of antioxidants from natural products.

    Science.gov (United States)

    Lu, Yanzhen; Wu, Nan; Fang, Yingtong; Shaheen, Nusrat; Wei, Yun

    2017-10-27

    Many natural products are rich in antioxidants which play an important role in preventing or postponing a variety of diseases, such as cardiovascular and inflammatory disease, diabetes as well as breast cancer. In this paper, an automatic on-line 2,2-diphenyl-1-picrylhydrazyl-high performance liquid chromatography (DPPH-HPLC) method was established for antioxidants screening with nine standards including organic acids (4-hydroxyphenylacetic acid, p-coumaric acid, ferulic acid, and benzoic acid), alkaloids (coptisine and berberine), and flavonoids (quercitrin, astragalin, and quercetin). The optimal concentration of DPPH was determined, and six potential antioxidants including 4-hydroxyphenylacetic acid, p-coumaric acid, ferulic acid, quercitrin, astragalin, and quercetin, and three non-antioxidants including benzoic acid, coptisine, and berberine, were successfully screened out and validated by conventional DPPH radical scavenging activity assay. The established method has been applied to the crude samples of Saccharum officinarum rinds, Coptis chinensis powders, and Malus pumila leaves, consecutively. Two potential antioxidant compounds from Saccharum officinarum rinds and five potential antioxidant compounds from Malus pumila eaves were rapidly screened out. Then these seven potential antioxidants were purified and identified as p-coumaric acid, ferulic acid, phloridzin, isoquercitrin, quercetin-3-xyloside, quercetin-3-arabinoside, and quercetin-3-rhamnoside using countercurrent chromatography combined with mass spectrometry and their antioxidant activities were further evaluated by conventional DPPH radical scavenging assay. The activity result was in accordance with that of the established method. This established method is cheap and automatic, and could be used as an efficient tool for high-throughput antioxidant screening from various complex natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Modeling Steroidogenesis Disruption Using High-Throughput ...

    Science.gov (United States)

    Environmental chemicals can elicit endocrine disruption by altering steroid hormone biosynthesis and metabolism (steroidogenesis) causing adverse reproductive and developmental effects. Historically, a lack of assays resulted in few chemicals having been evaluated for effects on steroidogenesis. The steroidogenic pathway is a series of hydroxylation and dehydrogenation steps carried out by CYP450 and hydroxysteroid dehydrogenase enzymes, yet the only enzyme in the pathway for which a high-throughput screening (HTS) assay has been developed is aromatase (CYP19A1), responsible for the aromatization of androgens to estrogens. Recently, the ToxCast HTS program adapted the OECD validated H295R steroidogenesis assay using human adrenocortical carcinoma cells into a high-throughput model to quantitatively assess the concentration-dependent (0.003-100 µM) effects of chemicals on 10 steroid hormones including progestagens, androgens, estrogens and glucocorticoids. These results, in combination with two CYP19A1 inhibition assays, comprise a large dataset amenable to clustering approaches supporting the identification and characterization of putative mechanisms of action (pMOA) for steroidogenesis disruption. In total, 514 chemicals were tested in all CYP19A1 and steroidogenesis assays. 216 chemicals were identified as CYP19A1 inhibitors in at least one CYP19A1 assay. 208 of these chemicals also altered hormone levels in the H295R assay, suggesting 96% sensitivity in the

  9. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

    Directory of Open Access Journals (Sweden)

    Canard Bruno

    2011-10-01

    Full Text Available Abstract Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4. Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

  10. SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

    Directory of Open Access Journals (Sweden)

    Velasco Riccardo

    2008-01-01

    Full Text Available Abstract Background Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP discovery and genotyping in grapevine (Vitis vinifera L.. However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs thus providing a valuable source for high-throughput genotyping methods. Results Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA methods were used for preparation of genomic DNA for the SNPlex assay. Conclusion Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA, is a good solution for future applications in well-equipped laboratories.

  11. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  12. High Throughput and Mechano-Active Platforms to Promote Cartilage Regeneration and Repair

    Science.gov (United States)

    Mohanraj, Bhavana

    Traumatic joint injuries initiate acute degenerative changes in articular cartilage that can lead to progressive loss of load-bearing function. As a result, patients often develop post-traumatic osteoarthritis (PTOA), a condition for which there currently exists no biologic interventions. To address this need, tissue engineering aims to mimic the structure and function of healthy, native counterparts. These constructs can be used to not only replace degenerated tissue, but also build in vitro, pre-clinical models of disease. Towards this latter goal, this thesis focuses on the design of a high throughput system to screen new therapeutics in a micro-engineered model of PTOA, and the development of a mechanically-responsive drug delivery system to augment tissue-engineered approaches for cartilage repair. High throughput screening is a powerful tool for drug discovery that can be adapted to include 3D tissue constructs. To facilitate this process for cartilage repair, we built a high throughput mechanical injury platform to create an engineered cartilage model of PTOA. Compressive injury of functionally mature constructs increased cell death and proteoglycan loss, two hallmarks of injury observed in vivo. Comparison of this response to that of native cartilage explants, and evaluation of putative therapeutics, validated this model for subsequent use in small molecule screens. A primary screen of 118 compounds identified a number of 'hits' and relevant pathways that may modulate pathologic signaling post-injury. To complement this process of therapeutic discovery, a stimuli-responsive delivery system was designed that used mechanical inputs as the 'trigger' mechanism for controlled release. The failure thresholds of these mechanically-activated microcapsules (MAMCs) were influenced by physical properties and composition, as well as matrix mechanical properties in 3D environments. TGF-beta released from the system upon mechano-activation stimulated stem cell

  13. Fabrication of combinatorial nm-planar electrode array for high throughput evaluation of organic semiconductors

    International Nuclear Information System (INIS)

    Haemori, M.; Edura, T.; Tsutsui, K.; Itaka, K.; Wada, Y.; Koinuma, H.

    2006-01-01

    We have fabricated a combinatorial nm-planar electrode array by using photolithography and chemical mechanical polishing processes for high throughput electrical evaluation of organic devices. Sub-nm precision was achieved with respect to the average level difference between each pair of electrodes and a dielectric layer. The insulating property between the electrodes is high enough to measure I-V characteristics of organic semiconductors. Bottom-contact field-effect-transistors (FETs) of pentacene were fabricated on this electrode array by use of molecular beam epitaxy. It was demonstrated that the array could be used as a pre-patterned device substrate for high throughput screening of the electrical properties of organic semiconductors

  14. DockoMatic 2.0: high throughput inverse virtual screening and homology modeling.

    Science.gov (United States)

    Bullock, Casey; Cornia, Nic; Jacob, Reed; Remm, Andrew; Peavey, Thomas; Weekes, Ken; Mallory, Chris; Oxford, Julia T; McDougal, Owen M; Andersen, Timothy L

    2013-08-26

    DockoMatic is a free and open source application that unifies a suite of software programs within a user-friendly graphical user interface (GUI) to facilitate molecular docking experiments. Here we describe the release of DockoMatic 2.0; significant software advances include the ability to (1) conduct high throughput inverse virtual screening (IVS); (2) construct 3D homology models; and (3) customize the user interface. Users can now efficiently setup, start, and manage IVS experiments through the DockoMatic GUI by specifying receptor(s), ligand(s), grid parameter file(s), and docking engine (either AutoDock or AutoDock Vina). DockoMatic automatically generates the needed experiment input files and output directories and allows the user to manage and monitor job progress. Upon job completion, a summary of results is generated by Dockomatic to facilitate interpretation by the user. DockoMatic functionality has also been expanded to facilitate the construction of 3D protein homology models using the Timely Integrated Modeler (TIM) wizard. The wizard TIM provides an interface that accesses the basic local alignment search tool (BLAST) and MODELER programs and guides the user through the necessary steps to easily and efficiently create 3D homology models for biomacromolecular structures. The DockoMatic GUI can be customized by the user, and the software design makes it relatively easy to integrate additional docking engines, scoring functions, or third party programs. DockoMatic is a free comprehensive molecular docking software program for all levels of scientists in both research and education.

  15. Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method

    DEFF Research Database (Denmark)

    Ornskov, D; Kolmos, B; Bendix Horn, P

    2008-01-01

    2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab...... samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance...... pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97...

  16. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    2010-08-01

    Full Text Available High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  17. A high throughput array microscope for the mechanical characterization of biomaterials

    Science.gov (United States)

    Cribb, Jeremy; Osborne, Lukas D.; Hsiao, Joe Ping-Lin; Vicci, Leandra; Meshram, Alok; O'Brien, E. Tim; Spero, Richard Chasen; Taylor, Russell; Superfine, Richard

    2015-02-01

    In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

  18. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  19. High-throughput characterization methods for lithium batteries

    Directory of Open Access Journals (Sweden)

    Yingchun Lyu

    2017-09-01

    Full Text Available The development of high-performance lithium ion batteries requires the discovery of new materials and the optimization of key components. By contrast with traditional one-by-one method, high-throughput method can synthesize and characterize a large number of compositionally varying samples, which is able to accelerate the pace of discovery, development and optimization process of materials. Because of rapid progress in thin film and automatic control technologies, thousands of compounds with different compositions could be synthesized rapidly right now, even in a single experiment. However, the lack of rapid or combinatorial characterization technologies to match with high-throughput synthesis methods, limit the application of high-throughput technology. Here, we review a series of representative high-throughput characterization methods used in lithium batteries, including high-throughput structural and electrochemical characterization methods and rapid measuring technologies based on synchrotron light sources.

