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Sample records for automated high-content screening

  1. Designs and concept reliance of a fully automated high-content screening platform.

    PubMed

    Radu, Constantin; Adrar, Hosna Sana; Alamir, Ab; Hatherley, Ian; Trinh, Trung; Djaballah, Hakim

    2012-10-01

    High-content screening (HCS) is becoming an accepted platform in academic and industry screening labs and does require slightly different logistics for execution. To automate our stand-alone HCS microscopes, namely, an alpha IN Cell Analyzer 3000 (INCA3000), originally a Praelux unit hooked to a Hudson Plate Crane with a maximum capacity of 50 plates per run, and the IN Cell Analyzer 2000 (INCA2000), in which up to 320 plates could be fed per run using the Thermo Fisher Scientific Orbitor, we opted for a 4 m linear track system harboring both microscopes, plate washer, bulk dispensers, and a high-capacity incubator allowing us to perform both live and fixed cell-based assays while accessing both microscopes on deck. Considerations in design were given to the integration of the alpha INCA3000, a new gripper concept to access the onboard nest, and peripheral locations on deck to ensure a self-reliant system capable of achieving higher throughput. The resulting system, referred to as Hestia, has been fully operational since the new year, has an onboard capacity of 504 plates, and harbors the only fully automated alpha INCA3000 unit in the world. PMID:22797489

  2. Designs and Concept-Reliance of a Fully Automated High Content Screening Platform

    PubMed Central

    Radu, Constantin; Adrar, Hosna Sana; Alamir, Ab; Hatherley, Ian; Trinh, Trung; Djaballah, Hakim

    2013-01-01

    High content screening (HCS) is becoming an accepted platform in academic and industry screening labs and does require slightly different logistics for execution. To automate our stand alone HCS microscopes, namely an alpha IN Cell Analyzer 3000 (INCA3000) originally a Praelux unit hooked to a Hudson Plate Crane with a maximum capacity of 50 plates per run; and the IN Cell Analyzer 2000 (INCA2000) where up to 320 plates could be fed per run using the Thermo Fisher Scientific Orbitor, we opted for a 4 meter linear track system harboring both microscopes, plate washer, bulk dispensers, and a high capacity incubator allowing us to perform both live and fixed cell based assays while accessing both microscopes on deck. Considerations in design were given to the integration of the alpha INCA3000, a new gripper concept to access the onboard nest, and peripheral locations on deck to ensure a self reliant system capable of achieving higher throughput. The resulting system, referred to as Hestia, has been fully operational since the New Year, has an onboard capacity of 504 plates, and harbors the only fully automated alpha INCA3000 unit in the World. PMID:22797489

  3. Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment

    PubMed Central

    Norton, John T.; Titus, Steven A.; Dexter, Dwayne; Austin, Christopher P.; Zheng, Wei; Huang, Sui

    2010-01-01

    All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)–fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure. PMID:19762548

  4. Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

    PubMed

    Gosai, Sager J; Kwak, Joon Hyeok; Luke, Cliff J; Long, Olivia S; King, Dale E; Kovatch, Kevin J; Johnston, Paul A; Shun, Tong Ying; Lazo, John S; Perlmutter, David H; Silverman, Gary A; Pak, Stephen C

    2010-01-01

    The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms. PMID:21103396

  5. Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

    PubMed

    Monjaret, François; Fernandes, Mathieu; Duchemin-Pelletier, Eve; Argento, Amelie; Degot, Sébastien; Young, Joanne

    2016-04-01

    Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications. PMID:26385905

  6. Computer vision for high content screening.

    PubMed

    Kraus, Oren Z; Frey, Brendan J

    2016-01-01

    High Content Screening (HCS) technologies that combine automated fluorescence microscopy with high throughput biotechnology have become powerful systems for studying cell biology and drug screening. These systems can produce more than 100 000 images per day, making their success dependent on automated image analysis. In this review, we describe the steps involved in quantifying microscopy images and different approaches for each step. Typically, individual cells are segmented from the background using a segmentation algorithm. Each cell is then quantified by extracting numerical features, such as area and intensity measurements. As these feature representations are typically high dimensional (>500), modern machine learning algorithms are used to classify, cluster and visualize cells in HCS experiments. Machine learning algorithms that learn feature representations, in addition to the classification or clustering task, have recently advanced the state of the art on several benchmarking tasks in the computer vision community. These techniques have also recently been applied to HCS image analysis. PMID:26806341

  7. An automated high-content screening image analysis pipeline for the identification of selective autophagic inducers in human cancer cell lines.

    PubMed

    Kriston-Vizi, Janos; Lim, Ching Aeng; Condron, Peter; Chua, Kelvin; Wasser, Martin; Flotow, Horst

    2010-08-01

    Automated image processing is a critical and often rate-limiting step in high-content screening (HCS) workflows. The authors describe an open-source imaging-statistical framework with emphasis on segmentation to identify novel selective pharmacological inducers of autophagy. They screened a human alveolar cancer cell line and evaluated images by both local adaptive and global segmentation. At an individual cell level, region-growing segmentation was compared with histogram-derived segmentation. The histogram approach allowed segmentation of a sporadic-pattern foreground and hence the attainment of pixel-level precision. Single-cell phenotypic features were measured and reduced after assessing assay quality control. Hit compounds selected by machine learning corresponded well to the subjective threshold-based hits determined by expert analysis. Histogram-derived segmentation displayed robustness against image noise, a factor adversely affecting region growing segmentation. PMID:20547532

  8. High-content screening for biofilm assays.

    PubMed

    Peng, Fubing; Hoek, Eric M V; Damoiseaux, Robert

    2010-08-01

    The authors describe a novel high-throughput screening platform that provides rapid, reliable, quantitative assessment of biofilm formation and removal on engineered surfaces. Unlike traditional biofilm assays based on plate readers, this assay platform is based on high-content screening, which allows for multiplexing to simultaneously quantify the number of bacterial adhesions per unit area and the viability of adhered cells using fluorescent dye combinations. This platform is fully automated and has a throughput of more than 10,000 wells per day. The authors used this platform to examine the influence of different assay buffer systems on bacterial adhesion, viability, and removal on cross-linked polyvinyl alcohol coating films synthesized directly onto the bottoms of 384-well plates. The results indicated that water chemistry, bacteria cell type, and film chemistry combine to govern biofilm formation. In general, both reversible and irreversible bacterial adhesion increased with the extent of cross-linking in coating films, which correlates strongly with coating film cross-linking degree and hydrophobicity, which is closely related. The high-throughput platform offers a powerful tool for rapid evaluation of fouling-resistant coating films in addition to elucidation of fundamental mechanisms governing bacterial adhesion. PMID:20639506

  9. Developmental toxicity assay using high content screening of zebrafish embryos

    PubMed Central

    Lantz-McPeak, Susan; Guo, Xiaoqing; Cuevas, Elvis; Dumas, Melanie; Newport, Glenn D.; Ali, Syed F.; Paule, Merle G.; Kanungo, Jyotshna

    2016-01-01

    Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. PMID:24871937

  10. High-Content Screening for Quantitative Cell Biology.

    PubMed

    Mattiazzi Usaj, Mojca; Styles, Erin B; Verster, Adrian J; Friesen, Helena; Boone, Charles; Andrews, Brenda J

    2016-08-01

    High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future. PMID:27118708

  11. [High content screening in chemical biology: overview and main challenges].

    PubMed

    Brodin, Priscille; DelNery, Elaine; Soleilhac, Emmanuelle

    2015-02-01

    The last two decades have seen the development of high content screening (HCS) methodology and its adaptation for the evaluation of small molecules as drug candidates or their use as chemical tools for research purpose. HCS was initially set-up for the understanding of the mechanism of action of compounds by testing them on cell based-assays for pharmacological and toxicological studies. Since the last decade, the use of HCS has been extended to academic research laboratories and this technology has become the starting point for numerous projects aiming at the identification of molecular targets and cellular pathways for a given disease on which novel type of drugs could act. This screening approach relies on image capture of fluorescently labeled cells therefore generating a large amount of data that must be handled by appropriate automated image analysis methods and storage instrumentation. These latter in addition to the integration and data sharing are current challenges that HCS must still tackle. PMID:25744266

  12. Application of Imaging-Based Assays in Microplate Formats for High-Content Screening.

    PubMed

    Fogel, Adam I; Martin, Scott E; Hasson, Samuel A

    2016-01-01

    The use of multiparametric microscopy-based screens with automated analysis has enabled the large-scale study of biological phenomena that are currently not measurable by any other method. Collectively referred to as high-content screening (HCS), or high-content analysis (HCA), these methods rely on an expanding array of imaging hardware and software automation. Coupled with an ever-growing amount of diverse chemical matter and functional genomic tools, HCS has helped open the door to a new frontier of understanding cell biology through phenotype-driven screening. With the ability to interrogate biology on a cell-by-cell basis in highly parallel microplate-based platforms, the utility of HCS continues to grow as advancements are made in acquisition speed, model system complexity, data management, and analysis systems. This chapter uses an example of screening for genetic factors regulating mitochondrial quality control to exemplify the practical considerations in developing and executing high-content campaigns. PMID:27317002

  13. Automated Groundwater Screening

    SciTech Connect

    Taylor, Glenn A.; Collard, Leonard, B.

    2005-10-31

    The Automated Intruder Analysis has been extended to include an Automated Ground Water Screening option. This option screens 825 radionuclides while rigorously applying the National Council on Radiation Protection (NCRP) methodology. An extension to that methodology is presented to give a more realistic screening factor for those radionuclides which have significant daughters. The extension has the promise of reducing the number of radionuclides which must be tracked by the customer. By combining the Automated Intruder Analysis with the Automated Groundwater Screening a consistent set of assumptions and databases is used. A method is proposed to eliminate trigger values by performing rigorous calculation of the screening factor thereby reducing the number of radionuclides sent to further analysis. Using the same problem definitions as in previous groundwater screenings, the automated groundwater screening found one additional nuclide, Ge-68, which failed the screening. It also found that 18 of the 57 radionuclides contained in NCRP Table 3.1 failed the screening. This report describes the automated groundwater screening computer application.

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

    PubMed

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

    2015-09-01

    High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. PMID:25548139

  15. Metadata management for high content screening in OMERO

    PubMed Central

    Li, Simon; Besson, Sébastien; Blackburn, Colin; Carroll, Mark; Ferguson, Richard K.; Flynn, Helen; Gillen, Kenneth; Leigh, Roger; Lindner, Dominik; Linkert, Melissa; Moore, William J.; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Allan, Chris; Burel, Jean-Marie; Moore, Josh; Swedlow, Jason R.

    2016-01-01

    High content screening (HCS) experiments create a classic data management challenge—multiple, large sets of heterogeneous structured and unstructured data, that must be integrated and linked to produce a set of “final” results. These different data include images, reagents, protocols, analytic output, and phenotypes, all of which must be stored, linked and made accessible for users, scientists, collaborators and where appropriate the wider community. The OME Consortium has built several open source tools for managing, linking and sharing these different types of data. The OME Data Model is a metadata specification that supports the image data and metadata recorded in HCS experiments. Bio-Formats is a Java library that reads recorded image data and metadata and includes support for several HCS screening systems. OMERO is an enterprise data management application that integrates image data, experimental and analytic metadata and makes them accessible for visualization, mining, sharing and downstream analysis. We discuss how Bio-Formats and OMERO handle these different data types, and how they can be used to integrate, link and share HCS experiments in facilities and public data repositories. OME specifications and software are open source and are available at https://www.openmicroscopy.org. PMID:26476368

  16. Metadata management for high content screening in OMERO.

    PubMed

    Li, Simon; Besson, Sébastien; Blackburn, Colin; Carroll, Mark; Ferguson, Richard K; Flynn, Helen; Gillen, Kenneth; Leigh, Roger; Lindner, Dominik; Linkert, Melissa; Moore, William J; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Allan, Chris; Burel, Jean-Marie; Moore, Josh; Swedlow, Jason R

    2016-03-01

    High content screening (HCS) experiments create a classic data management challenge-multiple, large sets of heterogeneous structured and unstructured data, that must be integrated and linked to produce a set of "final" results. These different data include images, reagents, protocols, analytic output, and phenotypes, all of which must be stored, linked and made accessible for users, scientists, collaborators and where appropriate the wider community. The OME Consortium has built several open source tools for managing, linking and sharing these different types of data. The OME Data Model is a metadata specification that supports the image data and metadata recorded in HCS experiments. Bio-Formats is a Java library that reads recorded image data and metadata and includes support for several HCS screening systems. OMERO is an enterprise data management application that integrates image data, experimental and analytic metadata and makes them accessible for visualization, mining, sharing and downstream analysis. We discuss how Bio-Formats and OMERO handle these different data types, and how they can be used to integrate, link and share HCS experiments in facilities and public data repositories. OME specifications and software are open source and are available at https://www.openmicroscopy.org. PMID:26476368

  17. Automated macromolecular crystallization screening

    DOEpatents

    Segelke, Brent W.; Rupp, Bernhard; Krupka, Heike I.

    2005-03-01

    An automated macromolecular crystallization screening system wherein a multiplicity of reagent mixes are produced. A multiplicity of analysis plates is produced utilizing the reagent mixes combined with a sample. The analysis plates are incubated to promote growth of crystals. Images of the crystals are made. The images are analyzed with regard to suitability of the crystals for analysis by x-ray crystallography. A design of reagent mixes is produced based upon the expected suitability of the crystals for analysis by x-ray crystallography. A second multiplicity of mixes of the reagent components is produced utilizing the design and a second multiplicity of reagent mixes is used for a second round of automated macromolecular crystallization screening. In one embodiment the multiplicity of reagent mixes are produced by a random selection of reagent components.

  18. Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

    PubMed Central

    Martinez, Eric; Cantet, Franck; Bonazzi, Matteo

    2015-01-01

    Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches. PMID:25992686

  19. A high-content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling.

    PubMed

    Saydmohammed, Manush; Vollmer, Laura L; Onuoha, Ezenwa Obi; Vogt, Andreas; Tsang, Michael

    2011-09-01

    Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF. PMID:21932436

  20. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

    PubMed

    Leet, Jessica K; Lindberg, Casey D; Bassett, Luke A; Isales, Gregory M; Yozzo, Krystle L; Raftery, Tara D; Volz, David C

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism

  1. High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia

    PubMed Central

    Leet, Jessica K.; Lindberg, Casey D.; Bassett, Luke A.; Isales, Gregory M.; Yozzo, Krystle L.; Raftery, Tara D.; Volz, David C.

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis

  2. Online Phenotype Discovery in High-Content RNAi Screens using Gap Statistics

    NASA Astrophysics Data System (ADS)

    Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen T. C.

    2007-11-01

    Discovering and identifying novel phenotypes from images inputting online is a major challenge in high-content RNA interference (RNAi) screens. Discovered phenotypes should be visually distinct from existing ones and make biological sense. An online phenotype discovery method featuring adaptive phenotype modeling and iterative cluster merging using gap statistics is proposed. The method works well on discovering new phenotypes adaptively when applied to both of synthetic data sets and RNAi high content screen (HCS) images with ground truth labels.

  3. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos.

    PubMed

    Raftery, Tara D; Isales, Gregory M; Yozzo, Krystle L; Volz, David C

    2014-01-01

    Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants. PMID:24328182

  4. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells.

    PubMed

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C; Brauer, Patrick M; Zúñiga-Pflücker, Juan C; Leber, Brian; Spaner, David E; Andrews, David W

    2016-08-18

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax. PMID:27297795

  5. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells

    PubMed Central

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C.; Brauer, Patrick M.; Zúñiga-Pflücker, Juan C.; Leber, Brian; Spaner, David E.

    2016-01-01

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax. PMID:27297795

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

    EPA Science Inventory

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

  7. High content screening of ToxCast compounds using Vala Sciences’ complex cell culturing systems (SOT)

    EPA Science Inventory

    US EPA’s ToxCast research program evaluates bioactivity for thousands of chemicals utilizing high-throughput screening assays to inform chemical testing decisions. Vala Sciences provides high content, multiplexed assays that utilize quantitative cell-based digital image analysis....

  8. Automation of antimicrobial activity screening.

    PubMed

    Forry, Samuel P; Madonna, Megan C; López-Pérez, Daneli; Lin, Nancy J; Pasco, Madeleine D

    2016-03-01

    Manual and automated methods were compared for routine screening of compounds for antimicrobial activity. Automation generally accelerated assays and required less user intervention while producing comparable results. Automated protocols were validated for planktonic, biofilm, and agar cultures of the oral microbe Streptococcus mutans that is commonly associated with tooth decay. Toxicity assays for the known antimicrobial compound cetylpyridinium chloride (CPC) were validated against planktonic, biofilm forming, and 24 h biofilm culture conditions, and several commonly reported toxicity/antimicrobial activity measures were evaluated: the 50 % inhibitory concentration (IC50), the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC). Using automated methods, three halide salts of cetylpyridinium (CPC, CPB, CPI) were rapidly screened with no detectable effect of the counter ion on antimicrobial activity. PMID:26970766

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

    PubMed Central

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

    2014-01-01

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

  10. Mineotaur: a tool for high-content microscopy screen sharing and visual analytics.

    PubMed

    Antal, Bálint; Chessel, Anatole; Carazo Salas, Rafael E

    2015-01-01

    High-throughput/high-content microscopy-based screens are powerful tools for functional genomics, yielding intracellular information down to the level of single-cells for thousands of genotypic conditions. However, accessing their data requires specialized knowledge and most often that data is no longer analyzed after initial publication. We describe Mineotaur ( http://www.mineotaur.org ), a open-source, downloadable web application that allows easy online sharing and interactive visualisation of large screen datasets, facilitating their dissemination and further analysis, and enhancing their impact. PMID:26679168

  11. Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines.