  20. Quadruplex MAPH: improvement of throughput in high-resolution copy number screening.

    Science.gov (United States)

    Tyson, Jess; Majerus, Tamsin Mo; Walker, Susan; Armour, John Al

    2009-09-28

    Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms.

  1. Rapid evaporative ionization mass spectrometry for high-throughput screening in food analysis: The case of boar taint.

    Science.gov (United States)

    Verplanken, Kaat; Stead, Sara; Jandova, Renata; Poucke, Christof Van; Claereboudt, Jan; Bussche, Julie Vanden; Saeger, Sarah De; Takats, Zoltan; Wauters, Jella; Vanhaecke, Lynn

    2017-07-01

    Boar taint is a contemporary off-odor present in meat of uncastrated male pigs. As European Member States intend to abandon surgical castration of pigs by 2018, this off-odor has gained a lot of research interest. In this study, rapid evaporative ionization mass spectrometry (REIMS) was explored for the rapid detection of boar taint in neck fat. Untargeted screening of samples (n=150) enabled discrimination between sow, tainted and untainted boars. The obtained OPLS-DA models showed excellent classification accuracy, i.e. 99% and 100% for sow and boar samples or solely boar samples, respectively. Furthermore, the obtained models demonstrated excellent validation characteristics (R 2 (Y)=0.872-0.969; Q 2 (Y)=0.756-0.917), which were confirmed by CV-ANOVA (phighly accurate and high-throughput (<10s) classification of tainted and untainted boar samples was achieved, rendering REIMS a promising technique for predictive modelling in food safety and quality applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening.

    Science.gov (United States)

    Chung, Thomas D Y; Sergienko, Eduard; Millán, José Luis

    2010-04-27

    The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

  3. Molecular recognition and self-assembly special feature: A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin.

    Science.gov (United States)

    Shabbir, Shagufta H; Regan, Clinton J; Anslyn, Eric V

    2009-06-30

    A general approach to high-throughput screening of enantiomeric excess (ee) and concentration was developed by using indicator displacement assays (IDAs), and the protocol was then applied to the vicinal diol hydrobenzoin. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic acid-based receptors were screened by using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an artificial neural network to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified.

  4. High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

    Science.gov (United States)

    Rohde, Christopher B.; Zeng, Fei; Gilleland, Cody; Samara, Chrysanthi; Yanik, Mehmet F.

    2009-02-01

    In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.

  5. Noise Reduction in High-Throughput Gene Perturbation Screens

    Science.gov (United States)

    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...

  6. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  7. High content screening in microfluidic devices

    Science.gov (United States)

    Cheong, Raymond; Paliwal, Saurabh; Levchenko, Andre

    2011-01-01

    Importance of the field Miniaturization is key to advancing the state-of-the-art in high content screening (HCS), in order to enable dramatic cost savings through reduced usage of expensive biochemical reagents and to enable large-scale screening on primary cells. Microfluidic technology offers the potential to enable HCS to be performed with an unprecedented degree of miniaturization. Areas covered in this review This perspective highlights a real-world example from the authors’ work of HCS assays implemented in a highly miniaturized microfluidic format. Advantages of this technology are discussed, including cost savings, high throughput screening on primary cells, improved accuracy, the ability to study complex time-varying stimuli, and ease of automation, integration, and scaling. What the reader will gain The reader will understand the capabilities of a new microfluidics-based platform for HCS, and the advantages it provides over conventional plate-based HCS. Take home message Microfluidics technology will drive significant advancements and broader usage and applicability of HCS in drug discovery. PMID:21852997

  8. Development of scalable high throughput fermentation approaches for physiological characterisation of yeast and filamentous fungi

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen

    producing the heterologous model polyketide, 6-methylsalicylic acid (6-MSA). An automated methodology for high throughput screening focusing on growth rates, together with a fully automated method for quantitative physiological characterisation in microtiter plates, was established for yeast. Full...

  9. Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry.

    Science.gov (United States)

    Aqai, Payam; Blesa, Natalia Gómez; Major, Hilary; Pedotti, Mattia; Varani, Luca; Ferrero, Valentina E V; Haasnoot, Willem; Nielen, Michel W F

    2013-11-01

    A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

  10. Development of a STAT3 reporter prostate cancer cell line for high throughput screening of STAT3 activators and inhibitors

    International Nuclear Information System (INIS)

    Chau, My N.; Banerjee, Partha P.

    2008-01-01

    STAT3 is constitutively activated in several cancers, including prostate cancer, and is therefore, a potential target for cancer therapy. DU-145 prostate cancer cells were stably co-transfected with STAT3 reporter and puromycin resistant plasmids to create a stable STAT3 reporter cell line that can be used for high throughput screening of STAT3 modulators. The applicability of this cell line was tested with two known activators and inhibitors of STAT3. As expected, EGF and IL-6 increased STAT3 reporter activity and enhanced the nuclear localization of phosphorylated STAT3 (pSTAT3); whereas Cucurbitacin I and AG490 decreased STAT3 reporter activity dose and time-dependently and reduced the localization of pSTAT3 in the nuclei of prostate cancer cells. Given the importance of STAT3 in cancer initiation and progression, the development of a stable STAT3 reporter cell line in prostate cancer cells provides a rapid, sensitive, and cost effective method for the screening of potential STAT3 modulators.

  11. High-throughput migration modelling for estimating exposure to chemicals in food packaging in screening and prioritization tools.

    Science.gov (United States)

    Ernstoff, Alexi S; Fantke, Peter; Huang, Lei; Jolliet, Olivier

    2017-11-01

    Specialty software and simplified models are often used to estimate migration of potentially toxic chemicals from packaging into food. Current models, however, are not suitable for emerging applications in decision-support tools, e.g. in Life Cycle Assessment and risk-based screening and prioritization, which require rapid computation of accurate estimates for diverse scenarios. To fulfil this need, we develop an accurate and rapid (high-throughput) model that estimates the fraction of organic chemicals migrating from polymeric packaging materials into foods. Several hundred step-wise simulations optimised the model coefficients to cover a range of user-defined scenarios (e.g. temperature). The developed model, operationalised in a spreadsheet for future dissemination, nearly instantaneously estimates chemical migration, and has improved performance over commonly used model simplifications. When using measured diffusion coefficients the model accurately predicted (R 2  = 0.9, standard error (S e ) = 0.5) hundreds of empirical data points for various scenarios. Diffusion coefficient modelling, which determines the speed of chemical transfer from package to food, was a major contributor to uncertainty and dramatically decreased model performance (R 2  = 0.4, S e  = 1). In all, this study provides a rapid migration modelling approach to estimate exposure to chemicals in food packaging for emerging screening and prioritization approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Quadruplex MAPH: improvement of throughput in high-resolution copy number screening

    Directory of Open Access Journals (Sweden)

    Walker Susan

    2009-09-01

    Full Text Available Abstract Background Copy number variation (CNV in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Results Multiplex Amplifiable Probe Hybridisation (MAPH is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH" that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC samples. Conclusion QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms.

  13. High-throughput combinatorial chemical bath deposition: The case of doping Cu (In, Ga) Se film with antimony

    Science.gov (United States)

    Yan, Zongkai; Zhang, Xiaokun; Li, Guang; Cui, Yuxing; Jiang, Zhaolian; Liu, Wen; Peng, Zhi; Xiang, Yong

    2018-01-01

    The conventional methods for designing and preparing thin film based on wet process remain a challenge due to disadvantages such as time-consuming and ineffective, which hinders the development of novel materials. Herein, we present a high-throughput combinatorial technique for continuous thin film preparation relied on chemical bath deposition (CBD). The method is ideally used to prepare high-throughput combinatorial material library with low decomposition temperatures and high water- or oxygen-sensitivity at relatively high-temperature. To check this system, a Cu(In, Ga)Se (CIGS) thin films library doped with 0-19.04 at.% of antimony (Sb) was taken as an example to evaluate the regulation of varying Sb doping concentration on the grain growth, structure, morphology and electrical properties of CIGS thin film systemically. Combined with the Energy Dispersive Spectrometer (EDS), X-ray Photoelectron Spectroscopy (XPS), automated X-ray Diffraction (XRD) for rapid screening and Localized Electrochemical Impedance Spectroscopy (LEIS), it was confirmed that this combinatorial high-throughput system could be used to identify the composition with the optimal grain orientation growth, microstructure and electrical properties systematically, through accurately monitoring the doping content and material composition. According to the characterization results, a Sb2Se3 quasi-liquid phase promoted CIGS film-growth model has been put forward. In addition to CIGS thin film reported here, the combinatorial CBD also could be applied to the high-throughput screening of other sulfide thin film material systems.