    PubMed

    Kang, Jungseog; Hsu, Chien-Hsiang; Wu, Qi; Liu, Shanshan; Coster, Adam D; Posner, Bruce A; Altschuler, Steven J; Wu, Lani F

    2016-01-01

    High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them--the ORACL--best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy by means of orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power. PMID:26655497

  12. Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines

    PubMed Central

    Wu, Qi; Liu, Shanshan; Coster, Adam D.; Posner, Bruce A.; Altschuler, Steven J.; Wu, Lani F.

    2015-01-01

    High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them—the ORACL—best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy via orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power. PMID:26655497

  13. High Content Screening in Zebrafish Speeds up Hazard Ranking of Transition Metal Oxide Nanoparticles

    PubMed Central

    Lin, Sijie; Zhao, Yan; Xia, Tian; Meng, Huan; Zhaoxia, Ji; Liu, Rong; George, Saji; Xiong, Sijing; Wang, Xiang; Zhang, Haiyuan; Pokhrel, Suman; Mädler, Lutz; Damoiseaux, Robert; Lin, Shuo; Nel, Andre E.

    2014-01-01

    Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO and Co3O4) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependant hatching interference in the embryos, with the exception of Co3O4 which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn2+. This hypothesis is based on the presence of metal–sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA sensitive. In conclusion, we demonstrate that high content imaging of embryo development, morphological abnormalities and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo. PMID:21851096

  14. Trait Variability of Cancer Cells Quantified by High-Content Automated Microscopy of Single Cells

    PubMed Central

    Quaranta, Vito; Tyson, Darren R.; Garbett, Shawn P.; Weidow, Brandy; Harris, Mark P.; Georgescu, Walter

    2010-01-01

    Mapping quantitative cell traits (QCT) to underlying molecular defects is a central challenge in cancer research because heterogeneity at all biological scales, from genes to cells to populations, is recognized as the main driver of cancer progression and treatment resistance. A major roadblock to a multiscale framework linking cell to signaling to genetic cancer heterogeneity is the dearth of large-scale, single-cell data on QCT-such as proliferation, death sensitivity, motility, metabolism, and other hallmarks of cancer. High-volume single-cell data can be used to represent cell-to-cell genetic and nongenetic QCT variability in cancer cell populations as averages, distributions, and statistical subpopulations. By matching the abundance of available data on cancer genetic and molecular variability, QCT data should enable quantitative mapping of phenotype to genotype in cancer. This challenge is being met by high-content automated microscopy (HCAM), based on the convergence of several technologies including computerized microscopy, image processing, computation, and heterogeneity science. In this chapter, we describe an HCAM workflow that can be set up in a medium size interdisciplinary laboratory, and its application to produce high-throughput QCT data for cancer cell motility and proliferation. This type of data is ideally suited to populate cell-scale computational and mathematical models of cancer progression for quantitatively and predictively evaluating cancer drug discovery and treatment. PMID:19897088

  15. High Content Screening for Modulators of Cardiac Differentiation in Human Pluripotent Stem Cells

    PubMed Central

    Spiering, Sean; Davidovics, Herman; Bushway, Paul J.; Mercola, Mark; Willems, Erik

    2016-01-01

    Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation. PMID:25618335

  16. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    PubMed

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

  17. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

    PubMed Central

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

  18. Gamma-H2AX foci counting: image processing and control software for high-content screening

    NASA Astrophysics Data System (ADS)

    Barber, P. R.; Locke, R. J.; Pierce, G. P.; Rothkamm, K.; Vojnovic, B.

    2007-02-01

    Phosphorylation of the chromatin protein H2AX (forming γH2AX) is implicated in the repair of DNA double strand breaks (DSB's); a large number of H2AX molecules become phosphorylated at the sites of DSB's. Fluorescent staining of the cell nuclei for γH2AX, via an antibody, visualises the formation of these foci, allowing the quantification of DNA DSB's and forming the basis for a sensitive biological dosimeter of ionising radiation. We describe an automated fluorescence microscopy system, including automated image processing, to count γH2AX foci. The image processing is performed by a Hough transform based algorithm, CHARM, which has wide applicability for the detection and analysis of cells and cell colonies. This algorithm and its applications for cell nucleus and foci detection will be described. The system also relies heavily on robust control software, written using multi-threaded cbased modules in LabWindows/CVI that adapt to the timing requirements of a particular experiment for optimised slide/plate scanning and mosaicing, making use of modern multi-core processors. The system forms the basis of a general purpose high-content screening platform with wide ranging applications in live and fixed cell imaging and tissue micro arrays, that in future, can incorporate spectrally and time-resolved information.

  19. Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line

    PubMed Central

    Alonso-Padilla, Julio; Cotillo, Ignacio; Presa, Jesús L.; Cantizani, Juan; Peña, Imanol; Bardera, Ana I.; Martín, Jose J.; Rodriguez, Ana

    2015-01-01

    Background Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. Methodology/Principal Findings Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. Conclusions/Significance We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite. PMID:25615687

  20. Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

    PubMed Central

    Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

    2010-01-01

    We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

  1. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening.

    PubMed

    Daniel, James A; Malladi, Chandra S; Kettle, Emma; McCluskey, Adam; Robinson, Phillip J

    2012-08-01

    Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis. PMID:22767087

  2. Discovery of New Anti-Schistosomal Hits by Integration of QSAR-Based Virtual Screening and High Content Screening.

    PubMed

    Neves, Bruno J; Dantas, Rafael F; Senger, Mario R; Melo-Filho, Cleber C; Valente, Walter C G; de Almeida, Ana C M; Rezende-Neto, João M; Lima, Elid F C; Paveley, Ross; Furnham, Nicholas; Muratov, Eugene; Kamentsky, Lee; Carpenter, Anne E; Braga, Rodolpho C; Silva-Junior, Floriano P; Andrade, Carolina Horta

    2016-08-11

    Schistosomiasis is a debilitating neglected tropical disease, caused by flatworms of Schistosoma genus. The treatment relies on a single drug, praziquantel (PZQ), making the discovery of new compounds extremely urgent. In this work, we integrated QSAR-based virtual screening (VS) of Schistosoma mansoni thioredoxin glutathione reductase (SmTGR) inhibitors and high content screening (HCS) aiming to discover new antischistosomal agents. Initially, binary QSAR models for inhibition of SmTGR were developed and validated using the Organization for Economic Co-operation and Development (OECD) guidance. Using these models, we prioritized 29 compounds for further testing in two HCS platforms based on image analysis of assay plates. Among them, 2-[2-(3-methyl-4-nitro-5-isoxazolyl)vinyl]pyridine and 2-(benzylsulfonyl)-1,3-benzothiazole, two compounds representing new chemical scaffolds have activity against schistosomula and adult worms at low micromolar concentrations and therefore represent promising antischistosomal hits for further hit-to-lead optimization. PMID:27396732

  3. DetecTiff: a novel image analysis routine for high-content screening microscopy.

    PubMed

    Gilbert, Daniel F; Meinhof, Till; Pepperkok, Rainer; Runz, Heiko

    2009-09-01

    In this article, the authors describe the image analysis software DetecTiff, which allows fully automated object recognition and quantification from digital images. The core module of the LabView-based routine is an algorithm for structure recognition that employs intensity thresholding and size-dependent particle filtering from microscopic images in an iterative manner. Detected structures are converted into templates, which are used for quantitative image analysis. DetecTiff enables processing of multiple detection channels and provides functions for template organization and fast interpretation of acquired data. The authors demonstrate the applicability of DetecTiff for automated analysis of cellular uptake of fluorescence-labeled low-density lipoproteins as well as diverse other image data sets from a variety of biomedical applications. Moreover, the performance of DetecTiff is compared with preexisting image analysis tools. The results show that DetecTiff can be applied with high consistency for automated quantitative analysis of image data (e.g., from large-scale functional RNAi screening projects). PMID:19641223

  4. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    PubMed

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  5. A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3.

    PubMed

    Lahtela, Jenni; Corson, Laura B; Hemmes, Annabrita; Brauer, Matthew J; Koopal, Sonja; Lee, James; Hunsaker, Thomas L; Jackson, Peter K; Verschuren, Emmy W

    2013-02-15

    Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated β-galactosidase, DNA damage and p53 or p16 (INK4a) expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated. PMID:23324396

  6. Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

    PubMed Central

    Wu, Chaohong; Schulte, Joost; Sepp, Katharine J.; Littleton, J. Troy

    2011-01-01

    Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model. PMID:20405243

  7. A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

    PubMed Central

    Lahtela, Jenni; Corson, Laura B.; Hemmes, Annabrita; Brauer, Matthew J.; Koopal, Sonja; Lee, James; Hunsaker, Thomas L.; Jackson, Peter K.; Verschuren, Emmy W.

    2013-01-01

    Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated β-galactosidase, DNA damage and p53 or p16INK4a expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated. PMID:23324396

  8. High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device.

    PubMed

    Kim, Min Jung; Lee, Su Chul; Pal, Sukdeb; Han, Eunyoung; Song, Joon Myong

    2011-01-01

    Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity. PMID:21060932

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

    EPA Science Inventory

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

  10. FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

    PubMed Central

    Kumar, Sunil; Alibhai, Dominic; Margineanu, Anca; Laine, Romain; Kennedy, Gordon; McGinty, James; Warren, Sean; Kelly, Douglas; Alexandrov, Yuriy; Munro, Ian; Talbot, Clifford; Stuckey, Daniel W; Kimberly, Christopher; Viellerobe, Bertrand; Lacombe, Francois; Lam, Eric W-F; Taylor, Harriet; Dallman, Margaret J; Stamp, Gordon; Murray, Edward J; Stuhmeier, Frank; Sardini, Alessandro; Katan, Matilda; Elson, Daniel S; Neil, Mark A A; Dunsby, Chris; French, Paul M W

    2011-01-01

    A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated. PMID:21337485

  11. The myImageAnalysis Project: A Web-Based Application for High-Content Screening

    PubMed Central

    Szafran, Adam T.

    2014-01-01

    Abstract A major challenge faced by screening centers developing image-based assays is the wide range of assays needed compared to the limited resources that are available to effectively analyze and manage them. To overcome this limitation, we have developed the web-based myImageAnalysis (mIA) application, integrated with an open database connectivity compliant database and powered by Pipeline Pilot (PLP) that incorporates dataset tracking, scheduling and archiving, image analysis, and data reporting. For system administrators, mIA provides automated methods for managing and archiving data. For the biologist, this application allows those without any programming or image analysis experience to quickly develop, validate, and share results of complex image-based assays. Further, the structure of the application within PLP allows those with experience in PLP programming to easily add additional analysis tools as required. The tools within mIA allow users to assess basic (cell count, protein per cell, protein subcellular localization) and more advanced (engineered cell lines analysis, cell toxicity) biological image-based assays that employ advanced statistics and provides key assay performance metrics. PMID:24547743

  12. The myImageAnalysis project: a web-based application for high-content screening.

    PubMed

    Szafran, Adam T; Mancini, Michael A

    2014-01-01

    A major challenge faced by screening centers developing image-based assays is the wide range of assays needed compared to the limited resources that are available to effectively analyze and manage them. To overcome this limitation, we have developed the web-based myImageAnalysis (mIA) application, integrated with an open database connectivity compliant database and powered by Pipeline Pilot (PLP) that incorporates dataset tracking, scheduling and archiving, image analysis, and data reporting. For system administrators, mIA provides automated methods for managing and archiving data. For the biologist, this application allows those without any programming or image analysis experience to quickly develop, validate, and share results of complex image-based assays. Further, the structure of the application within PLP allows those with experience in PLP programming to easily add additional analysis tools as required. The tools within mIA allow users to assess basic (cell count, protein per cell, protein subcellular localization) and more advanced (engineered cell lines analysis, cell toxicity) biological image-based assays that employ advanced statistics and provides key assay performance metrics. PMID:24547743

  13. Cell-Based Assay Design for High-Content Screening of Drug Candidates.

    PubMed

    Nierode, Gregory; Kwon, Paul S; Dordick, Jonathan S; Kwon, Seok-Joon

    2016-02-01

    To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner. PMID:26428732

  14. High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes

    PubMed Central

    Grimm, Fabian Alexander; Iwata, Yasuhiro; Sirenko, Oksana; Bittner, Michael

    2015-01-01

    Abstract Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity. PMID:26539751

  15. A high-content screening platform with fluorescent chemical probes for the discovery of first-in-class therapeutics.

    PubMed

    Jo, Ala; Jung, Jinjoo; Kim, Eunha; Park, Seung Bum

    2016-06-14

    Phenotypic screening has emerged as a promising approach to discover novel first-in-class therapeutic agents. Rapid advances in phenotypic screening systems facilitate a high-throughput unbiased evaluation of compound libraries. However, limited sets of phenotypic changes are utilized in high-content screening, which require extensive genetic engineering. Therefore, it is critical to develop new chemical probes that can reflect phenotypic changes in any type of cells, especially primary cells, tissues, and organisms. Herein, we introduce our continuous efforts in the development of fluorescent bioprobes and their application to phenotypic screening. In addition, we emphasize the importance of the phenotype-based approach in conjunction with target identification at an early stage of research to accelerate the discovery of therapeutics with new modes of action. PMID:27166145

  16. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    SciTech Connect

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H.; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content

  17. Automated screening of pigmentary skin neoplasms

    NASA Astrophysics Data System (ADS)

    Kudrin, Konstantin G.; Matorin, Oleg V.; Reshetov, Igor V.

    2015-01-01

    We have analysed the clinical symptoms and the malignization signs of pigmented skin neoplasms. We have estimated the complex of clinical parameters which could be measured for the purpose of skin screening diagnostic via digital image processing. Allowable errors of clinical parameter characterization have been calculated, and the origin of these errors has been discussed. Proposed technique for automated screening of pigmentary skin neoplasms should become an effective tool for early skin diagnostics.

  18. Identification of small-molecule HSF1 amplifiers by high content screening in protection of cells from stress induced injury.

    PubMed

    Zhang, Bin; Au, Qingyan; Yoon, Il Sang; Tremblay, Marie-Helene; Yip, Gary; Zhou, Yuefen; Barber, Jack R; Ng, Shi Chung

    2009-12-18

    Small molecule amplifiers of heat shock response have shown promising results in rescuing stress related injury through chaperone amplification. Herein, we report the results of a high content target-based primary screening of several known bioactive libraries. Screening resulted in the identification of three potent gedunin derivatives and a sappanone A derivative. Western blot results confirmed compound-induced activation of HSF1 and increased expression level of HSP70. These compounds rescued cells from cell death caused by proteasome inhibitor MG-132 and RNAi knockdown of HSF1 significantly reversed the cytoprotective effects, confirming an HSF1-dependent mechanism of action. These HSF1 amplifiers were tested in two mammalian cell based models of Huntington's disease (HD) and found to improve survival. Therefore, these screening hits may have therapeutic potential for HD and possibly other protein conformational disorders. PMID:19852939

  19. High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.

    PubMed

    Green, Jon M; Metz, Jeremy; Lee, Okhyun; Trznadel, Maciej; Takesono, Aya; Brown, A Ross; Owen, Stewart F; Kudoh, Tetsuhiro; Tyler, Charles R

    2016-06-21

    Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates. PMID:27227508

  20. Neuronal models for evaluation of proliferation in vitro using high content screening

    EPA Science Inventory

    In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental proc...

  1. A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

    PubMed

    Kubben, Nard; Brimacombe, Kyle R; Donegan, Megan; Li, Zhuyin; Misteli, Tom

    2016-03-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery. PMID:26341717

  2. Robust Classification of Small-Molecule Mechanism of Action Using a Minimalist High-Content Microscopy Screen and Multidimensional Phenotypic Trajectory Analysis

    PubMed Central

    Twarog, Nathaniel R.; Low, Jonathan A.; Currier, Duane G.; Miller, Greg; Chen, Taosheng; Shelat, Anang A.

    2016-01-01

    Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2’-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate

  3. Novel High Content Screen Detects Compounds That Promote Neurite Regeneration from Cochlear Spiral Ganglion Neurons.

    PubMed

    Whitlon, Donna S; Grover, Mary; Dunne, Sara F; Richter, Sonja; Luan, Chi-Hao; Richter, Claus-Peter

    2015-01-01

    The bipolar spiral ganglion neurons (SGN) carry sound information from cochlear hair cells to the brain. After noise, antibiotic or toxic insult to the cochlea, damage to SGN and/or hair cells causes hearing impairment. Damage ranges from fiber and synapse degeneration to dysfunction and loss of cells. New interventions to regenerate peripheral nerve fibers could help reestablish transfer of auditory information from surviving or regenerated hair cells or improve results from cochlear implants, but the biochemical mechanisms to target are largely unknown. Presently, no drugs exist that are FDA approved to stimulate the regeneration of SGN nerve fibers. We designed an original phenotypic assay to screen 440 compounds of the NIH Clinical Collection directly on dissociated mouse spiral ganglia. The assay detected one compound, cerivastatin, that increased the length of regenerating neurites. The effect, mimicked by other statins at different optimal concentrations, was blocked by geranylgeraniol. These results demonstrate the utility of screening small compound libraries on mixed cultures of dissociated primary ganglia. The success of this screen narrows down a moderately sized library to a single compound which can be elevated to in-depth in vivo studies, and highlights a potential new molecular pathway for targeting of hearing loss drugs. PMID:26521685

  4. Novel High Content Screen Detects Compounds That Promote Neurite Regeneration from Cochlear Spiral Ganglion Neurons

    PubMed Central

    Whitlon, Donna S.; Grover, Mary; Dunne, Sara F.; Richter, Sonja; Luan, Chi-Hao; Richter, Claus-Peter

    2015-01-01

    The bipolar spiral ganglion neurons (SGN) carry sound information from cochlear hair cells to the brain. After noise, antibiotic or toxic insult to the cochlea, damage to SGN and/or hair cells causes hearing impairment. Damage ranges from fiber and synapse degeneration to dysfunction and loss of cells. New interventions to regenerate peripheral nerve fibers could help reestablish transfer of auditory information from surviving or regenerated hair cells or improve results from cochlear implants, but the biochemical mechanisms to target are largely unknown. Presently, no drugs exist that are FDA approved to stimulate the regeneration of SGN nerve fibers. We designed an original phenotypic assay to screen 440 compounds of the NIH Clinical Collection directly on dissociated mouse spiral ganglia. The assay detected one compound, cerivastatin, that increased the length of regenerating neurites. The effect, mimicked by other statins at different optimal concentrations, was blocked by geranylgeraniol. These results demonstrate the utility of screening small compound libraries on mixed cultures of dissociated primary ganglia. The success of this screen narrows down a moderately sized library to a single compound which can be elevated to in-depth in vivo studies, and highlights a potential new molecular pathway for targeting of hearing loss drugs. PMID:26521685

  5. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

    PubMed

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-05-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  6. High-content screening identifies a role for Na(+) channels in insulin production.

    PubMed

    Szabat, Marta; Modi, Honey; Ramracheya, Reshma; Girbinger, Vroni; Chan, Forson; Lee, Jason T C; Piske, Micah; Kamal, Sepehr; Carol Yang, Yu Hsuan; Welling, Andrea; Rorsman, Patrik; Johnson, James D

    2015-12-01

    Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production. PMID:27019722

  7. High-content screening identifies a role for Na+ channels in insulin production

    PubMed Central

    Szabat, Marta; Modi, Honey; Ramracheya, Reshma; Girbinger, Vroni; Chan, Forson; Lee, Jason T. C.; Piske, Micah; Kamal, Sepehr; Carol Yang, Yu Hsuan; Welling, Andrea; Rorsman, Patrik; Johnson, James D.

    2015-01-01

    Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production. PMID:27019722

  8. High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

    PubMed Central

    Marescotti, Diego; Gonzalez Suarez, Ignacio; Acali, Stefano; Johne, Stephanie; Laurent, Alexandra; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C.

    2016-01-01

    Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2 For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound's toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC. Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (< 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator. This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents. PMID:27228213

  9. High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC).

    PubMed

    Marescotti, Diego; Gonzalez Suarez, Ignacio; Acali, Stefano; Johne, Stephanie; Laurent, Alexandra; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C

    2016-01-01

    Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA). For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound's toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC. Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (< 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator. This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents. PMID:27228213

  10. A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion

    PubMed Central

    Circu, Magdalena L.; Dykes, Samantha S.; Carroll, Jennifer; Kelly, Kinsey; Galiano, Floyd; Greer, Adam; Cardelli, James; El-Osta, Hazem

    2016-01-01

    Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear

  11. Identification of modulators of autophagic flux in an image-based high content siRNA screen.

    PubMed

    Hale, Christopher M; Cheng, Qingwen; Ortuno, Danny; Huang, Ming; Nojima, Dana; Kassner, Paul D; Wang, Songli; Ollmann, Michael M; Carlisle, Holly J

    2016-04-01

    Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration. PMID:27050463

  12. Automated Propulsion Data Screening demonstration system

    NASA Astrophysics Data System (ADS)

    Hoyt, W. Andes; Choate, Timothy D.; Whitehead, Bruce A.

    1995-05-01

    A fully-instrumented firing of a propulsion system typically generates a very large quantity of data. In the case of the Space Shuttle Main Engine (SSME), data analysis from ground tests and flights is currently a labor-intensive process. Human experts spend a great deal of time examining the large volume of sensor data generated by each engine firing. These experts look for any anomalies in the data which might indicate engine conditions warranting further investigation. The contract effort was to develop a 'first-cut' screening system for application to SSME engine firings that would identify the relatively small volume of data which is unusual or anomalous in some way. With such a system, limited and expensive human resources could focus on this small volume of unusual data for thorough analysis. The overall project objective was to develop a fully operational Automated Propulsion Data Screening (APDS) system with the capability of detecting significant trends and anomalies in transient and steady-state data. However, the effort limited screening of transient data to ground test data for throttle-down cases typical of the 3-g acceleration, and for engine throttling required to reach the maximum dynamic pressure limits imposed on the Space Shuttle. This APDS is based on neural networks designed to detect anomalies in propulsion system data that are not part of the data used for neural network training. The delivered system allows engineers to build their own screening sets for application to completed or planned firings of the SSME. ERC developers also built some generic screening sets that NASA engineers could apply immediately to their data analysis efforts.

  13. Automated Propulsion Data Screening demonstration system

    NASA Technical Reports Server (NTRS)

    Hoyt, W. Andes; Choate, Timothy D.; Whitehead, Bruce A.

    1995-01-01

    A fully-instrumented firing of a propulsion system typically generates a very large quantity of data. In the case of the Space Shuttle Main Engine (SSME), data analysis from ground tests and flights is currently a labor-intensive process. Human experts spend a great deal of time examining the large volume of sensor data generated by each engine firing. These experts look for any anomalies in the data which might indicate engine conditions warranting further investigation. The contract effort was to develop a 'first-cut' screening system for application to SSME engine firings that would identify the relatively small volume of data which is unusual or anomalous in some way. With such a system, limited and expensive human resources could focus on this small volume of unusual data for thorough analysis. The overall project objective was to develop a fully operational Automated Propulsion Data Screening (APDS) system with the capability of detecting significant trends and anomalies in transient and steady-state data. However, the effort limited screening of transient data to ground test data for throttle-down cases typical of the 3-g acceleration, and for engine throttling required to reach the maximum dynamic pressure limits imposed on the Space Shuttle. This APDS is based on neural networks designed to detect anomalies in propulsion system data that are not part of the data used for neural network training. The delivered system allows engineers to build their own screening sets for application to completed or planned firings of the SSME. ERC developers also built some generic screening sets that NASA engineers could apply immediately to their data analysis efforts.

  14. A high-content, multiplexed screen in human breast cancer cells identifies profilin-1 inducers with anti-migratory activities.

    PubMed

    Joy, Marion E; Vollmer, Laura L; Hulkower, Keren; Stern, Andrew M; Peterson, Cameron K; Boltz, R C Dutch; Roy, Partha; Vogt, Andreas

    2014-01-01

    Profilin-1 (Pfn-1) is a ubiquitously expressed actin-binding protein that is essential for normal cell proliferation and migration. In breast cancer and several other adenocarcinomas, Pfn-1 expression is downregulated when compared to normal tissues. Previous studies from our laboratory have shown that genetically modulating Pfn-1 expression significantly impacts proliferation, migration, and invasion of breast cancer cells in vitro, and mammary tumor growth, dissemination, and metastatic colonization in vivo. Therefore, small molecules that can modulate Pfn-1 expression could have therapeutic potential in the treatment of metastatic breast cancer. The overall goal of this study was to perform a multiplexed phenotypic screen to identify compounds that inhibit cell motility through upregulation of Pfn-1. Screening of a test cassette of 1280 compounds with known biological activities on an Oris™ Pro 384 cell migration platform identified several agents that increased Pfn-1 expression greater than two-fold over vehicle controls and exerted anti-migratory effects in the absence of overt cytotoxicity in MDA-MB-231 human breast cancer cells. Concentration-response confirmation and orthogonal follow-up assays identified two bona fide inducers of Pfn-1, purvalanol and tyrphostin A9, that confirmed in single-cell motility assays and Western blot analyses. SiRNA-mediated knockdown of Pfn-1 abrogated the inhibitory effect of tyrphostin A9 on cell migration, suggesting Pfn-1 is mechanistically linked to tyrphostin A9's anti-migratory activity. The data illustrate the utility of the high-content cell motility assay to discover novel targeted anti-migratory agents by integrating functional phenotypic analyses with target-specific readouts in a single assay platform. PMID:24520372

  15. A Multistep High-Content Screening Approach to Identify Novel Functionally Relevant Target Genes in Pancreatic Cancer

    PubMed Central

    Buchholz, Malte; Honstein, Tatjana; Kirchhoff, Sandra; Kreider, Ramona; Schmidt, Harald; Sipos, Bence; Gress, Thomas M.

    2015-01-01

    In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of ‘reverse transfection array’ technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from “druggable” gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented. PMID:25849100

  16. Effects of 60-GHz millimeter waves on neurite outgrowth in PC12 cells using high-content screening.

    PubMed

    Haas, Alexis J; Le Page, Yann; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-04-01

    Technologies for wireless telecommunication systems using millimeter waves (MMW) will be widely deployed in the near future. Forthcoming applications in this band, especially around 60GHz, are mainly developed for high data-rate local and body-centric telecommunications. At those frequencies, electromagnetic radiations have a very shallow penetration into biological tissues, making skin keratinocytes, and free nerve endings of the upper dermis the main targets of MMW. Only a few studies assessed the impact of MMW on neuronal cells, and none of them investigated a possible effect on neuronal differentiation. We used a neuron-like cell line (PC12), which undergoes neuronal differentiation when treated with the neuronal growth factor (NGF). PC12 cells were exposed at 60.4GHz for 24h, at an incident power density averaged over the cell monolayer of 10mW/cm(2). Using a large scale cell-by-cell analysis based on high-content screening microscopy approach, we assessed potential effects of MMW on PC12 neurite outgrowth and cytoskeleton protein expression. No differences were found in protein expression of the neuronal marker β3-tubulin nor in internal expression control β-tubulin. On the other hand, our data showed a slight increase, although insignificant, in neurite outgrowth, induced by MMW exposure. However, experimental controls demonstrated that this increase was related to heating. PMID:26921450

  17. A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

    PubMed Central

    Iantomasi, Raffaella; Veyron-Churlet, Romain; Deboosère, Nathalie; Landry, Valérie; Baulard, Alain; Brodin, Priscille

    2014-01-01

    Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells. PMID:24473237

  18. A High-Content, Phenotypic Screen Identifies Fluorouridine as an Inhibitor of Pyoverdine Biosynthesis and Pseudomonas aeruginosa Virulence

    PubMed Central

    Kirienko, Daniel R.; Revtovich, Alexey V.

    2016-01-01

    ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and

  19. A High-Content, Phenotypic Screen Identifies Fluorouridine as an Inhibitor of Pyoverdine Biosynthesis and Pseudomonas aeruginosa Virulence.

    PubMed

    Kirienko, Daniel R; Revtovich, Alexey V; Kirienko, Natalia V

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host

  20. Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus

    PubMed Central

    Shinde, Sunita N.; Hua, Yun; Shun, Tong Ying; Lazo, John S.; Day, Billy W.

    2012-01-01

    Abstract Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to

  1. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening.

    PubMed

    O'Brien, P J; Irwin, W; Diaz, D; Howard-Cofield, E; Krejsa, C M; Slaughter, M R; Gao, B; Kaludercic, N; Angeline, A; Bernardi, P; Brain, P; Hougham, C

    2006-09-01

    To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium

  2. Mimer: an automated spreadsheet-based crystallization screening system

    PubMed Central

    Brodersen, Ditlev Egeskov; Andersen, Gregers Rom; Andersen, Christian Brix Folsted

    2013-01-01

    In this paper, a simple low-cost alternative to large commercial systems for preparing macromolecular crystallization conditions is described. Using an intuitive spreadsheet-based approach, the system allows the rapid calculation of relevant pipetting volumes given known stock-solution concentrations and incorporates the automatic design of custom crystallization screens via the incomplete-factorial and grid-screen approaches. Automated dispensing of the resulting crystallization screens is achieved using a generic and relatively inexpensive liquid handler. PMID:23832216

  3. Automated screening for biological weapons in homeland defense.

    PubMed

    Emanuel, Peter A; Fruchey, Isaac R; Bailey, Andrew M; Dang, Jessica L; Niyogi, Kakoli; Roos, Jason W; Cullin, David; Emanuel, Diana C

    2005-01-01

    Biological threat detection programs that collect air samples and monitor for large-scale release of biowarfare agents generate large numbers of samples that must be quickly and accurately screened for the presence of biological agents. An impediment to the rapid analysis of large numbers of environmental biological samples is that manual laboratory processes are time-consuming and require resources to maintain infrastructure, trained personnel, and adequate supplies of testing reagents. An ideal screening system would be capable of processing multiple samples rapidly, cost-effectively, and with minimal personnel. In the present study, we evaluated the Automated Biological Agent Testing System (ABATS) to explore the capability of automation to increase sample throughput, maximize system accuracy, and reduce the analysis costs associated with biological threat agent screening in environmental samples. This study demonstrates the utility of this concept and the potential of an automated system to address the growing environmental monitoring needs of the United States. PMID:15853454

  4. High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

    PubMed Central

    Silva-Miranda, Mayra; Breiman, Adrien; Asehnoune, Karim; Barros-Aguirre, David; Pethe, Kevin; Ewann, Fanny; Brodin, Priscille; Ballell-Pages, Lluís

    2014-01-01

    Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC50s between the activities of the compounds under extracellular and granuloma conditions. PMID:25348525

  5. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS

    EPA Science Inventory

    The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse c...

  6. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  7. Screening for Chemical Effects on Neuronal Proliferation and Neurite Outgrowth Using High-Content/High-Throughput Microscopy

    EPA Science Inventory

    The need to develop novel screening methods for developmental neurotoxicity in order to alleviate the demands of cost, time, and animals required for in vivo toxicity studies is well recognized. Accordingly, the U.S. EPA launched the ToxCast research program in 2007 to develop c...

  8. A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells

    PubMed Central

    Panarella, Angela; Bexiga, Mariana G.; Galea, George; O’ Neill, Elaine D.; Salvati, Anna; Dawson, Kenneth A.; Simpson, Jeremy C.

    2016-01-01

    Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies. PMID:27374232

  9. Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries

    PubMed Central

    Kurita, Kenji L.; Glassey, Emerson; Linington, Roger G.

    2015-01-01

    Traditional natural products discovery using a combination of live/dead screening followed by iterative bioassay-guided fractionation affords no information about compound structure or mode of action until late in the discovery process. This leads to high rates of rediscovery and low probabilities of finding compounds with unique biological and/or chemical properties. By integrating image-based phenotypic screening in HeLa cells with high-resolution untargeted metabolomics analysis, we have developed a new platform, termed Compound Activity Mapping, that is capable of directly predicting the identities and modes of action of bioactive constituents for any complex natural product extract library. This new tool can be used to rapidly identify novel bioactive constituents and provide predictions of compound modes of action directly from primary screening data. This approach inverts the natural products discovery process from the existing ‟grind and find” model to a targeted, hypothesis-driven discovery model where the chemical features and biological function of bioactive metabolites are known early in the screening workflow, and lead compounds can be rationally selected based on biological and/or chemical novelty. We demonstrate the utility of the Compound Activity Mapping platform by combining 10,977 mass spectral features and 58,032 biological measurements from a library of 234 natural products extracts and integrating these two datasets to identify 13 clusters of fractions containing 11 known compound families and four new compounds. Using Compound Activity Mapping we discovered the quinocinnolinomycins, a new family of natural products with a unique carbon skeleton that cause endoplasmic reticulum stress. PMID:26371303

  10. Automated Primary Care Screening in Pediatric Waiting Rooms

    PubMed Central

    Carroll, Aaron E.; Downs, Stephen M.

    2012-01-01

    BACKGROUND AND OBJECTIVE: Implementing US Preventive Services Task Force and American Academy of Pediatrics preventive service guidelines within the short duration of a visit is difficult because identifying which of a large number of guidelines apply to a particular patient is impractical. Clinical decision support system integrated with electronic medical records offer a good strategy for implementing screening in waiting rooms. Our objective was to determine rates of positive risk screens during typical well-care visits among children and adolescents in a primary care setting. METHODS: Child Health Improvement through Computer Automation (CHICA) is a pediatric clinical decision support system developed by our research group. CHICA encodes clinical guidelines as medical logic modules to generate scanable paper forms: the patient screening form to collect structured data from patient families in the waiting room and the physician worksheet to provide physician assessments at each visit. By using visit as a unit of analysis from CHICA’s database, we have determined positive risk screen rates in our population. RESULTS: From a cohort of 16 963 patients, 408 601 questions were asked in 31 843 visits. Of the questions asked, 362 363 (89%) had a response. Of those, 39 176 (11%) identified positive risk screens in both the younger children and the adolescent age groups. CONCLUSIONS: By automating the process of screening and alerting the physician to those who screened positive, we have significantly decreased the burden of identifying relevant guidelines and screening of patient families in our clinics. PMID:22508925

  11. Automated Human Screening for Detecting Concealed Knowledge

    ERIC Educational Resources Information Center

    Twyman, Nathan W.

    2012-01-01

    Screening individuals for concealed knowledge has traditionally been the purview of professional interrogators investigating a crime. But the ability to detect when a person is hiding important information would be of high value to many other fields and functions. This dissertation proposes design principles for and reports on an implementation…

  12. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.