  14. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

    Directory of Open Access Journals (Sweden)

    Yeon Sook Cho

    Full Text Available Interleukin-7 (IL-7 is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

  15. An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

    Directory of Open Access Journals (Sweden)

    Meirson T

    2017-05-01

    Full Text Available Tomer Meirson, Abraham O Samson, Hava Gil-Henn Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Abstract: The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2 is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. Keywords: virtual screen, efficiency metrics, MM-GBSA, molecular dynamics

  16. Kansas Power Plants

    Data.gov (United States)

    Kansas Data Access and Support Center — The Kansas Power Plants database depicts, as point features, the locations of the various types of power plant locations in Kansas. The locations of the power plants...

  17. High-content screening of small compounds on human embryonic stem cells.

    Science.gov (United States)

    Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

    2010-08-01

    Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

  18. Combinatorial materials synthesis and high-throughput screening: an integrated materials chip approach to mapping phase diagrams and discovery and optimization of functional materials.

    Science.gov (United States)

    Xiang, X D

    Combinatorial materials synthesis methods and high-throughput evaluation techniques have been developed to accelerate the process of materials discovery and optimization and phase-diagram mapping. Analogous to integrated circuit chips, integrated materials chips containing thousands of discrete different compositions or continuous phase diagrams, often in the form of high-quality epitaxial thin films, can be fabricated and screened for interesting properties. Microspot x-ray method, various optical measurement techniques, and a novel evanescent microwave microscope have been used to characterize the structural, optical, magnetic, and electrical properties of samples on the materials chips. These techniques are routinely used to discover/optimize and map phase diagrams of ferroelectric, dielectric, optical, magnetic, and superconducting materials.

  19. High-throughput screening using pseudotyped lentiviral particles: a strategy for the identification of HIV-1 inhibitors in a cell-based assay.

    Science.gov (United States)

    Garcia, Jean-Michel; Gao, Anhui; He, Pei-Lan; Choi, Joyce; Tang, Wei; Bruzzone, Roberto; Schwartz, Olivier; Naya, Hugo; Nan, Fa-Jun; Li, Jia; Altmeyer, Ralf; Zuo, Jian-Ping

    2009-03-01

    Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

  20. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

    Science.gov (United States)

    Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

    2015-11-20

    Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

  2. High-throughput crystal-optimization strategies in the South Paris Yeast Structural Genomics Project: one size fits all?

    Science.gov (United States)

    Leulliot, Nicolas; Trésaugues, Lionel; Bremang, Michael; Sorel, Isabelle; Ulryck, Nathalie; Graille, Marc; Aboulfath, Ilham; Poupon, Anne; Liger, Dominique; Quevillon-Cheruel, Sophie; Janin, Joël; van Tilbeurgh, Herman

    2005-06-01

    Crystallization has long been regarded as one of the major bottlenecks in high-throughput structural determination by X-ray crystallography. Structural genomics projects have addressed this issue by using robots to set up automated crystal screens using nanodrop technology. This has moved the bottleneck from obtaining the first crystal hit to obtaining diffraction-quality crystals, as crystal optimization is a notoriously slow process that is difficult to automatize. This article describes the high-throughput optimization strategies used in the Yeast Structural Genomics project, with selected successful examples.

  3. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

    Energy Technology Data Exchange (ETDEWEB)

    Avonto, Cristina; Chittiboyina, Amar G. [National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States); Rua, Diego [The Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD 20740 (United States); Khan, Ikhlas A., E-mail: ikhan@olemiss.edu [National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States); Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States)

    2015-12-01

    Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow

  4. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

    International Nuclear Information System (INIS)

    Avonto, Cristina; Chittiboyina, Amar G.; Rua, Diego; Khan, Ikhlas A.

    2015-01-01

    Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow

  5. A high-throughput colorimetric assay for glucose detection based on glucose oxidase-catalyzed enlargement of gold nanoparticles

    Science.gov (United States)

    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

  6. High throughput screening of phenoxy carboxylic acids with dispersive solid phase extraction followed by direct analysis in real time mass spectrometry.

    Science.gov (United States)

    Wang, Jiaqin; Zhu, Jun; Si, Ling; Du, Qi; Li, Hongli; Bi, Wentao; Chen, David Da Yong

    2017-12-15

    A high throughput, low environmental impact methodology for rapid determination of phenoxy carboxylic acids (PCAs) in water samples was developed by combing dispersive solid phase extraction (DSPE) using velvet-like graphitic carbon nitride (V-g-C 3 N 4 ) and direct analysis in real time mass spectrometry (DART-MS). Due to the large surface area and good dispersity of V-g-C 3 N 4 , the DSPE of PCAs in water was completed within 20 s, and the elution of PCAs was accomplished in 20 s as well using methanol. The eluents were then analyzed and quantified using DART ionization source coupled to a high resolution mass spectrometer, where an internal standard was added in the samples. The limit of detection ranged from 0.5 ng L -1 to 2 ng L -1 on the basis of 50 mL water sample; the recovery 79.9-119.1%; and the relative standard deviation 0.23%-9.82% (≥5 replicates). With the ease of use and speed of DART-MS, the whole protocol can complete within mere minutes, including sample preparation, extraction, elution, detection and quantitation. The methodology developed here is simple, fast, sensitive, quantitative, requiring little sample preparation and consuming significantly less toxic organic solvent, which can be used for high throughput screening of PCAs and potentially other contaminants in water. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

    Science.gov (United States)

    Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

    2016-02-01

    In the last two decades, market were due to cardiac toxicity, where unintended interactions with ion channels disrupt the heart's normal electrical function. Consequently, all new drugs must undergo preclinical testing for cardiac liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

  8. Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

    Science.gov (United States)

    Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

    2016-01-01

    The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

  9. Rapid recombination mapping for high-throughput genetic screens in Drosophila.

    Science.gov (United States)

    Sapiro, Anne L; Ihry, Robert J; Buhr, Derek L; Konieczko, Kevin M; Ives, Sarah M; Engstrom, Anna K; Wleklinski, Nicholas P; Kopish, Kristin J; Bashirullah, Arash

    2013-12-09

    Mutagenesis screens are a staple of classical genetics. Chemical-induced mutations, however, are often difficult and time-consuming to identify. Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster. This method requires only two generations and a total of six crosses in vials to estimate the genetic map position of the responsible lesion with high accuracy. This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing. We have used this approach to successfully map a collection of mutations from an ethyl methanesulfonate-based mutagenesis screen on the third chromosome. We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available. By facilitating the rapid identification of mutated genes, our mapping strategy removes a primary obstacle to the widespread use of powerful chemical mutagenesis screens to understand fundamental biological phenomena.

  10. Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay.

    Directory of Open Access Journals (Sweden)

    Ikechukwu Okoli

    2009-09-01

    Full Text Available Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans-C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.

  11. Infra-red thermography for high throughput field phenotyping in Solanum tuberosum.

    Directory of Open Access Journals (Sweden)

    Ankush Prashar

    Full Text Available The rapid development of genomic technology has made high throughput genotyping widely accessible but the associated high throughput phenotyping is now the major limiting factor in genetic analysis of traits. This paper evaluates the use of thermal imaging for the high throughput field phenotyping of Solanum tuberosum for differences in stomatal behaviour. A large multi-replicated trial of a potato mapping population was used to investigate the consistency in genotypic rankings across different trials and across measurements made at different times of day and on different days. The results confirmed a high degree of consistency between the genotypic rankings based on relative canopy temperature on different occasions. Genotype discrimination was enhanced both through normalising data by expressing genotype temperatures as differences from image means and through the enhanced replication obtained by using overlapping images. A Monte Carlo simulation approach was used to confirm the magnitude of genotypic differences that it is possible to discriminate. The results showed a clear negative association between canopy temperature and final tuber yield for this population, when grown under ample moisture supply. We have therefore established infrared thermography as an easy, rapid and non-destructive screening method for evaluating large population trials for genetic analysis. We also envisage this approach as having great potential for evaluating plant response to stress under field conditions.

  12. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  13. Evaluation of e-liquid toxicity using an open-source high-throughput screening assay

    Science.gov (United States)

    Keating, James E.; Zorn, Bryan T.; Kochar, Tavleen K.; Wolfgang, Matthew C.; Glish, Gary L.; Tarran, Robert

    2018-01-01

    The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography–mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition. PMID:29584716

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

    Science.gov (United States)

    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 ...