    PubMed

    Hoesli, Corinne A; Johnson, James D; Piret, James M

    2012-01-01

    The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells. PMID:22442738

  13. Automated Telephone Screening Survey for Depression on a University Campus.

    ERIC Educational Resources Information Center

    Portnoy, Robert N.

    On college and university campuses across the United States, depression has taken a huge toll on the academic and personal productivity of students, faculty, and staff. The results of a university's automated telephone screening survey for depression are reported here. Callers were recruited through a variety of media, including advertising and…

  14. Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.

    PubMed

    Martin, Julio

    2010-09-01

    Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electrical impedance, are versatile platforms for detecting phenotypic changes in both engineered and native cells. Their non-invasive nature allows for the kinetic monitoring of multiple real-time cellular responses to external stimuli, as well as for the use of successive pharmacological challenges. The temporal signature recorded for a particular stimulus is characteristic of the cell type and the signaling pathway activated upon binding of a ligand to its receptor. Cellular label-free technology is an important technical advance in the study of functional pharmacological selectivity. Described in this overview are some of the hurdles encountered in modern drug discovery and the ways in which label-free technologies can be used to overcome these obstacles. PMID:22294376

  15. Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis.

    PubMed

    Grimm, T; Grimm, M; Klat, R; Neubauer, A; Palela, M; Neubauer, P

    2012-05-01

    The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10-15% and an increased glucose release by increasing the enzyme content to 15 U L(-1). The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD(600)) of approximately 40 AU (corresponding to over 60 g L(-1) wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches. PMID:22258642

  16. Automated protein-ligand interaction screening by mass spectrometry.

    PubMed

    Maple, Hannah J; Garlish, Rachel A; Rigau-Roca, Laura; Porter, John; Whitcombe, Ian; Prosser, Christine E; Kennedy, Jeff; Henry, Alistair J; Taylor, Richard J; Crump, Matthew P; Crosby, John

    2012-01-26

    Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening. PMID:22148839

  17. 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.

    PubMed

    Johnston, Paul A; 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; Wang, Zhou

    2016-05-01

    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

  18. A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

    PubMed

    Rhim, Ji Heon; Luo, Xiangjian; Xu, Xiaoyun; Gao, Dongbing; Zhou, Tieling; Li, Fuhai; Qin, Lidong; Wang, Ping; Xia, Xiaofeng; Wong, Stephen T C

    2015-01-01

    Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs. PMID:26542303

  19. A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells

    PubMed Central

    Rhim, Ji heon; Luo, Xiangjian; Xu, Xiaoyun; Gao, Dongbing; Zhou, Tieling; Li, Fuhai; Qin, Lidong; Wang, Ping; Xia, Xiaofeng; Wong, Stephen T. C.

    2015-01-01

    Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs. PMID:26542303

  20. In vitro optimization of EtNBS-PDT against hypoxic tumor environments with a tiered, high-content, 3D model optical screening platform.

    PubMed

    Klein, Oliver J; Bhayana, Brijesh; Park, Yong Jin; Evans, Conor L

    2012-11-01

    Hypoxia and acidosis are widely recognized as major contributors to the development of treatment resistant cancer. For patients with disseminated metastatic lesions, such as most women with ovarian cancer (OvCa), the progression to treatment resistant disease is almost always fatal. Numerous therapeutic approaches have been developed to eliminate treatment resistant carcinoma, including novel biologic, chemo, radiation, and photodynamic therapy (PDT) regimens. Recently, PDT using the cationic photosensitizer EtNBS was found to be highly effective against therapeutically unresponsive hypoxic and acidic OvCa cellular populations in vitro. To optimize this treatment regimen, we developed a tiered, high-content, image-based screening approach utilizing a biologically relevant OvCa 3D culture model to investigate a small library of side-chain modified EtNBS derivatives. The uptake, localization, and photocytotoxicity of these compounds on both the cellular and nodular levels were observed to be largely mediated by their respective ethyl side chain chemical alterations. In particular, EtNBS and its hydroxyl-terminated derivative (EtNBS-OH) were found to have similar pharmacological parameters, such as their nodular localization patterns and uptake kinetics. Interestingly, these two molecules were found to induce dramatically different therapeutic outcomes: EtNBS was found to be more effective in killing the hypoxic, nodule core cells with superior selectivity, while EtNBS-OH was observed to trigger widespread structural degradation of nodules. This breakdown of the tumor architecture can improve the therapeutic outcome and is known to synergistically enhance the antitumor effects of front-line chemotherapeutic regimens. These results, which would not have been predicted or observed using traditional monolayer or in vivo animal screening techniques, demonstrate the powerful capabilities of 3D in vitro screening approaches for the selection and optimization of therapeutic

  1. Fully automated diabetic retinopathy screening using morphological component analysis.

    PubMed

    Imani, Elaheh; Pourreza, Hamid-Reza; Banaee, Touka

    2015-07-01

    Diabetic retinopathy is the major cause of blindness in the world. It has been shown that early diagnosis can play a major role in prevention of visual loss and blindness. This diagnosis can be made through regular screening and timely treatment. Besides, automation of this process can significantly reduce the work of ophthalmologists and alleviate inter and intra observer variability. This paper provides a fully automated diabetic retinopathy screening system with the ability of retinal image quality assessment. The novelty of the proposed method lies in the use of Morphological Component Analysis (MCA) algorithm to discriminate between normal and pathological retinal structures. To this end, first a pre-screening algorithm is used to assess the quality of retinal images. If the quality of the image is not satisfactory, it is examined by an ophthalmologist and must be recaptured if necessary. Otherwise, the image is processed for diabetic retinopathy detection. In this stage, normal and pathological structures of the retinal image are separated by MCA algorithm. Finally, the normal and abnormal retinal images are distinguished by statistical features of the retinal lesions. Our proposed system achieved 92.01% sensitivity and 95.45% specificity on the Messidor dataset which is a remarkable result in comparison with previous work. PMID:25863517

  2. High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical's bio-activation with regards hepatotoxicity.

    PubMed

    Peyre, Ludovic; de Sousa, Georges; Barcellini-Couget, Sylvie; Luzy, Anne-Pascale; Zucchini-Pascal, Nathalie; Rahmani, Roger

    2015-10-01

    Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals. PMID:26239606

  3. Pre-Treatment of platinum resistant ovarian cancer cells with an MMP-9/MMP-2 inhibitor prior to cisplatin enhances cytotoxicity as determined by high content screening.

    PubMed

    Laios, Alexandros; Mohamed, Bashir M; Kelly, Lynn; Flavin, Richard; Finn, Stephen; McEvoy, Lynda; Gallagher, Michael; Martin, Cara; Sheils, Orla; Ring, Martina; Davies, Anthony; Lawson, Margaret; Gleeson, Noreen; D'Arcy, Tom; d'Adhemar, Charles; Norris, Lucy; Langhe, Ream; Saadeh, Feras Abu; O'Leary, John J; O'Toole, Sharon A

    2013-01-01

    Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated. PMID:23340649

  4. A screened automated structural search with semiempirical methods

    NASA Astrophysics Data System (ADS)

    Ota, Yukihiro; Ruiz-Barragan, Sergi; Machida, Masahiko; Shiga, Motoyuki

    2016-03-01

    We developed an interface program between a program suite for an automated search of chemical reaction pathways, GRRM, and a program package of semiempirical methods, MOPAC. A two-step structural search is proposed as an application of this interface program. A screening test is first performed by semiempirical calculations. Subsequently, a reoptimization procedure is done by ab initio or density functional calculations. We apply this approach to ion adsorption on cellulose. The computational efficiency is also shown for a GRRM search. The interface program is suitable for the structural search of large molecular systems for which semiempirical methods are applicable.

  5. High-throughput automated refolding screening of inclusion bodies

    PubMed Central

    Vincentelli, Renaud; Canaan, Stéphane; Campanacci, Valérie; Valencia, Christel; Maurin, Damien; Frassinetti, Frédéric; Scappucini-Calvo, Loréna; Bourne, Yves; Cambillau, Christian; Bignon, Christophe

    2004-01-01

    One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals. PMID:15388864

  6. Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening.

    PubMed

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Frizzell, Caroline; Verhaegen, Steven; Ropstad, Erik; Connolly, Lisa

    2016-03-01

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p'-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2h and 48h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC+Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br+Cl, PFC+Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment. PMID:26772051

  7. An Intelligent Phonocardiography for Automated Screening of Pediatric Heart Diseases.

    PubMed

    Sepehri, Amir A; Kocharian, Armen; Janani, Azin; Gharehbaghi, Arash

    2016-01-01

    This paper presents a robust device for automated screening of pediatric heart diseases based on our unique processing method in murmur characterization; the Arash-Band method. The present study modifies the Arash-Band method and employs output of the modified method in conjunction with the two other original techniques to extract indicative feature vectors for the screening. The extracted feature vectors are classified by using the support vector machine method. Results show that the proposed modifications significantly enhances performance of the Arash-Band in terms of the both accuracy and sensitivity as the corresponding effect sizes are sufficiently large. The proposed algorithm has been incorporated into an Android-based tablet to constitute an intelligent phonocardiogram with the automatic screening capability. In order to obtain confidence interval of the accuracy and sensitivity, an inferable statistical test is applied on our database containing the phonocardiogram signals recorded from 263 of the referrals to a hospital. The expected value of the accuracy/sensitivity is estimated to be 87.45 % / 87.29 % with a 95 % confidence interval of (80.19 % - 92.47 %) / (76.01 % - 95.78 %) exhibiting superior performance than a pediatric cardiologist who relies on conventional or even computer-assisted auscultation. PMID:26573653

  8. Automated Recommendation for Cervical Cancer Screening and Surveillance

    PubMed Central

    Wagholikar, Kavishwar B; MacLaughlin, Kathy L; Casey, Petra M; Kastner, Thomas M; Henry, Michael R; Hankey, Ronald A; Peters, Steve G; Greenes, Robert A; Chute, Christopher G; Liu, Hongfang; Chaudhry, Rajeev

    2014-01-01

    Because of the complexity of cervical cancer prevention guidelines, clinicians often fail to follow best-practice recommendations. Moreover, existing clinical decision support (CDS) systems generally recommend a cervical cytology every three years for all female patients, which is inappropriate for patients with abnormal findings that require surveillance at shorter intervals. To address this problem, we developed a decision tree-based CDS system that integrates national guidelines to provide comprehensive guidance to clinicians. Validation was performed in several iterations by comparing recommendations generated by the system with those of clinicians for 333 patients. The CDS system extracted relevant patient information from the electronic health record and applied the guideline model with an overall accuracy of 87%. Providers without CDS assistance needed an average of 1 minute 39 seconds to decide on recommendations for management of abnormal findings. Overall, our work demonstrates the feasibility and potential utility of automated recommendation system for cervical cancer screening and surveillance. PMID:25368505

  9. Automated recommendation for cervical cancer screening and surveillance.

    PubMed

    Wagholikar, Kavishwar B; MacLaughlin, Kathy L; Casey, Petra M; Kastner, Thomas M; Henry, Michael R; Hankey, Ronald A; Peters, Steve G; Greenes, Robert A; Chute, Christopher G; Liu, Hongfang; Chaudhry, Rajeev

    2014-01-01

    Because of the complexity of cervical cancer prevention guidelines, clinicians often fail to follow best-practice recommendations. Moreover, existing clinical decision support (CDS) systems generally recommend a cervical cytology every three years for all female patients, which is inappropriate for patients with abnormal findings that require surveillance at shorter intervals. To address this problem, we developed a decision tree-based CDS system that integrates national guidelines to provide comprehensive guidance to clinicians. Validation was performed in several iterations by comparing recommendations generated by the system with those of clinicians for 333 patients. The CDS system extracted relevant patient information from the electronic health record and applied the guideline model with an overall accuracy of 87%. Providers without CDS assistance needed an average of 1 minute 39 seconds to decide on recommendations for management of abnormal findings. Overall, our work demonstrates the feasibility and potential utility of automated recommendation system for cervical cancer screening and surveillance. PMID:25368505

  10. Current trends and perspectives for automated screening of cardiac murmurs

    PubMed Central

    Marascio, Giuseppe; Modesti, Pietro Amedeo

    2013-01-01

    Although in high income countries rheumatic heart disease is now rare, it remains a major burden in low and middle income countries. In these world areas, physicians and expert sonographers are rare, and screening campaigns are usually performed by nomadic caregivers who can only recognise patients in an advanced phase of heart failure with high economic and social costs. Therefore, great interest exists regarding the possibility of developing a simple, low-cost procedure for screening valvular heart disease. With the development of computer science, the cardiac sound signal can be analysed in an automatic way. More precisely, a panel of features characterising the acoustic signal are extracted and sent to a decision-making software able to provide the final diagnosis. Although no system is currently available in the market, the rapid evolution of these technologies recently led to the activation of clinical trials. The aim of this note is to review the state of advancement of this technology (trends in feature selection and automatic diagnostic strategies), data available regarding performance of the technology in the clinical setting and finally what obstacles still need to be overcome before automated systems can be clinically/commercially viable. PMID:27326133

  11. Current trends and perspectives for automated screening of cardiac murmurs.

    PubMed

    Marascio, Giuseppe; Modesti, Pietro Amedeo

    2013-01-01

    Although in high income countries rheumatic heart disease is now rare, it remains a major burden in low and middle income countries. In these world areas, physicians and expert sonographers are rare, and screening campaigns are usually performed by nomadic caregivers who can only recognise patients in an advanced phase of heart failure with high economic and social costs. Therefore, great interest exists regarding the possibility of developing a simple, low-cost procedure for screening valvular heart disease. With the development of computer science, the cardiac sound signal can be analysed in an automatic way. More precisely, a panel of features characterising the acoustic signal are extracted and sent to a decision-making software able to provide the final diagnosis. Although no system is currently available in the market, the rapid evolution of these technologies recently led to the activation of clinical trials. The aim of this note is to review the state of advancement of this technology (trends in feature selection and automatic diagnostic strategies), data available regarding performance of the technology in the clinical setting and finally what obstacles still need to be overcome before automated systems can be clinically/commercially viable. PMID:27326133

  12. Automated video screening for unattended background monitoring in dynamic environments.

    SciTech Connect

    Carlson, Jeffrey J.

    2004-03-01

    This report addresses the development of automated video-screening technology to assist security forces in protecting our homeland against terrorist threats. A threat of specific interest to this project is the covert placement and subsequent remote detonation of bombs (e.g., briefcase bombs) inside crowded public facilities. Different from existing video motion detection systems, the video-screening technology described in this report is capable of detecting changes in the static background of an otherwise, dynamic environment - environments where motion and human activities are persistent. Our goal was to quickly detect changes in the background - even under conditions when the background is visible to the camera less than 5% of the time. Instead of subtracting the background to detect movement or changes in a scene, we subtracted the dynamic scene variations to produce an estimate of the static background. Subsequent comparisons of static background estimates are used to detect changes in the background. Detected changes can be used to alert security forces of the presence and location of potential threats. The results of this research are summarized in two MS Power-point presentations included with this report.

  13. Multiplexing high-content flow (HCF) and quantitative high-throughput screening (qHTS) to identify compounds capable of decreasing cell viability, activating caspase 3/7, expressing annexin V, and changing mitochondrial membrane integrity.

    PubMed

    Mathews, Lesley A; Keller, Jonathan M; McKnight, Crystal; Michael, Sam; Shinn, Paul; Liu, Dongbo; Staudt, Louis M; Thomas, Craig J; Ferrer, Marc

    2013-01-01

    High-content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high-throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large-scale screens. When assays can be miniaturized to a 384- or 1536-well microplate format, it is possible to implement dose-response-based high-throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL). PMID:24391083

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

    PubMed Central

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

    2012-01-01

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

  15. Automated touch screen device for recording complex rodent behaviors

    PubMed Central

    Mabrouk, O.S.; Dripps, I.J.; Ramani, S.; Chang, C.; Han, J.L.; Rice, KC; Jutkiewicz, E.M.

    2016-01-01

    Background Monitoring mouse behavior is a critical step in the development of modern pharmacotherapies. New Method Here we describe the application of a novel method that utilizes a touch display computer (tablet) and software to detect, record, and report fine motor behaviors. A consumer-grade tablet device is placed in the bottom of a specially made acrylic cage allowing the animal to walk on the device (MouseTrapp). We describe its application in open field (for general locomotor studies) which measures step lengths and velocity. The device can perform light-dark (anxiety) tests by illuminating half of the screen and keeping the other half darkened. A divider is built into the lid of the device allowing the animal free access to either side. Results Treating mice with amphetamine and the delta opioid peptide receptor agonist SNC80 stimulated locomotor activity on the device. Amphetamine increased step velocity but not step length during its peak effect (40–70 min after treatment), thus indicating detection of subtle amphetamine-induced effects. Animals showed a preference (74% of time spent) for the darkened half compared to the illuminated side. Comparison with Existing Method Animals were videotaped within the chamber to compare quadrant crosses to detected motion on the device. The slope, duration and magnitude of quadrant crosses tightly correlated with overall locomotor activity as detected by Mousetrapp. Conclusions We suggest that modern touch display devices such as MouseTrapp will be an important step toward automation of behavioral analyses for characterizing phenotypes and drug effects. PMID:24952323

  16. Assessment of Automated Disease Detection in Diabetic Retinopathy Screening Using Two-Field Photography

    PubMed Central

    Goatman, Keith; Charnley, Amanda; Webster, Laura; Nussey, Stephen

    2011-01-01

    Aim To assess the performance of automated disease detection in diabetic retinopathy screening using two field mydriatic photography. Methods Images from 8,271 sequential patient screening episodes from a South London diabetic retinopathy screening service were processed by the Medalytix iGrading™ automated grading system. For each screening episode macular-centred and disc-centred images of both eyes were acquired and independently graded according to the English national grading scheme. Where discrepancies were found between the automated result and original manual grade, internal and external arbitration was used to determine the final study grades. Two versions of the software were used: one that detected microaneurysms alone, and one that detected blot haemorrhages and exudates in addition to microaneurysms. Results for each version were calculated once using both fields and once using the macula-centred field alone. Results Of the 8,271 episodes, 346 (4.2%) were considered unassessable. Referable disease was detected in 587 episodes (7.1%). The sensitivity of the automated system for detecting unassessable images ranged from 97.4% to 99.1% depending on configuration. The sensitivity of the automated system for referable episodes ranged from 98.3% to 99.3%. All the episodes that included proliferative or pre-proliferative retinopathy were detected by the automated system regardless of configuration (192/192, 95% confidence interval 98.0% to 100%). If implemented as the first step in grading, the automated system would have reduced the manual grading effort by between 2,183 and 3,147 patient episodes (26.4% to 38.1%). Conclusion Automated grading can safely reduce the workload of manual grading using two field, mydriatic photography in a routine screening service. PMID:22174741

  17. A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

    PubMed Central

    Danovi, Davide; Folarin, Amos; Gogolok, Sabine; Ender, Christine; Elbatsh, Ahmed M. O.; Engström, Pär G.; Stricker, Stefan H.; Gagrica, Sladjana; Georgian, Ana; Yu, Ding; U, Kin Pong; Harvey, Kevin J.; Ferretti, Patrizia; Paddison, Patrick J.; Preston, Jane E.; Abbott, N. Joan; Bertone, Paul; Smith, Austin; Pollard, Steven M.

    2013-01-01

    Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value. PMID:24204733

  18. Longitudinal, Quantitative Monitoring of Therapeutic Response in 3D In Vitro Tumor Models with OCT for High-Content Therapeutic Screening

    PubMed Central

    Klein, O. J.; Jung, Y. K.; Evans, C. L.

    2013-01-01

    In vitro three-dimensional models of cancer have the ability to recapitulate many features of tumors found in vivo, including cell-cell and cell-matrix interactions, microenvironments that become hypoxic and acidic, and other barriers to effective therapy. These model tumors can be large, highly complex, heterogeneous, and undergo time-dependent growth and treatment response processes that are difficult to track and quantify using standard imaging tools. Optical coherence tomography is an optical ranging technique that is ideally suited for visualizing, monitoring, and quantifying the growth and treatment response dynamics occurring in these informative model systems. By optimizing both optical coherence tomography and 3D culture systems, it is possible to continuously and non-perturbatively monitor advanced in vitro models without the use of labels over the course of hours and days. In this article, we describe approaches and methods for creating and carrying out quantitative therapeutic screens with in vitro 3D cultures using optical coherence tomography to gain insights into therapeutic mechanisms and build more effective treatment regimens. PMID:24013042

  19. High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

    PubMed Central

    Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

    2014-01-01

    Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active

  20. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    PubMed

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV). PMID:26032261

  1. Automated screening of propulsion system test data by neural networks, phase 1

    NASA Technical Reports Server (NTRS)

    Hoyt, W. Andes; Whitehead, Bruce A.

    1992-01-01

    The evaluation of propulsion system test and flight performance data involves reviewing an extremely large volume of sensor data generated by each test. An automated system that screens large volumes of data and identifies propulsion system parameters which appear unusual or anomalous will increase the productivity of data analysis. Data analysts may then focus on a smaller subset of anomalous data for further evaluation of propulsion system tests. Such an automated data screening system would give NASA the benefit of a reduction in the manpower and time required to complete a propulsion system data evaluation. A phase 1 effort to develop a prototype data screening system is reported. Neural networks will detect anomalies based on nominal propulsion system data only. It appears that a reasonable goal for an operational system would be to screen out 95 pct. of the nominal data, leaving less than 5 pct. needing further analysis by human experts.

  2. Automated Telephone Calls To Enhance Colorectal Cancer Screening: An Economic Analysis from a Randomized Trial

    PubMed Central

    Smith, David H.; Feldstein, Adrianne C.; Perrin, Nancy; Rosales, A. Gabriela; Mosen, David M.; Liles, Elizabeth G.; Schneider, Jennifer L.; Lafata, Jennifer E.; Myers, Ronald E.; Glasgow, Russell E.

    2013-01-01

    Colorectal cancer screening has been shown to be a cost-effective intervention, but uncertainty remains over the most cost-effective methods for increasing screening rates. We used data from a pragmatic randomized controlled trial to estimate the cost-effectiveness of an automated telephone intervention from a managed care perspective. Intervention group patients received calls for fecal occult blood testing (FOBT) screening. Electronic medical records confirmed whether a patient had completed screening. We searched patient’s electronic medical record for any screening (defined as FOBT, flexible sigmoidoscopy, double contrast barium enema, or colonoscopy) during follow-up. Intervention costs included project implementation and management, telephone calls, patient identification and tracking. Costs of screening included FOBT (kits, mailing and processing) and any completed screening tests during follow-up. We estimated the incremental cost-effectiveness ratio (ICER) of the cost per additional screen. Results At 6 months average costs per patient in the intervention group were $37 (25% screened) and $34 (19% screened) in the control groups. The ICER at 6 months was $40 per additional screen. The probability of cost-effectiveness was 0.49, 0.84 and 0.99 for willingness to pay thresholds of $40, $100 and $200, respectively. Similar results were seen at 9 months. Screening rates and cost-effectiveness differed by age. A greater increase in FOBT testing was seen for patients aged 70 years and over (45 per 100 intervention, 33 per 100 control) compared with younger patients (25 per 100 intervention, 21 per 100 control). The intervention was dominant (lower costs and greater proportion of patients screened) for patients aged 70 years and over and was $73 per additional screen for younger patients. Discussion A patient-directed, automated phone calling increased screening rates by about 6% and costs by $3 per patient. The ICER we report is less than half what other

  3. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

    PubMed

    McDonough, Patrick M; Ingermanson, Randall S; Loy, Patricia A; Koon, Erick D; Whittaker, Ross; Laris, Casey A; Hilton, Jeffrey M; Nicoll, James B; Buehrer, Benjamin M; Price, Jeffrey H

    2011-06-01

    Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes. PMID:21186937

  4. Comparison of automated docking programs as virtual screening tools.

    PubMed

    Cummings, Maxwell D; DesJarlais, Renee L; Gibbs, Alan C; Mohan, Venkatraman; Jaeger, Edward P

    2005-02-24

    The performance of several commercially available docking programs is compared in the context of virtual screening. Five different protein targets are used, each with several known ligands. The simulated screening deck comprised 1000 molecules from a cleansed version of the MDL drug data report and 49 known ligands. For many of the known ligands, crystal structures of the relevant protein-ligand complexes were available. We attempted to run experiments with each docking method that were as similar as possible. For a given docking method, hit rates were improved versus what would be expected for random selection for most protein targets. However, the ability to prioritize known ligands on the basis of docking poses that resemble known crystal structures is both method- and target-dependent. PMID:15715466

  5. AMMOS: Automated Molecular Mechanics Optimization tool for in silico Screening

    PubMed Central

    Pencheva, Tania; Lagorce, David; Pajeva, Ilza; Villoutreix, Bruno O; Miteva, Maria A

    2008-01-01

    Background Virtual or in silico ligand screening combined with other computational methods is one of the most promising methods to search for new lead compounds, thereby greatly assisting the drug discovery process. Despite considerable progresses made in virtual screening methodologies, available computer programs do not easily address problems such as: structural optimization of compounds in a screening library, receptor flexibility/induced-fit, and accurate prediction of protein-ligand interactions. It has been shown that structural optimization of chemical compounds and that post-docking optimization in multi-step structure-based virtual screening approaches help to further improve the overall efficiency of the methods. To address some of these points, we developed the program AMMOS for refining both, the 3D structures of the small molecules present in chemical libraries and the predicted receptor-ligand complexes through allowing partial to full atom flexibility through molecular mechanics optimization. Results The program AMMOS carries out an automatic procedure that allows for the structural refinement of compound collections and energy minimization of protein-ligand complexes using the open source program AMMP. The performance of our package was evaluated by comparing the structures of small chemical entities minimized by AMMOS with those minimized with the Tripos and MMFF94s force fields. Next, AMMOS was used for full flexible minimization of protein-ligands complexes obtained from a mutli-step virtual screening. Enrichment studies of the selected pre-docked complexes containing 60% of the initially added inhibitors were carried out with or without final AMMOS minimization on two protein targets having different binding pocket properties. AMMOS was able to improve the enrichment after the pre-docking stage with 40 to 60% of the initially added active compounds found in the top 3% to 5% of the entire compound collection. Conclusion The open source AMMOS

  6. A High Content Assay to Assess Cellular Fitness

    PubMed Central

    Antczak, Christophe; Mahida, Jeni P.; Singh, Chanpreet; Calder, Paul A.; Djaballah, Hakim

    2013-01-01

    A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection. PMID:23957721

  7. Automated computational screening of the thiol reactivity of substituted alkenes.

    PubMed

    Smith, Jennifer M; Rowley, Christopher N

    2015-08-01

    Electrophilic olefins can react with the S-H moiety of cysteine side chains. The formation of a covalent adduct through this mechanism can result in the inhibition of an enzyme. The reactivity of an olefin towards cysteine depends on its functional groups. In this study, 325 reactions of thiol-Michael-type additions to olefins were modeled using density functional theory. All combinations of ethenes with hydrogen, methyl ester, amide, and cyano substituents were included. An automated workflow was developed to perform the construction, conformation search, minimization, and calculation of molecular properties for the reactant, carbanion intermediate, and thioether products for a model reaction of the addition of methanethiol to the electrophile. Known cysteine-reactive electrophiles present in the database were predicted to react exergonically with methanethiol through a carbanion with a stability in the 30-40 kcal mol(-1) range. 13 other compounds in our database that are also present in the PubChem database have similar properties. Natural bond orbital parameters were computed and regression analysis was used to determine the relationship between properties of the olefin electronic structure and the product and intermediate stability. The stability of the intermediates is very sensitive to electronic effects on the carbon where the anionic charge is centered. The stability of the products is more sensitive to steric factors. PMID:26159564

  8. A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

    PubMed

    Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel

    2015-04-01

    For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. PMID:25548140

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

    PubMed Central

    Smith, Clyde A.; Cohen, Aina E.

    2008-01-01

    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 (SSRL), a National User Facility which 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 Automated Mounter, or SAM) 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. PMID:19956359

  10. Screening for cervical cancer using automated analysis of PAP-smears.

    PubMed

    Bengtsson, Ewert; Malm, Patrik

    2014-01-01

    Cervical cancer is one of the most deadly and common forms of cancer among women if no action is taken to prevent it, yet it is preventable through a simple screening test, the so-called PAP-smear. This is the most effective cancer prevention measure developed so far. But the visual examination of the smears is time consuming and expensive and there have been numerous attempts at automating the analysis ever since the test was introduced more than 60 years ago. The first commercial systems for automated analysis of the cell samples appeared around the turn of the millennium but they have had limited impact on the screening costs. In this paper we examine the key issues that need to be addressed when an automated analysis system is developed and discuss how these challenges have been met over the years. The lessons learned may be useful in the efforts to create a cost-effective screening system that could make affordable screening for cervical cancer available for all women globally, thus preventing most of the quarter million annual unnecessary deaths still caused by this disease. PMID:24772188

  11. Screening for Cervical Cancer Using Automated Analysis of PAP-Smears

    PubMed Central

    Malm, Patrik

    2014-01-01

    Cervical cancer is one of the most deadly and common forms of cancer among women if no action is taken to prevent it, yet it is preventable through a simple screening test, the so-called PAP-smear. This is the most effective cancer prevention measure developed so far. But the visual examination of the smears is time consuming and expensive and there have been numerous attempts at automating the analysis ever since the test was introduced more than 60 years ago. The first commercial systems for automated analysis of the cell samples appeared around the turn of the millennium but they have had limited impact on the screening costs. In this paper we examine the key issues that need to be addressed when an automated analysis system is developed and discuss how these challenges have been met over the years. The lessons learned may be useful in the efforts to create a cost-effective screening system that could make affordable screening for cervical cancer available for all women globally, thus preventing most of the quarter million annual unnecessary deaths still caused by this disease. PMID:24772188

  12. Comparison of Automated Treponemal and Nontreponemal Test Algorithms as First-Line Syphilis Screening Assays

    PubMed Central

    Chung, Jae-Woo; Park, Seong Yeon; Chae, Seok Lae

    2016-01-01

    Background Automated Mediace Treponema pallidum latex agglutination (TPLA) and Mediace rapid plasma reagin (RPR) assays are used by many laboratories for syphilis diagnosis. This study compared the results of the traditional syphilis screening algorithm and a reverse algorithm using automated Mediace RPR or Mediace TPLA as first-line screening assays in subjects undergoing a health checkup. Methods Samples from 24,681 persons were included in this study. We routinely performed Mediace RPR and Mediace TPLA simultaneously. Results were analyzed according to both the traditional algorithm and reverse algorithm. Samples with discordant results on the reverse algorithm (e.g., positive Mediace TPLA, negative Mediace RPR) were tested with Treponema pallidum particle agglutination (TPPA). Results Among the 24,681 samples, 30 (0.1%) were found positive by traditional screening, and 190 (0.8%) by reverse screening. The identified syphilis rate and overall false-positive rate according to the traditional algorithm were lower than those according to the reverse algorithm (0.07% and 0.05% vs. 0.64% and 0.13%, respectively). A total of 173 discordant samples were tested with TPPA by using the reverse algorithm, of which 140 (80.9%) were TPPA positive. Conclusions Despite the increased false-positive results in populations with a low prevalence of syphilis, the reverse algorithm detected 140 samples with treponemal antibody that went undetected by the traditional algorithm. The reverse algorithm using Mediace TPLA as a screening test is more sensitive for the detection of syphilis. PMID:26522755

  13. Clinical performance evaluation of a novel, automated chemiluminescent immunoassay, QUANTA Flash CTD Screen Plus.

    PubMed

    Bentow, Chelsea; Lakos, Gabriella; Rosenblum, Rachel; Bryant, Cassandra; Seaman, Andrea; Mahler, Michael

    2015-02-01

    The QUANTA Flash(®) CTD Screen Plus is a chemiluminescent immunoassay (CIA) for the detection of the major antinuclear antibodies (ANA) on the BIO-FLASH(®) platform. NOVA View(®) is an automated fluorescence microscope that acquires digital images of indirect immunofluorescent assay (IFA) slides. Our goal was to evaluate the clinical performance of the two automated systems and compare their performance to that of traditional IFA. Sera from patients with systemic autoimmune rheumatic diseases (SARD, n = 178), along with disease and healthy controls (n = 204), were tested with the CTD CIA and with NOVA Lite(®) HEp-2 ANA, using both the manual method of reading the IFA slides and the NOVA View instrument. The CTD CIA showed 78.1% sensitivity for SARD, coupled with 94.1% specificity. Manual IFA and NOVA View showed somewhat higher sensitivity (81.5 and 84.8% in SARD, respectively), but significantly lower specificity (79.4 and 64.7%, respectively). Both automated systems displayed somewhat different performance, due to the different principals of ANA detection: IFA with NOVA View digital image interpretation had higher sensitivity, while the CTD CIA showed higher specificity. With the added benefits of full automation, the new CTD CIA is an attractive alternative to traditional ANA screening. PMID:25420962

  14. High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

    PubMed

    Ketley, Ami; Chen, Catherine Z; Li, Xin; Arya, Sukrat; Robinson, Thelma E; Granados-Riveron, Javier; Udosen, Inyang; Morris, Glenn E; Holt, Ian; Furling, Denis; Chaouch, Soraya; Haworth, Ben; Southall, Noel; Shinn, Paul; Zheng, Wei; Austin, Christopher P; Hayes, Christopher J; Brook, J David

    2014-03-15

    Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM. PMID:24179176

  15. Automated assessment of bilateral breast volume asymmetry as a breast cancer biomarker during mammographic screening

    SciTech Connect

    Williams, Alex C; Hitt, Austin N; Voisin, Sophie; Tourassi, Georgia

    2013-01-01

    The biological concept of bilateral symmetry as a marker of developmental stability and good health is well established. Although most individuals deviate slightly from perfect symmetry, humans are essentially considered bilaterally symmetrical. Consequently, increased fluctuating asymmetry of paired structures could be an indicator of disease. There are several published studies linking bilateral breast size asymmetry with increased breast cancer risk. These studies were based on radiologists manual measurements of breast size from mammographic images. We aim to develop a computerized technique to assess fluctuating breast volume asymmetry in screening mammograms and investigate whether it correlates with the presence of breast cancer. Using a large database of screening mammograms with known ground truth we applied automated breast region segmentation and automated breast size measurements in CC and MLO views using three well established methods. All three methods confirmed that indeed patients with breast cancer have statistically significantly higher fluctuating asymmetry of their breast volumes. However, statistically significant difference between patients with cancer and benign lesions was observed only for the MLO views. The study suggests that automated assessment of global bilateral asymmetry could serve as a breast cancer risk biomarker for women undergoing mammographic screening. Such biomarker could be used to alert radiologists or computer-assisted detection (CAD) systems to exercise increased vigilance if higher than normal cancer risk is suspected.