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

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background 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. Results 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 Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

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

    Science.gov (United States)

    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

  17. Printing Proteins as Microarrays for High-Throughput Function Determination

    Science.gov (United States)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  18. High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

    Science.gov (United States)

    Landry, James P; Fei, Yiyan; Zhu, X D

    2011-12-01

    Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

  19. A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Gai; Nash, Peter J.; Johnson, Britney; Pietzsch, Colette; Ilagan, Ma. Xenia G.; Bukreyev, Alexander; Basler, Christopher F.; Bowlin, Terry L.; Moir, Donald T.; Leung, Daisy W.; Amarasinghe, Gaya K. (WU-MED); (GSU); (Texas-MED); (Microbiotix)

    2017-01-24

    The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.

  20. High throughput sample processing and automated scoring

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2014-10-01

    Full Text Available The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to high throughput are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. High throughput methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies, and automation gives more uniform sample treatment and less dependence on operator performance. The high throughput modifications now available vary largely in their versatility, capacity, complexity and costs. The bottleneck for further increase of throughput appears to be the scoring.

  1. Identification of Novel "Inks" for 3D Printing Using High-Throughput Screening: Bioresorbable Photocurable Polymers for Controlled Drug Delivery.

    Science.gov (United States)

    Louzao, Iria; Koch, Britta; Taresco, Vincenzo; Ruiz-Cantu, Laura; Irvine, Derek J; Roberts, Clive J; Tuck, Christopher; Alexander, Cameron; Hague, Richard; Wildman, Ricky; Alexander, Morgan R

    2018-02-28

    A robust methodology is presented to identify novel biomaterials suitable for three-dimensional (3D) printing. Currently, the application of additive manufacturing is limited by the availability of functional inks, especially in the area of biomaterials; this is the first time when this method is used to tackle this problem, allowing hundreds of formulations to be readily assessed. Several functional properties, including the release of an antidepressive drug (paroxetine), cytotoxicity, and printability, are screened for 253 new ink formulations in a high-throughput format as well as mechanical properties. The selected candidates with the desirable properties are successfully scaled up using 3D printing into a range of object architectures. A full drug release study and degradability and tensile modulus experiments are presented on a simple architecture to validating the suitability of this methodology to identify printable inks for 3D printing devices with bespoke properties.

  2. Computational toxicology as implemented by the U.S. EPA: providing high throughput decision support tools for screening and assessing chemical exposure, hazard and risk.

    Science.gov (United States)

    Kavlock, Robert; Dix, David

    2010-02-01

    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. Environmental Protection Agency EPA is developing robust and flexible computational tools that can be applied to the thousands of chemicals in commerce, and contaminant mixtures found in air, water, and hazardous-waste sites. The Office of Research and Development (ORD) Computational Toxicology Research Program (CTRP) is composed of three main elements. The largest component is the National Center for Computational Toxicology (NCCT), which was established in 2005 to coordinate research on chemical screening and prioritization, informatics, and systems modeling. The second element consists of related activities in the National Health and Environmental Effects Research Laboratory (NHEERL) and the National Exposure Research Laboratory (NERL). The third and final component consists of academic centers working on various aspects of computational toxicology and funded by the U.S. EPA Science to Achieve Results (STAR) program. Together these elements form the key components in the implementation of both the initial strategy, A Framework for a Computational Toxicology Research Program (U.S. EPA, 2003), and the newly released The U.S. Environmental Protection Agency's Strategic Plan for Evaluating the Toxicity of Chemicals (U.S. EPA, 2009a). Key intramural projects of the CTRP include digitizing legacy toxicity testing information toxicity reference database (ToxRefDB), predicting toxicity (ToxCast) and exposure (ExpoCast), and creating virtual liver (v-Liver) and virtual embryo (v-Embryo) systems models. U.S. EPA-funded STAR centers are also providing bioinformatics, computational toxicology data and models, and developmental toxicity data and models. The models and underlying data are being made publicly

  3. Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

    Directory of Open Access Journals (Sweden)

    Santos Carla S

    2012-11-01

    Full Text Available Abstract Background Pine wilt disease (PWD, caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus, damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN and Pinus pinea (less susceptible to PWN. Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species

  4. Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

    Science.gov (United States)

    2012-01-01

    Background Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. Conclusions Defense-related genes

  5. High-throughput computational search for strengthening precipitates in alloys

    International Nuclear Information System (INIS)

    Kirklin, S.; Saal, James E.; Hegde, Vinay I.; Wolverton, C.

    2016-01-01

    The search for high-strength alloys and precipitation hardened systems has largely been accomplished through Edisonian trial and error experimentation. Here, we present a novel strategy using high-throughput computational approaches to search for promising precipitate/alloy systems. We perform density functional theory (DFT) calculations of an extremely large space of ∼200,000 potential compounds in search of effective strengthening precipitates for a variety of different alloy matrices, e.g., Fe, Al, Mg, Ni, Co, and Ti. Our search strategy involves screening phases that are likely to produce coherent precipitates (based on small lattice mismatch) and are composed of relatively common alloying elements. When combined with the Open Quantum Materials Database (OQMD), we can computationally screen for precipitates that either have a stable two-phase equilibrium with the host matrix, or are likely to precipitate as metastable phases. Our search produces (for the structure types considered) nearly all currently known high-strength precipitates in a variety of fcc, bcc, and hcp matrices, thus giving us confidence in the strategy. In addition, we predict a number of new, currently-unknown precipitate systems that should be explored experimentally as promising high-strength alloy chemistries.

  6. A cell-free fluorometric high-throughput screen for inhibitors of Rtt109-catalyzed histone acetylation.

    Directory of Open Access Journals (Sweden)

    Jayme L Dahlin

    Full Text Available The lysine acetyltransferase (KAT Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7% were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC50 values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could

  7. High Throughput Neuro-Imaging Informatics

    Directory of Open Access Journals (Sweden)

    Michael I Miller

    2013-12-01

    Full Text Available This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high dimensional neuroinformatic representations index containing O(E3-E4 discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high throughput machine learning methods for supporting (i cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii integration of image and non-image information for diagnosis and prognosis.

  8. High-throughput oxidation screen of antibody-drug conjugates by analytical protein A chromatography following IdeS digest.

    Science.gov (United States)

    Buecheler, Jakob W; Winzer, Matthias; Weber, Christian; Gieseler, Henning

    2018-05-01

    Oxidation of protein therapeutics is a major chemical degradation pathway which may impact bioactivity, serum half-life and stability. Therefore, oxidation is a relevant parameter which has to be monitored throughout formulation development. Methods such as HIC, RPLC and LC/MS achieve a separation of oxidized and non-oxidized species by differences in hydrophobicity. Antibody-drug conjugates (ADC) although are highly more complex due to the heterogeneity in linker, drug, drug-to-antibody ratio (DAR) and conjugation site. The analytical protein A chromatography can provide a simple and fast alternative to these common methods. A miniature analytical protein A chromatography method in combination with an IdeS digest was developed to analyse ADCs. The IdeS digest efficiency of an IgG1 was monitored using SEC-HPLC and non-reducing SDS-PAGE. An antibody-fluorescent dye conjugate was conjugated at different dye-to-antibody ratios as model construct to mimic an ADC. With IdeS, an almost complete digest of a model IgG1 can be achieved (digested protein amount >98%). This enables subsequent analytical protein A chromatography, which consequently eliminates any interference of payload with the stationary phase. A novel high-throughput method for an interchain cysteine-linked ADC oxidation screens during formulation development was developed. © 2018 Royal Pharmaceutical Society.

  9. High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

    2017-05-01

    The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  10. Multiple and high-throughput droplet reactions via combination of microsampling technique and microfluidic chip

    KAUST Repository

    Wu, Jinbo

    2012-11-20

    Microdroplets offer unique compartments for accommodating a large number of chemical and biological reactions in tiny volume with precise control. A major concern in droplet-based microfluidics is the difficulty to address droplets individually and achieve high throughput at the same time. Here, we have combined an improved cartridge sampling technique with a microfluidic chip to perform droplet screenings and aggressive reaction with minimal (nanoliter-scale) reagent consumption. The droplet composition, distance, volume (nanoliter to subnanoliter scale), number, and sequence could be precisely and digitally programmed through the improved sampling technique, while sample evaporation and cross-contamination are effectively eliminated. Our combined device provides a simple model to utilize multiple droplets for various reactions with low reagent consumption and high throughput. © 2012 American Chemical Society.

  11. High-throughput screening assay used in pharmacognosy: Selection, optimization and validation of methods of enzymatic inhibition by UV-visible spectrophotometry

    Directory of Open Access Journals (Sweden)

    Graciela Granados-Guzmán

    2014-02-01

    Full Text Available In research laboratories of both organic synthesis and extraction of natural products, every day a lot of products that can potentially introduce some biological activity are obtained. Therefore it is necessary to have in vitro assays, which provide reliable information for further evaluation in in vivo systems. From this point of view, in recent years has intensified the use of high-throughput screening assays. Such trials should be optimized and validated for accurate and precise results, i.e. reliable. The present review addresses the steps needed to develop and validate bioanalytical methods, emphasizing UV-Visible spectrophotometry as detection system. Particularly focuses on the selection of the method, the optimization to determine the best experimental conditions, validation, implementation of optimized and validated method to real samples, and finally maintenance and possible transfer it to a new laboratory.

  12. 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

    International Nuclear Information System (INIS)

    Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

    2016-01-01

    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 a 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 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

  13. Introducing Discrete Frequency Infrared Technology for High-Throughput Biofluid Screening

    Science.gov (United States)

    Hughes, Caryn; Clemens, Graeme; Bird, Benjamin; Dawson, Timothy; Ashton, Katherine M.; Jenkinson, Michael D.; Brodbelt, Andrew; Weida, Miles; Fotheringham, Edeline; Barre, Matthew; Rowlette, Jeremy; Baker, Matthew J.

    2016-02-01

    Accurate early diagnosis is critical to patient survival, management and quality of life. Biofluids are key to early diagnosis due to their ease of collection and intimate involvement in human function. Large-scale mid-IR imaging of dried fluid deposits offers a high-throughput molecular analysis paradigm for the biomedical laboratory. The exciting advent of tuneable quantum cascade lasers allows for the collection of discrete frequency infrared data enabling clinically relevant timescales. By scanning targeted frequencies spectral quality, reproducibility and diagnostic potential can be maintained while significantly reducing acquisition time and processing requirements, sampling 16 serum spots with 0.6, 5.1 and 15% relative standard deviation (RSD) for 199, 14 and 9 discrete frequencies respectively. We use this reproducible methodology to show proof of concept rapid diagnostics; 40 unique dried liquid biopsies from brain, breast, lung and skin cancer patients were classified in 2.4 cumulative seconds against 10 non-cancer controls with accuracies of up to 90%.

  14. A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC libraries using High Resolution Melt analysis

    Directory of Open Access Journals (Sweden)

    Caligari Peter DS

    2010-05-01

    Full Text Available Abstract Background The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR and High Resolution Melt (HRM analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

  15. Kansas Electric Transmission Lines

    Data.gov (United States)

    Kansas Data Access and Support Center — This data set is a digital representation of the EletcircTransmission lines for the State of Kansas as maintained by the Kansas Corporation Commission. Data is...

  16. High Throughput PBTK: Evaluating EPA’s Open-Source Data and Tools for Dosimetry and Exposure Reconstruction

    Science.gov (United States)

    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...

  17. Advances in Predictive Toxicology for Discovery Safety through High Content Screening.

    Science.gov (United States)

    Persson, Mikael; Hornberg, Jorrit J

    2016-12-19

    High content screening enables parallel acquisition of multiple molecular and cellular readouts. In particular the predictive toxicology field has progressed from the advances in high content screening, as more refined end points that report on cellular health can be studied in combination, at the single cell level, and in relatively high throughput. Here, we discuss how high content screening has become an essential tool for Discovery Safety, the discipline that integrates safety and toxicology in the drug discovery process to identify and mitigate safety concerns with the aim to design drug candidates with a superior safety profile. In addition to customized mechanistic assays to evaluate target safety, routine screening assays can be applied to identify risk factors for frequently occurring organ toxicities. We discuss the current state of high content screening assays for hepatotoxicity, cardiotoxicity, neurotoxicity, nephrotoxicity, and genotoxicity, including recent developments and current advances.

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

    Science.gov (United States)

    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.

  19. Comprehensive analysis of high-throughput screens with HiTSeekR

    DEFF Research Database (Denmark)

    List, Markus; Schmidt, Steffen; Christiansen, Helle

    2016-01-01

    TSeekR as a one-stop solution for chemical compound screens, siRNA knock-down and CRISPR/Cas9 knock-out screens, as well as microRNA inhibitor and -mimics screens. We chose three use cases that demonstrate the potential of HiTSeekR to fully exploit HTS screening data in quite heterogeneous contexts to generate...

  20. A High Throughput Model of Post-Traumatic Osteoarthritis using Engineered Cartilage Tissue Analogs

    Science.gov (United States)

    Mohanraj, Bhavana; Meloni, Gregory R.; Mauck, Robert L.; Dodge, George R.

    2014-01-01

    (1) Objective A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. (2) Method We utilized a high throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. (3) Results Engineered cartilage response to injury was strain dependent, with a 2-fold increase in GAG loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in LDH release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor (ZVF) was effective at reducing cell death, while the amphiphilic polymer (P188) and the free-radical scavenger (NAC) reduced GAG loss as compared to injury alone. (4) Conclusions The injury response in this engineered cartilage model replicated key features of the response from cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA. PMID:24999113

  1. In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening.

    Science.gov (United States)

    Gea, An; Stringano, Elisabetta; Brown, Ron H; Mueller-Harvey, Irene

    2011-01-26

    A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin ( Onobrychis viciifolia ) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6-113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

  2. High-throughput screening for various classes of doping agents using a new 'dilute-and-shoot' liquid chromatography-tandem mass spectrometry multi-target approach.

    Science.gov (United States)

    Guddat, S; Solymos, E; Orlovius, A; Thomas, A; Sigmund, G; Geyer, H; Thevis, M; Schänzer, W

    2011-01-01

    A new multi-target approach based on liquid chromatography--electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs--for example, diuretics, beta2-agonists--stimulants and narcotics are detectable at concentration levels far below the required limits. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work. Copyright © 2011 John

  3. Supplementary Material for: DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

    KAUST Repository

    Soufan, Othman

    2016-01-01

    Abstract Background Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemannâ Pick type C disease. Conclusion We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between

  4. Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

    Science.gov (United States)

    Rotem, Asaf; Garraway, Levi; Su, Mei-Ju; Basu, Anindita; Regev, Aviv; Struhl, Kevin

    2017-02-01

    Three-dimensional growth conditions reflect the natural environment of cancer cells and are crucial to be performed at drug screens. We developed a 3D assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the 50-year old benchmark assay-soft agar. Using GILA, we performed high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. This phenotypic approach is complementary to our genetic approach that utilizes single-cell RNA-sequencing of a patient sample to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein. Currently, we are dealing with a big challenge in our field- the limited number of cells that might be extracted from a biopsy. Small patient-derived samples are hard to test in the traditional multiwell plate and it will be helpful to minimize the culture area and the experimental system. We managed to design a suitable microfluidic device for limited number of cells and perform the assay using image analysis. We aim to test drugs on tumor cells, outside of the patient body- and recommend on the ideal treatment that is tailored to the individual. This device will help to minimize biopsy-sampling volumes and minimize interventions in the patient's tumor.

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

    Science.gov (United States)

    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.

  6. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    Science.gov (United States)

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  7. High-throughput spectrometer designs in a compact form-factor: principles and applications

    Science.gov (United States)

    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.

  8. 2005 Kansas Land Cover Patterns, Level IV, Kansas River Watershed

    Data.gov (United States)

    Kansas Data Access and Support Center — The 2005 Kansas Land Cover Patterns (KLCP) Mapping Initiative was a two-phase mapping endeavor that occurred over a three-year period (2007-2009). Note that while...

  9. Science programs in Kansas

    Science.gov (United States)

    Kramer, Ariele R.; Kelly, Brian P.

    2017-05-08

    The U.S. Geological Survey (USGS) is a non-regulatory Earth science agency within the Department of the Interior that provides impartial scientific information to describe and understand the health of our ecosystems and environment; minimize loss of life and property from natural disasters; manage water, biological, energy, and mineral resources; and enhance and protect our quality of life. The USGS cooperates with Federal, State, tribal, and local agencies in Kansas to deliver long-term data in real-time and interpretive reports describing what those data mean to the public and resource management agencies. USGS science programs in Kansas provide real-time groundwater monitoring at more than 23 locations; streamflow monitoring at more than 218 locations; water-quality and trends in the Little Arkansas and Kansas Rivers; inflows and outflows of sediment to/from reservoirs and in streams; harmful algal bloom research in the Kansas River, Milford Lake, and Cheney Reservoir; water-quantity and water-quality effects of artificial groundwater recharge for the Equus Beds Aquifer Storage and Recovery project near Wichita, Kansas; compilation of Kansas municipal and irrigation water-use data statewide; the occurrence, effects, and movement of environmental pesticides, antibiotics, algal toxins, and taste-and-odor compounds; and funding to the Kansas Water Resources Research Institute to further research and education through Kansas universities.

  10. Development and validation of a 48-target analytical method for high-throughput monitoring of genetically modified organisms.

    Science.gov (United States)

    Li, Xiaofei; Wu, Yuhua; Li, Jun; Li, Yunjing; Long, Likun; Li, Feiwu; Wu, Gang

    2015-01-05

    The rapid increase in the number of genetically modified (GM) varieties has led to a demand for high-throughput methods to detect genetically modified organisms (GMOs). We describe a new dynamic array-based high throughput method to simultaneously detect 48 targets in 48 samples on a Fludigm system. The test targets included species-specific genes, common screening elements, most of the Chinese-approved GM events, and several unapproved events. The 48 TaqMan assays successfully amplified products from both single-event samples and complex samples with a GMO DNA amount of 0.05 ng, and displayed high specificity. To improve the sensitivity of detection, a preamplification step for 48 pooled targets was added to enrich the amount of template before performing dynamic chip assays. This dynamic chip-based method allowed the synchronous high-throughput detection of multiple targets in multiple samples. Thus, it represents an efficient, qualitative method for GMO multi-detection.