  16. Automated assessment of bilateral breast volume asymmetry as a breast cancer biomarker during mammographic screening

    NASA Astrophysics Data System (ADS)

    Williams, Alex C.; Hitt, Austin; Voisin, Sophie; Tourassi, Georgia

    2013-03-01

    The biological concept of bilateral symmetry as a marker of developmental stability and good health is well established. Although most individuals deviate slightly from perfect symmetry, humans are essentially considered bilaterally symmetrical. Consequently, increased fluctuating asymmetry of paired structures could be an indicator of disease. There are several published studies linking bilateral breast size asymmetry with increased breast cancer risk. These studies were based on radiologists' manual measurements of breast size from mammographic images. We aim to develop a computerized technique to assess fluctuating breast volume asymmetry in screening mammograms and investigate whether it correlates with the presence of breast cancer. Using a large database of screening mammograms with known ground truth we applied automated breast region segmentation and automated breast size measurements in CC and MLO views using three well established methods. All three methods confirmed that indeed patients with breast cancer have statistically significantly higher fluctuating asymmetry of their breast volumes. However, statistically significant difference between patients with cancer and benign lesions was observed only for the MLO views. The study suggests that automated assessment of global bilateral asymmetry could serve as a breast cancer risk biomarker for women undergoing mammographic screening. Such biomarker could be used to alert radiologists or computer-assisted detection (CAD) systems to exercise increased vigilance if higher than normal cancer risk is suspected.

  17. Automated Screening for High-Frequency Hearing Loss

    PubMed Central

    MacKinnon, Robert C.; Jansen, Marije; Moore, David R.

    2014-01-01

    Objective: Hearing loss at high frequencies produces perceptual difficulties and is often an early sign of a more general hearing loss. This study reports the development and validation of two new speech-based hearing screening tests in English that focus on detecting hearing loss at frequencies above 2000 Hz. Design: The Internet-delivered, speech-in noise tests used closed target-word sets of digit triplets or consonant–vowel–consonant (CVC) words presented against a speech-shaped noise masker. The digit triplet test uses the digits 0 to 9 (excluding the disyllabic 7), grouped in quasi-random triplets. The CVC test uses simple words (e.g., “cat”) selected for the high-frequency spectral content of the consonants. During testing, triplets or CVC words were identified in an adaptive procedure to obtain the speech reception threshold (SRT) in noise. For these new, high-frequency (HF) tests, the noise was low-pass filtered to produce greater masking of the low-frequency speech components, increasing the sensitivity of the test for HF hearing loss. Individual test tokens (digits, CVCs) were first homogenized using a group of 10 normal-hearing (NH) listeners by equalizing intelligibility across tokens at several speech-in-noise levels. Both tests were then validated and standardized using groups of 24 NH listeners and 50 listeners with hearing impairment. Performance on the new high frequency digit triplet (HF-triplet) and CVC (HF-CVC) tests was compared with audiometric hearing loss, and with that on the unfiltered, broadband digit triplet test (BB-triplet) test, and the ASL (Adaptive Sentence Lists) speech-in-noise test. Results: The HF-triplet and HF-CVC test results (SRT) both correlated positively and highly with high-frequency audiometric hearing loss and with the ASL test. SRT for both tests as a function of high-frequency hearing loss increased at nearly three times the rate as that of the BB-triplet test. The intraindividual variability (SD) on the

  18. Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.

    PubMed

    Bleckmann, Maren; Schmelz, Stefan; Schinkowski, Christian; Scrima, Andrea; van den Heuvel, Joop

    2016-09-01

    Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor-, and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid-based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells

  19. Screening for atrial fibrillation with automated blood pressure measurement: Research evidence and practice recommendations.

    PubMed

    Verberk, Willem J; Omboni, Stefano; Kollias, Anastasios; Stergiou, George S

    2016-01-15

    Several guidelines recommend opportunistic screening for atrial fibrillation (AF) in subjects aged ≥ 65 years using pulse palpation during routine blood pressure (BP) measurement. However, this method has limited diagnostic accuracy. A specific algorithm for AF detection during automated BP measurement was developed and implemented in a novel oscillometric device (Microlife WatchBP Home-A). In 2013, the UK National Institute for Health and Care Excellence (NICE) recommended this device for AF screening during routine office BP measurement in primary care in subjects ≥ 65 years. A review and meta-analysis of the evidence on the diagnostic accuracy of this algorithm were performed. Six studies (n=2332) investigated the accuracy of AF detection using the Microlife BP monitor and estimated a pooled sensitivity at 0.98 (95% CI 0.95, 1.00) and specificity 0.92 (0.88, 0.96). Analysis of 4 studies (n=1126) showed more readings to improve specificity (from 0.86 to 0.91) and sensitivity (from 0.97 to 0.99). Taking 3 sequential readings with at least 2 detecting AF gave the highest diagnostic accuracy. A single study (n=139) of paroxysmal AF screening with home BP monitoring (3316 days) showed sensitivity 99% and specificity 93%. Another study (n=46) of AF screening with 24h ambulatory BP monitoring showed that AF detected in >15% of all readings has high probability of AF diagnosis requiring confirmation by 24h electrocardiography. AF detection with routine automated BP measurement is a reliable screening tool in the elderly, which requires confirmation by electrocardiography. Paroxysmal AF might also be detected by routine automated home or ambulatory BP monitoring. PMID:26547741

  20. High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

    PubMed

    Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

    2016-01-01

    High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. PMID:27367288

  1. Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds.

    PubMed

    Bilsland, Elizabeth; Sparkes, Andrew; Williams, Kevin; Moss, Harry J; de Clare, Michaela; Pir, Pinar; Rowland, Jem; Aubrey, Wayne; Pateman, Ron; Young, Mike; Carrington, Mark; King, Ross D; Oliver, Stephen G

    2013-02-01

    We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains' expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our 'hits' have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness. PMID:23446112

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

    SciTech Connect

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

    2009-05-26

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be 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.

  3. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  4. Classification of audiograms by sequential testing: reliability and validity of an automated behavioral hearing screening algorithm.

    PubMed

    Eilers, R E; Ozdamar, O; Steffens, M L

    1993-05-01

    In 1990, CAST (classification of audiograms by sequential testing) was proposed and developed as an automated, innovative approach to screening infant hearing using a modified Bayesian method. The method generated a four-frequency audiogram in a minimal number of test trials using VRA (visual reinforcement audiometry) techniques. Computer simulations were used to explore the properties (efficiency and accuracy) of the paradigm. The current work is designed to further test the utility of the paradigm with human infants and young children. Accordingly, infants and children between 6 months and 2 years of age were screened for hearing loss. The algorithm's efficacy was studied with respect to validity and reliability. Validity was evaluated by comparing CAST results with tympanometric data and outcomes of staircase-based testing. Test-retest reliability was also assessed. Results indicate that CAST is a valid, efficient, reliable, and potentially cost-effective screening method. PMID:8318708

  5. Automated measurement of Drosophila jump reflex habituation and its use for mutant screening.

    PubMed

    Sharma, Punita; Keane, John; O'Kane, Cahir J; Asztalos, Zoltan

    2009-08-30

    In habituation the probability of a behavioral response decreases with repeated presentations of a stimulus. This is a simple kind of learning since it involves an adaptive change in behavior due to experience. The present study describes a high-throughput semi-automated system to track movement of individual flies and score their jump response to repeated presentations of an odor. We find a decreased response on repeated presentations of odor, which a number of criteria suggest to be habituation. Tracking of up to sixteen flies simultaneously allows analysis of large numbers of flies for mutant screens. We demonstrate the use of the Autojump system for large-scale screens by conducting a pilot-scale screen of 150 P insert lines for habituation mutants. PMID:19520114

  6. Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening

    PubMed Central

    2014-01-01

    Background The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI). Methods Whole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out. Results The results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected. Conclusions Overall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks. PMID:25056295

  7. A Personalized Automated Messaging System to Improve Adherence to Prostate Cancer Screening: Research Protocol

    PubMed Central

    2012-01-01

    Background Public adherence to cancer screening guidelines is poor. Patient confusion over multiple recommendations and modalities for cancer screening has been found to be a major barrier to screening adherence. Such problems will only increase as screening guidelines and timetables become individualized. Objective We propose to increase compliance with cancer screening through two-way rich media mobile messaging based on personalized risk assessment. Methods We propose to develop and test a product that will store algorithms required to personalize cancer screening in a central database managed by a rule-based workflow engine, and implemented via messaging to the patient’s mobile phone. We will conduct a randomized controlled trial focusing on prostate cancer screening to study the hypothesis that mobile reminders improve adherence to screening guidelines. We will also explore a secondary hypothesis that patients who reply to the messaging reminders are more engaged and at lower risk of non-adherence. We will conduct a randomized controlled trial in a sample of males between 40 and 75 years (eligible for prostate cancer screening) who are willing to receive text messages, email, or automated voice messages. Participants will be recruited from a primary care clinic and asked to schedule prostate cancer screening at the clinic within the next 3 weeks. The intervention group will receive reminders and confirmation communications for making an appointment, keeping the appointment, and reporting the test results back to the investigators. Three outcomes will be evaluated: (1) the proportion of participants who make an appointment with a physician following a mobile message reminder, (2) the proportion of participants who keep the appointment, and (3) the proportion of participants who report the results of the screening (via text or Web). Results This is an ongoing project, supported by by a small business commercialization grant from the National Center for

  8. Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing

    PubMed Central

    2014-01-01

    Background The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. Results Here, we provide a cost effective method for the production of 96-well plate compatible zebrafish orientation tools using a desktop 3D printer. The printed tools enable the positioning and orientation of zebrafish embryos within cavities formed in agarose. Their applicability is demonstrated by acquiring lateral and dorsal views of zebrafish embryos arrayed within microtiter plates using an automated screening microscope. This enables the consistent visualization of morphological phenotypes and reporter gene expression patterns. Conclusions The designs are refined versions of previously demonstrated devices with added functionality and strongly reduced production costs. All corresponding 3D models are freely available and digital design can be easily shared electronically. In combination with the increasingly widespread usage of 3D printers, this provides access to the developed tools to a wide range of zebrafish users. Finally, the design files can serve as templates for other additive and subtractive fabrication methods. PMID:24886511

  9. Automated cervical precancerous cells screening system based on Fourier transform infrared spectroscopy features

    NASA Astrophysics Data System (ADS)

    Jusman, Yessi; Mat Isa, Nor Ashidi; Ng, Siew-Cheok; Hasikin, Khairunnisa; Abu Osman, Noor Azuan

    2016-07-01

    Fourier transform infrared (FTIR) spectroscopy technique can detect the abnormality of a cervical cell that occurs before the morphological change could be observed under the light microscope as employed in conventional techniques. This paper presents developed features extraction for an automated screening system for cervical precancerous cell based on the FTIR spectroscopy as a second opinion to pathologists. The automated system generally consists of the developed features extraction and classification stages. Signal processing techniques are used in the features extraction stage. Then, discriminant analysis and principal component analysis are employed to select dominant features for the classification process. The datasets of the cervical precancerous cells obtained from the feature selection process are classified using a hybrid multilayered perceptron network. The proposed system achieved 92% accuracy.

  10. Automated chip-on-carrier screening of a SOA integrated full band tunable laser (DSDBR)

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Dimitropoulos, George; Ward, Andrew J.; Yang, Guoyuan; Wu, Xuming

    2007-11-01

    Chip-on-carrier (CoC) sub-assemblies of Digital Ssupermode DBR (DSDBR) lasers are produced in high volume within Bookham manufacturing plants. These lasers can cover more than 100 ITU channels with a 50GHz channel range across the C or L band with a minimum 13dBm output power and 40dB side mode suppression ratio (SMSR). To guarantee a high quality and ensure a good yield, an automated screening process has been put in place at the CoC level to eliminate poor devices. Typical tuning maps and key performance features of the device are shown in this paper. We describe the general features of the tuning map, and indicate how a suitable operating point can be determined. The use of automated test kit is also described in this article. Finally, the performance of our device is presented in detail.

  11. A review of automated image understanding within 3D baggage computed tomography security screening.

    PubMed

    Mouton, Andre; Breckon, Toby P

    2015-01-01

    Baggage inspection is the principal safeguard against the transportation of prohibited and potentially dangerous materials at airport security checkpoints. Although traditionally performed by 2D X-ray based scanning, increasingly stringent security regulations have led to a growing demand for more advanced imaging technologies. The role of X-ray Computed Tomography is thus rapidly expanding beyond the traditional materials-based detection of explosives. The development of computer vision and image processing techniques for the automated understanding of 3D baggage-CT imagery is however, complicated by poor image resolutions, image clutter and high levels of noise and artefacts. We discuss the recent and most pertinent advancements and identify topics for future research within the challenging domain of automated image understanding for baggage security screening CT. PMID:26409422

  12. Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis.

    PubMed

    Rühmann, Broder; Schmid, Jochen; Sieber, Volker

    2016-01-01

    Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge

  13. Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

    PubMed Central

    Rühmann, Broder; Schmid, Jochen; Sieber, Volker

    2016-01-01

    Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge

  14. Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

    PubMed Central

    2010-01-01

    Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously

  15. High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell–Derived Cells

    PubMed Central

    Sirenko, Oksana; Hesley, Jayne; Rusyn, Ivan

    2014-01-01

    Abstract Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)–derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals. PMID:24229356

  16. Automated fabrication of back surface field silicon solar cells with screen printed wraparound contacts

    NASA Technical Reports Server (NTRS)

    Thornhill, J. W.

    1977-01-01

    The development of a process for fabricating 2 x 4 cm back surface field silicon solar cells having screen printed wraparound contacts is described. This process was specifically designed to be amenable for incorporation into the automated nonvacuum production line. Techniques were developed to permit the use of screen printing for producing improved back surface field structures, wraparound dielectric layers, and wraparound contacts. The optimized process sequence was then used to produce 1852 finished cells. Tests indicated an average conversion efficiency of 11% at AMO and 28 C, with an average degradation of maximum power output of 1.5% after boiling water immersion or thermal shock cycling. Contact adherence was satisfactory after these tests, as well as long term storage at high temperature and high humidity.

  17. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India.

    PubMed

    Rajput, Manoj Kumar

    2016-02-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  18. Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds.

    PubMed

    Bradow, James; Riley, Frank; Philippe, Laurence; Yan, Qi; Schuff, Brandon; Harris, Guy H

    2015-12-01

    A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes "on-demand" preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution-extrusion countercurrent chromatography was combined with non-dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass-directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow. PMID:26428946

  19. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India

    PubMed Central

    2016-01-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  20. Characterization and Optimization of a Novel Protein–Protein Interaction Biosensor High-Content Screening Assay to Identify Disruptors of the Interactions Between p53 and hDM2

    PubMed Central

    Dudgeon, Drew D.; Shinde, Sunita N.; Shun, Tong Ying; Lazo, John S.; Strock, Christopher J.; Giuliano, Kenneth A.; Taylor, D. Lansing; Johnston, Patricia A.

    2010-01-01

    Abstract We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein–protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further

  1. High-throughput screening of zebrafish embryos using automated heart detection and imaging.

    PubMed

    Spomer, Waldemar; Pfriem, Alexander; Alshut, Rüdiger; Just, Steffen; Pylatiuk, Christian

    2012-12-01

    Over the past decade, the zebrafish has become a key model organism in genetic screenings and drug discovery. A number of genes have been identified to affect the development of the shape and functioning of the heart, leading to zebrafish mutants with heart defects. The development of semiautomated microscopy systems has allowed for the investigation of drugs that reverse a disease phenotype on a larger scale. However, there is a lack of automated feature detection, and commercially available computer-aided microscopes are expensive. Screening of the zebrafish heart for drug discovery typically includes the identification of heart parameters, such as the frequency or fractional shortening. Until now, screening processes have been characterized by manual handling of the larvae and manual microscopy. Here, an intelligent robotic microscope is presented, which automatically identifies the orientation of a zebrafish in a micro well. A predefined region of interest, such as the heart, is detected automatically, and a video with higher magnification is recorded. Screening of a 96-well plate takes 35 to 55 min, depending on the length of the videos. Of the zebrafish hearts, 75% are recorded accurately without any user interaction. A description of the system, including the graphical user interface, is given. PMID:23053930

  2. Development and application of an automated, low-volume chromatography system for resin and condition screening.

    PubMed

    Teeters, Mark; Bezila, Daniel; Alred, Patricia; Velayudhan, Ajoy

    2008-10-01

    A small-volume chromatography system was developed for rapid resin and parameter screening and applied to the purification of a therapeutic monoclonal antibody from a key product-related impurity. Accounting for constraints in peripheral volume, gradient formation, column integrity, and fraction collection in microtiter plates, the resulting system employed 2-mL columns and was successfully integrated with plate-based methods for rapid sample analysis (e. g., use of automated liquid handlers, plate readers, and HPLC). Several cation-exchange chromatography resins were screened using automated programs and tailored gradients for the combination of a particular resin and a given antibody feedstock produced during Phase 1 development. Results from the tailored gradient runs were used to select a resin, and to arrive at efficient stepwise elution schedules for the chosen resin. By maintaining a constant residence time, final operating parameters were successfully scaled to representative bed heights and column diameters up to 2.6 cm (106 mL). This approach significantly improved throughput while reducing development time and material consumption. PMID:18543245

  3. An Automated Algorithm to Screen Massive Training Samples for a Global Impervious Surface Classification

    NASA Technical Reports Server (NTRS)

    Tan, Bin; Brown de Colstoun, Eric; Wolfe, Robert E.; Tilton, James C.; Huang, Chengquan; Smith, Sarah E.

    2012-01-01

    An algorithm is developed to automatically screen the outliers from massive training samples for Global Land Survey - Imperviousness Mapping Project (GLS-IMP). GLS-IMP is to produce a global 30 m spatial resolution impervious cover data set for years 2000 and 2010 based on the Landsat Global Land Survey (GLS) data set. This unprecedented high resolution impervious cover data set is not only significant to the urbanization studies but also desired by the global carbon, hydrology, and energy balance researches. A supervised classification method, regression tree, is applied in this project. A set of accurate training samples is the key to the supervised classifications. Here we developed the global scale training samples from 1 m or so resolution fine resolution satellite data (Quickbird and Worldview2), and then aggregate the fine resolution impervious cover map to 30 m resolution. In order to improve the classification accuracy, the training samples should be screened before used to train the regression tree. It is impossible to manually screen 30 m resolution training samples collected globally. For example, in Europe only, there are 174 training sites. The size of the sites ranges from 4.5 km by 4.5 km to 8.1 km by 3.6 km. The amount training samples are over six millions. Therefore, we develop this automated statistic based algorithm to screen the training samples in two levels: site and scene level. At the site level, all the training samples are divided to 10 groups according to the percentage of the impervious surface within a sample pixel. The samples following in each 10% forms one group. For each group, both univariate and multivariate outliers are detected and removed. Then the screen process escalates to the scene level. A similar screen process but with a looser threshold is applied on the scene level considering the possible variance due to the site difference. We do not perform the screen process across the scenes because the scenes might vary due to

  4. Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

    PubMed

    Froment, Jean; Thomas, Kevin V; Tollefsen, Knut Erik

    2016-08-01

    A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results. PMID:27085000

  5. Discovery of Overcoating Metal Oxides on Photoelectrode for Water Splitting by Automated Screening.

    PubMed

    Saito, Rie; Miseki, Yugo; Nini, Wang; Sayama, Kazuhiro

    2015-10-12

    We applied an automated semiconductor synthesis and screen system to discover overcoating film materials and optimize coating conditions on the BiVO4/WO3 composite photoelectrode to enhance stability and photocurrent. Thirteen metallic elements for overcoating oxides were examined with various coating amounts. The stability of the BiVO4/WO3 photoelectrode in a highly concentrated carbonate electrolyte aqueous solution was significantly improved by overcoating with Ta2O5 film, which was amorphous and porous when calcined at 550 °C. The photocurrent for the water oxidation reaction was only minimally inhibited by the presence of the Ta2O5 film on the BiVO4/WO3 photoelectrode. PMID:26325162

  6. Towards Exhaustive and Automated High-Throughput Screening for Crystalline Polymorphs

    PubMed Central

    2015-01-01

    Methods capable of exhaustively screening for crystal polymorphism remain an elusive goal in solid-state chemistry. Particularly promising among the new generation of approaches is polymer-induced heteronucleation (PIHn), a tool utilizing hundreds of unique polymers for granting kinetic access to polymorphs. Here PIHn is redeployed in a high density format in which 288 distinct polymers, each acting as a heteronucleant, are arrayed on one substrate. This format allows determining the outcome of thousands of crystallizations in an automated fashion with only a few milligrams of sample. This technology enables the study of a broader range of targets, including preclinical candidates, facilitating determination of polymorphism propensity much earlier in the drug development process. Here the efficacy of this approach is demonstrated using four pharmaceutically relevant compounds: acetaminophen, tolfenamic acid, ROY, and curcumin. PMID:24933573

  7. Automated measurement of mouse apolipoprotein B: convenient screening tool for mouse models of atherosclerosis.

    PubMed

    Levine, D M; Williams, K J

    1997-04-01

    Although mice are commonly used for studies of atherosclerosis, investigators have had no convenient way to quantify apolipoprotein (apo) B, the major protein of atherogenic lipoproteins, in this model. We now report an automated immunoturbidimetric assay for mouse apo B with an NCCLS imprecision study CV < 5%. Added hemoglobin up to 50 g/L did not interfere with the assay, nor did one freeze-thaw cycle of serum samples. Assay linearity extends to apo B concentrations of 325 mg/L. We have used the assay to determine serum apo B concentrations under several atherogenic conditions, including the apo E "knock-out" genotype and treatment with a high-cholesterol diet. Our assay can be used to survey inbred mouse strains for variants in apo B concentrations or regulation. Moreover, the mouse can now be used as a convenient small-animal model to screen compounds that may lower apo B concentrations. PMID:9105271

  8. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    PubMed

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  9. Delivering an Automated and Integrated Approach to Combination Screening Using Acoustic-Droplet Technology.

    PubMed

    Cross, Kevin; Craggs, Richard; Swift, Denise; Sitaram, Anesh; Daya, Sandeep; Roberts, Mark; Hawley, Shaun; Owen, Paul; Isherwood, Bev

    2016-02-01

    Drug combination testing in the pharmaceutical industry has typically been driven by late-stage opportunistic strategies rather than by early testing to identify drug combinations for clinical investigation that may deliver improved efficacy. A rationale for combinations exists across a number of diseases in which pathway redundancy or resistance to therapeutics are evident. However, early assays are complicated by the absence of both assay formats representative of disease biology and robust infrastructure to screen drug combinations in a medium-throughput capacity. When applying drug combination testing studies, it may be difficult to translate a study design into the required well contents for assay plates because of the number of compounds and concentrations involved. Dispensing these plates increases in difficulty as the number of compounds and concentration points increase and compounds are subsequently rolled onto additional labware. We describe the development of a software tool, in conjunction with the use of acoustic droplet technology, as part of a compound management platform, which allows the design of an assay incorporating combinations of compounds. These enhancements to infrastructure facilitate the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays. PMID:25835292

  10. Development of an Automated Microfluidic Reaction Platform for Multidimensional Screening: Reaction Discovery Employing Bicyclo[3.2.1]octanoid Scaffolds

    PubMed Central

    Goodell, John R.; McMullen, Jonathan P.; Zaborenko, Nikolay; Maloney, Jason R.; Ho, Chuan-Xing; Jensen, Klavs F.; Porco, John A.

    2010-01-01

    An automated, silicon-based microreactor system has been developed for rapid, low-volume, multidimensional reaction screening. Use of the microfluidic platform to identify transformations of densely functionalized bicyclo[3.2.1]octanoid scaffolds will be described. PMID:20560568

  11. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  12. Automated screening system for retinal health using bi-dimensional empirical mode decomposition and integrated index.

    PubMed

    Acharya, U Rajendra; Mookiah, Muthu Rama Krishnan; Koh, Joel E W; Tan, Jen Hong; Bhandary, Sulatha V; Rao, A Krishna; Fujita, Hamido; Hagiwara, Yuki; Chua, Chua Kuang; Laude, Augustinus

    2016-08-01

    Posterior Segment Eye Diseases (PSED) namely Diabetic Retinopathy (DR), glaucoma and Age-related Macular Degeneration (AMD) are the prime causes of vision loss globally. Vision loss can be prevented, if these diseases are detected at an early stage. Structural abnormalities such as changes in cup-to-disc ratio, Hard Exudates (HE), drusen, Microaneurysms (MA), Cotton Wool Spots (CWS), Haemorrhages (HA), Geographic Atrophy (GA) and Choroidal Neovascularization (CNV) in PSED can be identified by manual examination of fundus images by clinicians. However, manual screening is labour-intensive, tiresome and time consuming. Hence, there is a need to automate the eye screening. In this work Bi-dimensional Empirical Mode Decomposition (BEMD) technique is used to decompose fundus images into 2D Intrinsic Mode Functions (IMFs) to capture variations in the pixels due to morphological changes. Further, various entropy namely Renyi, Fuzzy, Shannon, Vajda, Kapur and Yager and energy features are extracted from IMFs. These extracted features are ranked using Chernoff Bound and Bhattacharyya Distance (CBBD), Kullback-Leibler Divergence (KLD), Fuzzy-minimum Redundancy Maximum Relevance (FmRMR), Wilcoxon, Receiver Operating Characteristics Curve (ROC) and t-test methods. Further, these ranked features are fed to Support Vector Machine (SVM) classifier to classify normal and abnormal (DR, AMD and glaucoma) classes. The performance of the proposed eye screening system is evaluated using 800 (Normal=400 and Abnormal=400) digital fundus images and 10-fold cross validation method. Our proposed system automatically identifies normal and abnormal classes with an average accuracy of 88.63%, sensitivity of 86.25% and specificity of 91% using 17 optimal features ranked using CBBD and SVM-Radial Basis Function (RBF) classifier. Moreover, a novel Retinal Risk Index (RRI) is developed using two significant features to distinguish two classes using single number. Such a system helps to reduce eye

  13. Automated, quantitative cognitive/behavioral screening of mice: for genetics, pharmacology, animal cognition and undergraduate instruction.

    PubMed

    Gallistel, C R; Balci, Fuat; Freestone, David; Kheifets, Aaron; King, Adam

    2014-01-01

    We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be

  14. Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

    PubMed Central

    Gallistel, C. R.; Balci, Fuat; Freestone, David; Kheifets, Aaron; King, Adam

    2014-01-01

    We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be

  15. Effects of Secondary Task Modality and Processing Code on Automation Trust and Utilization During Simulated Airline Luggage Screening

    NASA Technical Reports Server (NTRS)

    Phillips, Rachel; Madhavan, Poornima

    2010-01-01

    The purpose of this research was to examine the impact of environmental distractions on human trust and utilization of automation during the process of visual search. Participants performed a computer-simulated airline luggage screening task with the assistance of a 70% reliable automated decision aid (called DETECTOR) both with and without environmental distractions. The distraction was implemented as a secondary task in either a competing modality (visual) or non-competing modality (auditory). The secondary task processing code either competed with the luggage screening task (spatial code) or with the automation's textual directives (verbal code). We measured participants' system trust, perceived reliability of the system (when a target weapon was present and absent), compliance, reliance, and confidence when agreeing and disagreeing with the system under both distracted and undistracted conditions. Results revealed that system trust was lower in the visual-spatial and auditory-verbal conditions than in the visual-verbal and auditory-spatial conditions. Perceived reliability of the system (when the target was present) was significantly higher when the secondary task was visual rather than auditory. Compliance with the aid increased in all conditions except for the auditory-verbal condition, where it decreased. Similar to the pattern for trust, reliance on the automation was lower in the visual-spatial and auditory-verbal conditions than in the visual-verbal and auditory-spatial conditions. Confidence when agreeing with the system decreased with the addition of any kind of distraction; however, confidence when disagreeing increased with the addition of an auditory secondary task but decreased with the addition of a visual task. A model was developed to represent the research findings and demonstrate the relationship between secondary task modality, processing code, and automation use. Results suggest that the nature of environmental distractions influence

  16. Automated NMR Fragment Based Screening Identified a Novel Interface Blocker to the LARG/RhoA Complex

    PubMed Central

    Gao, Jia; Ma, Rongsheng; Wang, Wei; Wang, Na; Sasaki, Ryan; Snyderman, David; Wu, Jihui; Ruan, Ke

    2014-01-01

    The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to 15N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG. PMID:24505392

  17. Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

    NASA Astrophysics Data System (ADS)

    Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

    2011-03-01

    RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

  18. Development of a web-based tool for automated processing and cataloging of a unique combinatorial drug screen.

    PubMed

    Dalecki, Alex G; Wolschendorf, Frank

    2016-07-01

    Facing totally resistant bacteria, traditional drug discovery efforts have proven to be of limited use in replenishing our depleted arsenal of therapeutic antibiotics. Recently, the natural anti-bacterial properties of metal ions in synergy with metal-coordinating ligands have shown potential for generating new molecule candidates with potential therapeutic downstream applications. We recently developed a novel combinatorial screening approach to identify compounds with copper-dependent anti-bacterial properties. Through a parallel screening technique, the assay distinguishes between copper-dependent and independent activities against Mycobacterium tuberculosis with hits being defined as compounds with copper-dependent activities. These activities must then be linked to a compound master list to process and analyze the data and to identify the hit molecules, a labor intensive and mistake-prone analysis. Here, we describe a software program built to automate this analysis in order to streamline our workflow significantly. We conducted a small, 1440 compound screen against M. tuberculosis and used it as an example framework to build and optimize the software. Though specifically adapted to our own needs, it can be readily expanded for any small- to medium-throughput screening effort, parallel or conventional. Further, by virtue of the underlying Linux server, it can be easily adapted for chemoinformatic analysis of screens through packages such as OpenBabel. Overall, this setup represents an easy-to-use solution for streamlining processing and analysis of biological screening data, as well as offering a scaffold for ready functionality expansion. PMID:27117032

  19. A high-content, high-throughput siRNA screen identifies cyclin D2 as a potent regulator of muscle progenitor cell fusion and a target to enhance muscle regeneration.

    PubMed

    Khanjyan, Michael V; Yang, Jonathan; Kayali, Refik; Caldwell, Thomas; Bertoni, Carmen

    2013-08-15

    Cell-mediated regenerative approaches using muscle progenitor cells hold promises for the treatment of many forms of muscle disorders. Their applicability in the clinic, however, is hindered by the low levels of regeneration obtained after transplantation and the large number of cells required to achieve an effect. To better understand the mechanisms that regulate the temporal switch of replicating muscle progenitor cells into terminally differentiated cells and to develop new strategies that could enhance muscle regeneration, we have developed and performed a high-throughput screening (HTS) capable of identifying genes that play active roles during myogenesis. Secondary and tertiary screens were used to confirm the effects of RNAi in vitro and in vivo and to select for candidate hits that significantly increase regeneration into skeletal muscles. Downregulation of cyclin D2 (CCND2) was shown to dramatically enhance myogenic differentiation of muscle progenitor cells and to induce a robust regeneration after cell transplantation into skeletal muscles of dystrophin-deficient mice. Protein interaction network and pathway analysis revealed that CCND2 directly interacts with the cyclin-dependent kinase Cdk4 to inhibit phosphorylation of the retinoblastoma protein (pRb), thus blocking the activation of the myogenic switch during fusion. These studies identify CCND2 as a new key regulator of terminal differentiation in muscle progenitor cells and open new possibilities for the treatment of many forms of muscle disorders characterized by impaired regeneration and loss of muscle mass. PMID:23612904

  20. Development of a quantitative morphological assessment of toxicant-treated zebrafish larvae using brightfield imaging and high-content analysis.

    PubMed

    Deal, Samantha; Wambaugh, John; Judson, Richard; Mosher, Shad; Radio, Nick; Houck, Keith; Padilla, Stephanie

    2016-09-01

    One of the rate-limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time-consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high-content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V(TI) high-content image analysis platform. The basic visual assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (total area, width, spine length, head-tail length, length-width ratio, perimeter-area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high-content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans-retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26924781

  1. Automated high-content morphological analysis of muscle fiber histology.

    PubMed

    Miazaki, Mauro; Viana, Matheus P; Yang, Zhong; Comin, Cesar H; Wang, Yaming; da F Costa, Luciano; Xu, Xiaoyin

    2015-08-01

    In the search for a cure for many muscular disorders it is often necessary to analyze muscle fibers under a microscope. For this morphological analysis, we developed an image processing approach to automatically analyze and quantify muscle fiber images so as to replace today's less accurate and time-consuming manual method. Muscular disorders, that include cardiomyopathy, muscular dystrophies, and diseases of nerves that affect muscles such as neuropathy and myasthenia gravis, affect a large percentage of the population and, therefore, are an area of active research for new treatments. In research, the morphological features of muscle fibers play an important role as they are often used as biomarkers to evaluate the progress of underlying diseases and the effects of potential treatments. Such analysis involves assessing histopathological changes of muscle fibers as indicators for disease severity and also as a criterion in evaluating whether or not potential treatments work. However, quantifying morphological features is time-consuming, as it is usually performed manually, and error-prone. To replace this standard method, we developed an image processing approach to automatically detect and measure the cross-sections of muscle fibers observed under microscopy that produces faster and more objective results. As such, it is well-suited to processing the large number of muscle fiber images acquired in typical experiments, such as those from studies with pre-clinical models that often create many images. Tests on real images showed that the approach can segment and detect muscle fiber membranes and extract morphological features from highly complex images to generate quantitative results that are readily available for statistical analysis. PMID:26004825

  2. ClinicalTrials.gov as a Data Source for Semi-Automated Point-Of-Care Trial Eligibility Screening

    PubMed Central

    Pfiffner, Pascal B.; Oh, JiWon; Miller, Timothy A.; Mandl, Kenneth D.

    2014-01-01

    Background Implementing semi-automated processes to efficiently match patients to clinical trials at the point of care requires both detailed patient data and authoritative information about open studies. Objective To evaluate the utility of the ClinicalTrials.gov registry as a data source for semi-automated trial eligibility screening. Methods Eligibility criteria and metadata for 437 trials open for recruitment in four different clinical domains were identified in ClinicalTrials.gov. Trials were evaluated for up to date recruitment status and eligibility criteria were evaluated for obstacles to automated interpretation. Finally, phone or email outreach to coordinators at a subset of the trials was made to assess the accuracy of contact details and recruitment status. Results 24% (104 of 437) of trials declaring on open recruitment status list a study completion date in the past, indicating out of date records. Substantial barriers to automated eligibility interpretation in free form text are present in 81% to up to 94% of all trials. We were unable to contact coordinators at 31% (45 of 146) of the trials in the subset, either by phone or by email. Only 53% (74 of 146) would confirm that they were still recruiting patients. Conclusion Because ClinicalTrials.gov has entries on most US and many international trials, the registry could be repurposed as a comprehensive trial matching data source. Semi-automated point of care recruitment would be facilitated by matching the registry's eligibility criteria against clinical data from electronic health records. But the current entries fall short. Ultimately, improved techniques in natural language processing will facilitate semi-automated complex matching. As immediate next steps, we recommend augmenting ClinicalTrials.gov data entry forms to capture key eligibility criteria in a simple, structured format. PMID:25334031

  3. Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy.