  11. Rapid Recombination Mapping for High-Throughput Genetic Screens in Drosophila

    OpenAIRE

    Sapiro, Anne L.; Ihry, Robert J.; Buhr, Derek L.; Konieczko, Kevin M.; Ives, Sarah M.; Engstrom, Anna K.; Wleklinski, Nicholas P.; Kopish, Kristin J.; Bashirullah, Arash

    2013-01-01

    Mutagenesis screens are a staple of classical genetics. Chemical-induced mutations, however, are often difficult and time-consuming to identify. Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster. This method requires only two generations and a total of six crosses in vials to estimate the genetic map position of the responsible lesion with high accuracy. This genetic map positio...

  12. 2005 Kansas Land Cover Patterns, Level I, Kansas River Watershed

    Data.gov (United States)

    Kansas Data Access and Support Center — The Upper Kansas River Watershed Land Cover Patterns map represents Phase 1 of a two-phase mapping initiative occurring over a three-year period as part of a...

  13. High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

    Science.gov (United States)

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. Copyright © 2015 Zhou et al.

  14. Kansas Playa Wetlands

    Data.gov (United States)

    Kansas Data Access and Support Center — This digital dataset provides information about the distribution, areal extent, and morphometry of playa wetlands throughout western Kansas. Playa wetlands were...

  15. mIMT-visHTS: A novel method for multiplexing isobaric mass tagged datasets with an accompanying visualization high throughput screening tool for protein profiling.

    Science.gov (United States)

    Ricchiuto, Piero; Iwata, Hiroshi; Yabusaki, Katsumi; Yamada, Iwao; Pieper, Brett; Sharma, Amitabh; Aikawa, Masanori; Singh, Sasha A

    2015-10-14

    Isobaric mass tagging (IMT) methods enable the analysis of thousands of proteins simultaneously. We used tandem mass tagging reagents (TMT™) to monitor the relative changes in the proteome of the mouse macrophage cell line RAW264.7 at the same six time points after no stimulation (baseline phenotype), stimulation with interferon gamma (pro-inflammatory phenotype) or stimulation with interleukin-4 (anti-inflammatory phenotype). The combined TMT datasets yielded nearly 12,000 protein profiles for comparison. To facilitate this large analysis, we developed a novel method that combines or multiplexes the separate IMT (mIMT) datasets into a single super dataset for subsequent model-based clustering and co-regulation analysis. Specially designed visual High Throughput Screening (visHTS) software screened co-regulated proteins. visHTS generates an interactive and visually intuitive color-coded bullseye plot that enables users to browse the cluster outputs and identify co-regulated proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Rok Gaber

    2013-11-01

    Full Text Available To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.

  17. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

    Science.gov (United States)

    Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

    2013-01-01

    To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

  18. High-throughput screening and quantitation of guanidino and ureido compounds using liquid chromatography-drift tube ion mobility spectrometry-mass spectrometry

    International Nuclear Information System (INIS)

    Fan, Ruo-Jing; Zhang, Fang; Chen, Xiu-Ping; Qi, Wan-Shu; Guan, Qing; Sun, Tuan-Qi; Guo, Yin-Long

    2017-01-01

    The present work focused on the high-throughput screening and quantitation of guanidino compounds (GCs) and ureido compounds (UCs) in human thyroid tissues. The strategy employed benzylic rearrangement stable isotope labeling (BRSIL) for the sample preparation and then detection using liquid chromatography-drift tube ion mobility spectrometry-quadrupole time of flight mass spectrometry (LC-DTIMS-QTOF MS). A short reversed-phase LC realized an on-line desalting and a measurement cycle of 5.0 min. DTIMS separation enhanced the better specificity and selectivity for the benzil labeled GCs and UCs. The elevated mass resolution of QTOF MS enabled measure of the characteristic ions at accurate mass in MS and tandem MS spectra. Collision cross section (CCS) from DTIMS and accurate mass from QTOF MS were used as two qualifiers for the profiling and identification of GCs and UCs. In addition, an integral abundance arising from 3-D ion features (retention time, drift time, m/z) was applied to quantify the GCs and UCs in human thyroid tissues. The quantitative validation indicated good linearity (coefficient values ≥ 0.9981), good precision (1.0%–12.3% for intra-day and 0.9%–7.8% for inter-day) and good accuracy (91%–109%). The results demonstrated that the developed BRSIL coupled with LC-DTIMS-QTOF MS can be a powerful analysis platform to investigate GCs and UCs in human thyroid tissues. - Highlights: • The separation power of DTIMS-MS enhanced peak capacity, spectral clarity, and specificity of benzil labeled GCs and UCs. • Short-column LC for on-line desalting increased the throughput with a measurement cycle of 5.0 min. • CCS and accurate mass as a pair of qualifiers were used for the profiling and identification of GCs and UCs. • An integral abundance arising from 3-D ion features (RT, DT, m/z) was used as a novel quantifier for quantitation. • The developed method was applied to screen and quantify the GCs and UCs in human thyroid tissues.

  19. High-throughput screening and quantitation of guanidino and ureido compounds using liquid chromatography-drift tube ion mobility spectrometry-mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ruo-Jing [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Zhang, Fang, E-mail: fzhang@sioc.ac.cn [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Chen, Xiu-Ping; Qi, Wan-Shu [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Guan, Qing [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Sun, Tuan-Qi, E-mail: tuanqisun@163.com [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Guo, Yin-Long, E-mail: ylguo@sioc.ac.cn [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China)

    2017-04-08

    The present work focused on the high-throughput screening and quantitation of guanidino compounds (GCs) and ureido compounds (UCs) in human thyroid tissues. The strategy employed benzylic rearrangement stable isotope labeling (BRSIL) for the sample preparation and then detection using liquid chromatography-drift tube ion mobility spectrometry-quadrupole time of flight mass spectrometry (LC-DTIMS-QTOF MS). A short reversed-phase LC realized an on-line desalting and a measurement cycle of 5.0 min. DTIMS separation enhanced the better specificity and selectivity for the benzil labeled GCs and UCs. The elevated mass resolution of QTOF MS enabled measure of the characteristic ions at accurate mass in MS and tandem MS spectra. Collision cross section (CCS) from DTIMS and accurate mass from QTOF MS were used as two qualifiers for the profiling and identification of GCs and UCs. In addition, an integral abundance arising from 3-D ion features (retention time, drift time, m/z) was applied to quantify the GCs and UCs in human thyroid tissues. The quantitative validation indicated good linearity (coefficient values ≥ 0.9981), good precision (1.0%–12.3% for intra-day and 0.9%–7.8% for inter-day) and good accuracy (91%–109%). The results demonstrated that the developed BRSIL coupled with LC-DTIMS-QTOF MS can be a powerful analysis platform to investigate GCs and UCs in human thyroid tissues. - Highlights: • The separation power of DTIMS-MS enhanced peak capacity, spectral clarity, and specificity of benzil labeled GCs and UCs. • Short-column LC for on-line desalting increased the throughput with a measurement cycle of 5.0 min. • CCS and accurate mass as a pair of qualifiers were used for the profiling and identification of GCs and UCs. • An integral abundance arising from 3-D ion features (RT, DT, m/z) was used as a novel quantifier for quantitation. • The developed method was applied to screen and quantify the GCs and UCs in human thyroid tissues.

  20. High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

    Science.gov (United States)

    Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

    2012-05-01

    Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

  1. High Throughput Transcriptomics @ USEPA (Toxicology ...

    Science.gov (United States)

    The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.

  2. High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform.

    Science.gov (United States)

    Abalde-Cela, Sara; Gould, Anna; Liu, Xin; Kazamia, Elena; Smith, Alison G; Abell, Chris

    2015-05-06

    Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

  3. High-throughput evaluation of interactions between biomaterials, proteins and cells using patterned superhydrophobic substrates

    OpenAIRE

    Neto, Ana I.; Custódio, Catarina A.; Wenlong Song; Mano, J. F.

    2011-01-01

    We propose a new low cost platform for high-throughput analysis that permits screening the biological performance of independent combinations of biomaterials, cells and culture media. Patterned superhydrophobic flat substrates with controlled wettable spots are used to produce microarray chips for accelerated multiplexing evaluation. This work was partially supported by Fundação para a Ciência e Tecnologia (FCT) under project PTDC/FIS/68517/2006.

  4. NSC23925, identified in a high-throughput cell-based screen, reverses multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Zhenfeng Duan

    2009-10-01

    Full Text Available Multidrug resistance (MDR is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1 but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 microM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.

  5. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  6. Genecentric: a package to uncover graph-theoretic structure in high-throughput epistasis data

    OpenAIRE

    Gallant, Andrew; Leiserson, Mark DM; Kachalov, Maxim; Cowen, Lenore J; Hescott, Benjamin J

    2013-01-01

    Background New technology has resulted in high-throughput screens for pairwise genetic interactions in yeast and other model organisms. For each pair in a collection of non-essential genes, an epistasis score is obtained, representing how much sicker (or healthier) the double-knockout organism will be compared to what would be expected from the sickness of the component single knockouts. Recent algorithmic work has identified graph-theoretic patterns in this data that can indicate functional ...

  7. A high throughput DNA extraction method with high yield and quality

    Directory of Open Access Journals (Sweden)

    Xin Zhanguo

    2012-07-01

    Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

  8. Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells

    Science.gov (United States)

    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...

  9. Characterisation of powerful antioxidants and synthetic iron ligands, as protective agents against oxidative damages, using new high throughput screening assays

    International Nuclear Information System (INIS)

    Meunier, St.

    2002-12-01

    This work was devoted to the development of pertinent high throughput screening assays in the aim of studying oxidative stress. Three screening assays have been developed for the evaluation of protective agents toward ROS generated by gamma irradiation, UV or by a Fenton-like system. 24 natural extracts and a library of 120 pure compounds, containing among the most powerful antioxidants known to date, have been readily studied using, these new techniques. We found that two pulvinic acid derivatives possess excellent protective properties, and especially a pigment of fungus named norbadione A. Beyond its in vitro activity, this molecule displays remarkable biological properties. In the aim of studying an alternative pathway of protection against oxidation induced by iron, ligands able to modify the redox properties of this metal, have been synthesised. We have developed a parallel synthesis allowing the variation of the architecture, denticity, chelating moieties and hydrophobicity of iron chelates. Using this strategy, 47 potential Fe(III) ligands were obtained. Their protective capacities have been studied using a fourth screening assay, demonstrating the effectiveness of some ligands. Finally, the immunoassay technique called SPI-RAD has been used in order to study a particular consequence of drastic oxidative stress, namely covalent crosslinks between proteins. Our results demonstrate that these linkages occur in the presence of metals (FeII or CuII) and hydrogen peroxide, as well as in the presence of NO . radical. Moreover, it has been demonstrated that tyrosines residues and disulfide bridges play an important role in these phenomena. (author)

  10. Kansas Rivers TMDL

    Data.gov (United States)

    Kansas Data Access and Support Center — This data set includes all the streams in the Kansas 2006 Water Register that have established TMDLs as of October 17, 2006. The impairments and implementation...

  11. Using high throughput experimental data and in silico models to discover alternatives to toxic chromate corrosion inhibitors

    International Nuclear Information System (INIS)

    Winkler, D.A.; Breedon, M.; White, P.; Hughes, A.E.; Sapper, E.D.; Cole, I.

    2016-01-01

    Highlights: • We screened a large library of organic compounds as replacements for toxic chromates. • High throughput automated corrosion testing was used to assess inhibitor performance. • Robust, predictive machine learning models of corrosion inhibition were developed. • Models indicated molecular features contributing to performance of organic inhibitors. • We also showed that quantum chemistry descriptors do not correlate with performance. - Abstract: Restrictions on the use of toxic chromate-based corrosion inhibitors have created important issues for the aerospace and other industries. Benign alternatives that offer similar or superior performance are needed. We used high throughput experiments to assess 100 small organic molecules as potential inhibitors of corrosion in aerospace aluminium alloys AA2024 and AA7075. We generated robust, predictive, quantitative computational models of inhibitor efficiency at two pH values using these data. The models identified molecular features of inhibitor molecules that had the greatest impact on corrosion inhibition. Models can be used to discover better corrosion inhibitors by screening libraries of organic compounds for candidates with high corrosion inhibition.

  12. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident

    Science.gov (United States)

    Semenova, Vera A.; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from −4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = −0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. PMID:27814939

  13. Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells

    Science.gov (United States)

    Nguyen, Minh M.; Dar, Javid A.; Ai, Junkui; Wang, Yujuan; Masoodi, Khalid Z.; Shun, Tongying; Shinde, Sunita; Camarco, Daniel P.; Hua, Yun; Huryn, Donna M.; Wilson, Gabriela Mustata; Lazo, John S.; Nelson, Joel B.; Wipf, Peter

    2016-01-01

    Abstract Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide. PMID:27187604

  14. Multi-channel counter-current chromatography for high-throughput fractionation of natural products for drug discovery.

    Science.gov (United States)

    Wu, Shihua; Yang, Lu; Gao, Yuan; Liu, Xiaoyue; Liu, Feiyan

    2008-02-08

    A multi-channel counter-current chromatography (CCC) method has been designed and fabricated for the high-throughput fractionation of natural products without complications sometimes encountered with other conventional chromatographic systems, such as irreversible adsorptive constituent losses and deactivation, tailing of solute peaks and contamination. It has multiple independent CCC channels and each channel connects independent separation column(s) by parallel flow tubes, and thus the multi-channel CCC apparatus can achieve simultaneously two or more independent chromatographic processes. Furthermore, a high-throughput CCC fractionation method for natural products has been developed by a combination of a new three-channel CCC apparatus and conventional parallel chromatographic devices including pumps, sample injectors, effluent detectors and collectors, and its performance has been displayed on the fractionation of ethyl acetate extracts of three natural materials Solidago canadensis, Suillus placidus, and Trichosanthes kirilowii, which are found to be potent cytotoxic to tumor cell lines in the course of screening the antitumor candidates. By combination of biological screening programs and preparative high-performance liquid chromatography (HPLC) purification, 22.8 mg 6 beta-angeloyloxykolavenic acid and 29.4 mg 6 beta-tigloyloxykolavenic acid for S. canadensis, 25.3mg suillin for S. placidus, and 6.8 mg 23,24-dihydrocucurbitacin B for T. Kirilowii as their major cytotoxic principles were isolated from each 1000 mg crude ethyl acetate extract. Their chemical structures were characterized by electrospray ionization mass spectrometry, one- and two-dimensional nuclear magnetic resonance. The overall results indicate the multi-channel CCC is very useful for high-throughput fractionation of natural products for drug discovery in spite of the solvent balancing requirement and the lower resolution of the shorter CCC columns.

  15. Differentiating pathway-specific from nonspecific effects in high-throughput toxicity data: A foundation for prioritizing adverse outcome pathway development

    Science.gov (United States)

    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 ...

  16. Screening_mgmt: a Python module for managing screening data.

    Science.gov (United States)

    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.

  17. High Throughput Measurement of Locomotor Sensitization to Volatilized Cocaine in Drosophila melanogaster.

    Science.gov (United States)

    Filošević, Ana; Al-Samarai, Sabina; Andretić Waldowski, Rozi

    2018-01-01

    Drosophila melanogaster can be used to identify genes with novel functional roles in neuronal plasticity induced by repeated consumption of addictive drugs. Behavioral sensitization is a relatively simple behavioral output of plastic changes that occur in the brain after repeated exposures to drugs of abuse. The development of screening procedures for genes that control behavioral sensitization has stalled due to a lack of high-throughput behavioral tests that can be used in genetically tractable organism, such as Drosophila . We have developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. We validate FlyBong by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per) , Clock (Clk) , and cycle (cyc) . The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization

  18. High Throughput Measurement of Locomotor Sensitization to Volatilized Cocaine in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Ana Filošević

    2018-02-01

    Full Text Available Drosophila melanogaster can be used to identify genes with novel functional roles in neuronal plasticity induced by repeated consumption of addictive drugs. Behavioral sensitization is a relatively simple behavioral output of plastic changes that occur in the brain after repeated exposures to drugs of abuse. The development of screening procedures for genes that control behavioral sensitization has stalled due to a lack of high-throughput behavioral tests that can be used in genetically tractable organism, such as Drosophila. We have developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. We validate FlyBong by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per, Clock (Clk, and cycle (cyc. The locomotor sensitization that is present in timeless (tim and pigment dispersing factor (pdf mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor

  19. A high-throughput method for quantifying metabolically active yeast cells

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander

    2015-01-01

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved....... The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e.......g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts....

  20. DOVIS: A Tool for High-throughput Virtual Screening

    National Research Council Canada - National Science Library

    Jiang, Xiaohui; Kumar, Kamal; Wallqvist, Anders; Reifman, Jaques

    2007-01-01

    We developed a DOcking-based Virtual Screening (DO DOVIS) pipeline to predict how small molecules may interact with a given protein, so that we can rank a large database of molecules based on their predicted affinities to the protein...

  1. Efficient Management of High-Throughput Screening Libraries with SAVANAH

    DEFF Research Database (Denmark)

    List, Markus; Elnegaard, Marlene Pedersen; Schmidt, Steffen

    2017-01-01

    ) sample information from the library to experimental results from the assay plates. All results can be exported to the R statistical environment or piped into HiTSeekR (http://hitseekr.compbio.sdu.dk) for comprehensive follow-up analyses. In summary, SAVANAH supports the HTS community in managing...... for such screens are molecular libraries, that is, microtiter plates with solubilized reagents such as siRNAs, shRNAs, miRNA inhibitors or mimics, and sgRNAs, or small compounds, that is, drugs. These reagents are typically condensed to provide enough material for covering several screens. Library plates thus need...... to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects...

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

    Science.gov (United States)

    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.

  3. Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

    Directory of Open Access Journals (Sweden)

    Kensy Frank

    2009-06-01

    Full Text Available Abstract Background An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given. Results Bacterial and yeast biomass concentrations of up to 50 g/L cell dry weight could be linearly correlated to scattered light intensities. In media screening, the BioLector could clearly demonstrate its potential for detecting different biomass and product yields and deducing specific growth rates for quantitatively evaluating media and nutrients. Growth inhibition due to inappropriate buffer conditions could be detected by reduced growth rates and a temporary increase in NADH fluorescence. GFP served very well as reporter protein for investigating the promoter regulation under different carbon sources in yeast strains. A clone screening of 90 different GFP-expressing Hansenula polymorpha clones depicted the broad distribution of growth behavior and an even stronger distribution in GFP expression. The importance of mass transfer conditions could be demonstrated by varying filling volumes of an E. coli culture in 96 well MTP. The different filling volumes cause a deviation in the culture growth and acidification both monitored via scattered light intensities and the fluorescence of a pH indicator, respectively. Conclusion The BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode

  4. High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

    Science.gov (United States)

    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

  5. High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

    Science.gov (United States)

    Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

    2014-09-01

    Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. High-throughput measurement of polymer film thickness using optical dyes

    Science.gov (United States)

    Grunlan, Jaime C.; Mehrabi, Ali R.; Ly, Tien

    2005-01-01

    Optical dyes were added to polymer solutions in an effort to create a technique for high-throughput screening of dry polymer film thickness. Arrays of polystyrene films, cast from a toluene solution, containing methyl red or solvent green were used to demonstrate the feasibility of this technique. Measurements of the peak visible absorbance of each film were converted to thickness using the Beer-Lambert relationship. These absorbance-based thickness calculations agreed within 10% of thickness measured using a micrometer for polystyrene films that were 10-50 µm. At these thicknesses it is believed that the absorbance values are actually more accurate. At least for this solvent-based system, thickness was shown to be accurately measured in a high-throughput manner that could potentially be applied to other equivalent systems. Similar water-based films made with poly(sodium 4-styrenesulfonate) dyed with malachite green oxalate or congo red did not show the same level of agreement with the micrometer measurements. Extensive phase separation between polymer and dye resulted in inflated absorbance values and calculated thickness that was often more than 25% greater than that measured with the micrometer. Only at thicknesses below 15 µm could reasonable accuracy be achieved for the water-based films.

  7. High Throughput PBTK: Open-Source Data and Tools for ...

    Science.gov (United States)

    Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

  8. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

    Directory of Open Access Journals (Sweden)

    Bottomley Stephen P

    2006-03-01

    Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput

  9. Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

    Science.gov (United States)

    Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

    2015-03-01

    The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

  10. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  11. Zebrafish: A marvel of high-throughput biology for 21st century toxicology.

    Science.gov (United States)

    Bugel, Sean M; Tanguay, Robert L; Planchart, Antonio

    2014-09-07

    The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing.

  12. X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

    International Nuclear Information System (INIS)

    Kisselman, Gera; Qiu, Wei; Romanov, Vladimir; Thompson, Christine M.; Lam, Robert; Battaile, Kevin P.; Pai, Emil F.; Chirgadze, Nickolay Y.

    2011-01-01

    The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers

  13. X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

    Energy Technology Data Exchange (ETDEWEB)

    Kisselman, Gera; Qiu, Wei; Romanov, Vladimir; Thompson, Christine M.; Lam, Robert [Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario M5G 2C4 (Canada); Battaile, Kevin P. [Argonne National Laboratory, Argonne, Illinois 60439 (United States); Pai, Emil F.; Chirgadze, Nickolay Y., E-mail: nchirgad@uhnresearch.ca [Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario M5G 2C4 (Canada); University of Toronto, Toronto, Ontario M5S 1A8 (Canada)

    2011-06-01

    The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers

  14. Introducing Kansas Lava

    Science.gov (United States)

    Gill, Andy; Bull, Tristan; Kimmell, Garrin; Perrins, Erik; Komp, Ed; Werling, Brett

    Kansas Lava is a domain specific language for hardware description. Though there have been a number of previous implementations of Lava, we have found the design space rich, with unexplored choices. We use a direct (Chalmers style) specification of circuits, and make significant use of Haskell overloading of standard classes, leading to concise circuit descriptions. Kansas Lava supports both simulation (inside GHCi), and execution via VHDL, by having a dual shallow and deep embedding inside our Signal type. We also have a lightweight sized-type mechanism, allowing for MATLAB style matrix based specifications to be directly expressed in Kansas Lava.

  15. G protein-coupled receptor internalization assays in the high-content screening format.

    Science.gov (United States)

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  16. High throughput research and evaporation rate modeling for solvent screening for ethylcellulose barrier membranes in pharmaceutical applications.

    Science.gov (United States)

    Schoener, Cody A; Curtis-Fisk, Jaime L; Rogers, True L; Tate, Michael P

    2016-10-01

    Ethylcellulose is commonly dissolved in a solvent or formed into an aqueous dispersion and sprayed onto various dosage forms to form a barrier membrane to provide controlled release in pharmaceutical formulations. Due to the variety of solvents utilized in the pharmaceutical industry and the importance solvent can play on film formation and film strength it is critical to understand how solvent can influence these parameters. To systematically study a variety of solvent blends and how these solvent blends influence ethylcellulose film formation, physical and mechanical film properties and solution properties such as clarity and viscosity. Using high throughput capabilities and evaporation rate modeling, thirty-one different solvent blends composed of ethanol, isopropanol, acetone, methanol, and/or water were formulated, analyzed for viscosity and clarity, and narrowed down to four solvent blends. Brookfield viscosity, film casting, mechanical film testing and water permeation were also completed. High throughput analysis identified isopropanol/water, ethanol, ethanol/water and methanol/acetone/water as solvent blends with unique clarity and viscosity values. Evaporation rate modeling further rank ordered these candidates from excellent to poor interaction with ethylcellulose. Isopropanol/water was identified as the most suitable solvent blend for ethylcellulose due to azeotrope formation during evaporation, which resulted in a solvent-rich phase allowing the ethylcellulose polymer chains to remain maximally extended during film formation. Consequently, the highest clarity and most ductile films were formed. Employing high throughput capabilities paired with evaporation rate modeling allowed strong predictions between solvent interaction with ethylcellulose and mechanical film properties.

  17. Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Alquicer, Glenda; Sedlák, David; Byun, Y.; Pavlíček, Jiří; Stathis, M.; Rojas, C.; Slusher, B.; Pomper, M.G.; Bartůněk, Petr; Bařinka, Cyril

    2012-01-01

    Roč. 17, č. 8 (2012), s. 1030-1040 ISSN 1087-0571 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378050 Keywords : fluorescence polarization * glutamate carboxypeptidase II * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.207, year: 2012

  18. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

    Science.gov (United States)

    Semenova, Vera A; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. Published by Elsevier Ltd.

  19. AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

    Science.gov (United States)

    Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

    2017-09-29

    Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

  20. High throughput synthesis and characterization of the PbnNb2O5+n (0.5 < n < 4.1) system on a single chip

    International Nuclear Information System (INIS)

    Mirsaneh, Mehdi; Hayden, Brian E.; Miao Shu; Pokorny, Jan; Perini, Steve; Furman, Eugene; Lanagan, Michael T.; Ubic, Rick; Reaney, Ian M.

    2011-01-01

    Most high throughput studies focus on assessing the effect of composition within a single known fundamental structure type, such as perovskite. Here we demonstrate how high throughput synthesis and screening can be used to establish structure-property relations in the PbO-Nb 2 O 5 system, for which eight distinct fundamental structure types are known to exist. PbNb 4 O 11 , PbNb 2 O 6 and pyrochlore could be easily distinguished by X-ray diffraction (XRD). However, XRD was insensitive to distortions of the pyrochlore structure and instead Raman spectroscopy was utilized to determine changes in symmetry from cubic to rhombohedral as the PbO concentration increased. High throughput screening of the capacitance revealed permittivity (ε r ) maxima in the PbNb 4 O 11 (ε r = 700) and cubic pyrochlore phases (ε r = 450). The ε r of PbNb 4 O 11 has not to date been reported but the value for cubic pyrochlore is higher than that reported for bulk ceramics (ε r = 270). Initial high electric field studies also revealed exceptionally high tunability (four times that reported for bismuth zinc niobate-based pyrochlores) of the capacitance in the pyrochlore phase.