    PubMed

    Iannetti, Eligio F; Smeitink, Jan A M; Beyrath, Julien; Willems, Peter H G M; Koopman, Werner J H

    2016-09-01

    Mitochondria have a central role in cellular (patho)physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), calcein-AM (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are subjected to a quality control (QC) algorithm based upon principal component analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of ∼4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening. PMID:27560174

  4. High-content analysis of sequential events during the early phase of influenza A virus infection.

    PubMed

    Banerjee, Indranil; Yamauchi, Yohei; Helenius, Ari; Horvath, Peter

    2013-01-01

    Influenza A virus (IAV) represents a worldwide threat to public health by causing severe morbidity and mortality every year. Due to high mutation rate, new strains of IAV emerge frequently. These IAVs are often drug-resistant and require vaccine reformulation. A promising approach to circumvent this problem is to target host cell determinants crucial for IAV infection, but dispensable for the cell. Several RNAi-based screens have identified about one thousand cellular factors that promote IAV infection. However, systematic analyses to determine their specific functions are lacking. To address this issue, we developed quantitative, imaging-based assays to dissect seven consecutive steps in the early phases of IAV infection in tissue culture cells. The entry steps for which we developed the assays were: virus binding to the cell membrane, endocytosis, exposure to low pH in endocytic vacuoles, acid-activated fusion of viral envelope with the vacuolar membrane, nucleocapsid uncoating in the cytosol, nuclear import of viral ribonucleoproteins, and expression of the viral nucleoprotein. We adapted the assays to automated microscopy and optimized them for high-content screening. To quantify the image data, we performed both single and multi-parametric analyses, in combination with machine learning. By time-course experiments, we determined the optimal time points for each assay. Our quality control experiments showed that the assays were sufficiently robust for high-content analysis. The methods we describe in this study provide a powerful high-throughput platform to understand the host cell processes, which can eventually lead to the discovery of novel anti-pathogen strategies. PMID:23874633

  5. Evaluation of an Automated Information Extraction Tool for Imaging Data Elements to Populate a Breast Cancer Screening Registry.

    PubMed

    Lacson, Ronilda; Harris, Kimberly; Brawarsky, Phyllis; Tosteson, Tor D; Onega, Tracy; Tosteson, Anna N A; Kaye, Abby; Gonzalez, Irina; Birdwell, Robyn; Haas, Jennifer S

    2015-10-01

    Breast cancer screening is central to early breast cancer detection. Identifying and monitoring process measures for screening is a focus of the National Cancer Institute's Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) initiative, which requires participating centers to report structured data across the cancer screening continuum. We evaluate the accuracy of automated information extraction of imaging findings from radiology reports, which are available as unstructured text. We present prevalence estimates of imaging findings for breast imaging received by women who obtained care in a primary care network participating in PROSPR (n = 139,953 radiology reports) and compared automatically extracted data elements to a "gold standard" based on manual review for a validation sample of 941 randomly selected radiology reports, including mammograms, digital breast tomosynthesis, ultrasound, and magnetic resonance imaging (MRI). The prevalence of imaging findings vary by data element and modality (e.g., suspicious calcification noted in 2.6% of screening mammograms, 12.1% of diagnostic mammograms, and 9.4% of tomosynthesis exams). In the validation sample, the accuracy of identifying imaging findings, including suspicious calcifications, masses, and architectural distortion (on mammogram and tomosynthesis); masses, cysts, non-mass enhancement, and enhancing foci (on MRI); and masses and cysts (on ultrasound), range from 0.8 to1.0 for recall, precision, and F-measure. Information extraction tools can be used for accurate documentation of imaging findings as structured data elements from text reports for a variety of breast imaging modalities. These data can be used to populate screening registries to help elucidate more effective breast cancer screening processes. PMID:25561069

  6. Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

    PubMed Central

    Kota, Krishna P.; Eaton, Brett; Lane, Douglas; Ulrich, Melanie; Ulrich, Ricky; Peyser, Brian D.; Robinson, Camenzind G.; Jaissle, James G.; Pegoraro, Gianluca; Bavari, Sina; Panchal, Rekha G.

    2013-01-01

    The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics. PMID:23383093

  7. A timetable organizer for the planning and implementation of screenings in manual or semi-automation mode.

    PubMed

    Goktug, Asli N; Chai, Sergio C; Chen, Taosheng

    2013-09-01

    We have designed an Excel spreadsheet to facilitate the planning and execution of screenings performed manually or in semi-automation mode, following a sequential set of events. Many assays involve multiple steps, often including time-sensitive stages, thus complicating the proper implementation to ensure that all plates are treated equally to achieve reliable outcomes. The spreadsheet macro presented in this study analyzes and breaks down the timings for all tasks, calculates the limitation in the number of plates that suit the desired parameters, and allows for optimization based on tolerance of time delay and equal treatment of plates when possible. The generated Gantt charts allow for visual inspection of the screening process and provide timings in a tabulated form to assist the user to conduct the experiments as projected by the software. The program can be downloaded from http://sourceforge.net/projects/sams-hts/. PMID:23653394

  8. Constraint factor graph cut–based active contour method for automated cellular image segmentation in RNAi screening

    PubMed Central

    CHEN, C.; LI, H.; ZHOU, X.; WONG, S. T. C.

    2010-01-01

    Summary Image-based, high throughput genome-wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time-consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome-wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale-adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi-channel image screening data. PMID:18445146

  9. Validation of an automated screening method for persistent organic contaminants in fats and oils by GC×GC-ToFMS.

    PubMed

    López, Patricia; Tienstra, Marc; Lommen, Arjen; Mol, Hans G J

    2016-11-15

    An screening method, comprised of straightforward sample treatment based on silica clean-up, GC×GC-ToFMS detection and automated data processing with the non-proprietary free downloadable software MetAlignID, has been successfully validated with respect to false negatives for the sum PCB 28, 52, 101, 138, 153 and 180), for the sum of BDE 28, 47, 99, 100, 153, 154 and 183, for the four markers of PAHs and for a number of emerging brominated flame retardants. A screening detection limit (SDL) equal to or lower than the maximum regulatory level was always achieved. MetAlignID considerably decreased the time needed for data treatment from 20 to 5min/file. Automated identification of the signature mass spectral patterns was applied to identify chlorinated- and brominated-containing substances with more than two halogen atoms, and PAH derivates. Although the success rate was variable and needs to be further improved, the tool was considered to be of added value. PMID:27283679

  10. Feasibility of Using Low-Cost Motion Capture for Automated Screening of Shoulder Motion Limitation after Breast Cancer Surgery

    PubMed Central

    Gritsenko, Valeriya; Dailey, Eric; Kyle, Nicholas; Taylor, Matt; Whittacre, Sean; Swisher, Anne K.

    2015-01-01

    Objective To determine if a low-cost, automated motion analysis system using Microsoft Kinect could accurately measure shoulder motion and detect motion impairments in women following breast cancer surgery. Design Descriptive study of motion measured via 2 methods. Setting Academic cancer center oncology clinic. Participants 20 women (mean age = 60 yrs) were assessed for active and passive shoulder motions during a routine post-operative clinic visit (mean = 18 days after surgery) following mastectomy (n = 4) or lumpectomy (n = 16) for breast cancer. Interventions Participants performed 3 repetitions of active and passive shoulder motions on the side of the breast surgery. Arm motion was recorded using motion capture by Kinect for Windows sensor and on video. Goniometric values were determined from video recordings, while motion capture data were transformed to joint angles using 2 methods (body angle and projection angle). Main Outcome Measure Correlation of motion capture with goniometry and detection of motion limitation. Results Active shoulder motion measured with low-cost motion capture agreed well with goniometry (r = 0.70–0.80), while passive shoulder motion measurements did not correlate well. Using motion capture, it was possible to reliably identify participants whose range of shoulder motion was reduced by 40% or more. Conclusions Low-cost, automated motion analysis may be acceptable to screen for moderate to severe motion impairments in active shoulder motion. Automatic detection of motion limitation may allow quick screening to be performed in an oncologist's office and trigger timely referrals for rehabilitation. PMID:26076031

  11. Sequential operation droplet array: an automated microfluidic platform for picoliter-scale liquid handling, analysis, and screening.

    PubMed

    Zhu, Ying; Zhang, Yun-Xia; Cai, Long-Fei; Fang, Qun

    2013-07-16

    This contribution describes a sequential operation droplet array (SODA) system, a fully automated droplet-based microfluidic system capable of performing picoliter-scale liquid manipulation, analysis, and screening. The SODA system was built using a tapered capillary-syringe pump module and a two-dimensional (2D) oil-covered droplet array installed on an x-y-z translation stage. With the system, we developed a novel picoliter-scale droplet depositing technique for forming a 2D picoliter-droplet array. On this basis, an automated droplet manipulation method with picoliter precision was established using the programmable combination of the capillary-based liquid aspirating-depositing and the moving of the oil-covered droplet array, the so-called "aspirating-depositing-moving" (ADM) method. Differing from the previously reported droplet systems based on microchips, microcapillaries, or digital microfluidics, this method can achieve complete and flexible droplet manipulations, including droplet assembling, generation, indexing, transferring, splitting, and fusion in the picoliter range, endowing the present system with ultralow sample/reagent consumptions and substantial versatility in analysis and screening for multiple different samples. To demonstrate its feasibility and versatility, we applied the SODA system in multiple experiments required in drug screening, including the screening of inhibitors for capases-1 from a chemical library, the measurement of IC50 values for the identified inhibitors, and the screening of the synergistic effect of multiple inhibitors. In the experiments, the consumptions of samples and reagents are only 60-180 pL for each droplet microreactor, which are commonly 3-5 orders of magnitude lower than those of conventional multiwell plate systems, and 1-2 orders of magnitude lower than other droplet-based microfluidic systems for multiple sample screening. The ability of the SODA system in performing complicated and multistep droplet

  12. Automated urine screening devices make urine sediment microscopy in diagnostic laboratories economically viable.

    PubMed

    Zaman, Zahur

    2015-11-01

    Automated urinalysis devices are reproducible, accurate and faster than the standard manual microscopy. Economic analysis has shown that decreases in turn-around-time and labour cost savings offered by these devices make them more economic than manual microscopy. PMID:26057217

  13. An automated screening technique for the detection of sickle-cell haemoglobin

    PubMed Central

    Canning, D. M.; Crane, R. S.; Huntsman, R. G.; Yawson, G. I.

    1972-01-01

    An automated technique is described which is capable of detecting sickle-cell haemoglobin and differentiating the sickle-cell trait from sickle-cell anaemia. The method is based upon the Itano solubility test and utilizes Technicon equipment. Images PMID:5028640

  14. Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis.

    PubMed

    An, Philip; Winters, Dwight; Walker, Kenneth W

    2016-04-01

    Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process. PMID:26678961

  15. An automated tuberculosis screening strategy combining X-ray-based computer-aided detection and clinical information

    PubMed Central

    Melendez, Jaime; Sánchez, Clara I.; Philipsen, Rick H. H. M.; Maduskar, Pragnya; Dawson, Rodney; Theron, Grant; Dheda, Keertan; van Ginneken, Bram

    2016-01-01

    Lack of human resources and radiological interpretation expertise impair tuberculosis (TB) screening programmes in TB-endemic countries. Computer-aided detection (CAD) constitutes a viable alternative for chest radiograph (CXR) reading. However, no automated techniques that exploit the additional clinical information typically available during screening exist. To address this issue and optimally exploit this information, a machine learning-based combination framework is introduced. We have evaluated this framework on a database containing 392 patient records from suspected TB subjects prospectively recruited in Cape Town, South Africa. Each record comprised a CAD score, automatically computed from a CXR, and 12 clinical features. Comparisons with strategies relying on either CAD scores or clinical information alone were performed. Our results indicate that the combination framework outperforms the individual strategies in terms of the area under the receiving operating characteristic curve (0.84 versus 0.78 and 0.72), specificity at 95% sensitivity (49% versus 24% and 31%) and negative predictive value (98% versus 95% and 96%). Thus, it is believed that combining CAD and clinical information to estimate the risk of active disease is a promising tool for TB screening. PMID:27126741

  16. An automated tuberculosis screening strategy combining X-ray-based computer-aided detection and clinical information.

    PubMed

    Melendez, Jaime; Sánchez, Clara I; Philipsen, Rick H H M; Maduskar, Pragnya; Dawson, Rodney; Theron, Grant; Dheda, Keertan; van Ginneken, Bram

    2016-01-01

    Lack of human resources and radiological interpretation expertise impair tuberculosis (TB) screening programmes in TB-endemic countries. Computer-aided detection (CAD) constitutes a viable alternative for chest radiograph (CXR) reading. However, no automated techniques that exploit the additional clinical information typically available during screening exist. To address this issue and optimally exploit this information, a machine learning-based combination framework is introduced. We have evaluated this framework on a database containing 392 patient records from suspected TB subjects prospectively recruited in Cape Town, South Africa. Each record comprised a CAD score, automatically computed from a CXR, and 12 clinical features. Comparisons with strategies relying on either CAD scores or clinical information alone were performed. Our results indicate that the combination framework outperforms the individual strategies in terms of the area under the receiving operating characteristic curve (0.84 versus 0.78 and 0.72), specificity at 95% sensitivity (49% versus 24% and 31%) and negative predictive value (98% versus 95% and 96%). Thus, it is believed that combining CAD and clinical information to estimate the risk of active disease is a promising tool for TB screening. PMID:27126741

  17. ASSESSMENT OF SYNAPSE FORMATION IN RAT PRIMARY NEURAL CELL CULTURE USING HIGH CONTENT MICROSCOPY.

    EPA Science Inventory

    Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of high content screening (HCS) to dete...

  18. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system

    PubMed Central

    Yoshimoto, Nobuo; Tatematsu, Kenji; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D.; Fujii, Ikuo; Kondo, Akihiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2014-01-01

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 106 peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening. PMID:24577528

  19. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

    PubMed

    Yoshimoto, Nobuo; Tatematsu, Kenji; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D; Fujii, Ikuo; Kondo, Akihiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2014-01-01

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening. PMID:24577528

  20. Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry

    PubMed Central

    Stolker, Alida A. M.; Peters, Ruud J. B.; Zuiderent, Richard; DiBussolo, Joseph M.

    2010-01-01

    There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 µg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 µg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained. PMID:20379812

  1. Evaluation of automated direct sample introduction with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for the screening analysis of dioxins of fish oil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An automated direct sample introduction technique coupled to comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (DSI-GC×GC/TOF-MS) was applied for the development of a relatively fast and easy analytical screening method for 17 polychlorinated dibenzo-p-dioxins/dibenzo...

  2. Automated cloud screening of AVHRR imagery using split-and-merge clustering

    NASA Technical Reports Server (NTRS)

    Gallaudet, Timothy C.; Simpson, James J.

    1991-01-01

    Previous methods to segment clouds from ocean in AVHRR imagery have shown varying degrees of success, with nighttime approaches being the most limited. An improved method of automatic image segmentation, the principal component transformation split-and-merge clustering (PCTSMC) algorithm, is presented and applied to cloud screening of both nighttime and daytime AVHRR data. The method combines spectral differencing, the principal component transformation, and split-and-merge clustering to sample objectively the natural classes in the data. This segmentation method is then augmented by supervised classification techniques to screen clouds from the imagery. Comparisons with other nighttime methods demonstrate its improved capability in this application. The sensitivity of the method to clustering parameters is presented; the results show that the method is insensitive to the split-and-merge thresholds.

  3. Application of high-content image analysis for quantitatively estimating lipid accumulation in oleaginous yeasts with potential for use in biodiesel production.

    PubMed

    Capus, Aurélie; Monnerat, Marianne; Ribeiro, Luiz Carlos; de Souza, Wanderley; Martins, Juliana Lopes; Sant'Anna, Celso

    2016-03-01

    Biodiesel from oleaginous microorganisms is a viable substitute for a fossil fuel. Current methods for microorganism lipid productivity evaluation do not analyze lipid dynamics in single cells. Here, we described a high-content image analysis (HCA) as a promising strategy for screening oleaginous microorganisms for biodiesel production, while generating single-cell lipid dynamics data in large cell density. Rhodotorula slooffiae yeast were grown in standard (CTL) or lipid trigger medium (LTM), and lipid droplet (LD) accumulation was analyzed in deconvolved confocal microscopy images of cells stained with the lipophilic fluorescent Nile red (NR) dye using automated cell and LD segmentation. The 'vesicle segmentation' method yielded valid morphometric results for limited lipid accumulation in smaller LDs (CTL samples) and for high lipid accumulation in larger LDs (LTM samples), and detected LD localization changes. Thus, HCA can be used to analyze the lipid accumulation patterns likely to be encountered in screens for biodiesel production. PMID:26744805

  4. A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

    PubMed Central

    Kota, Krishna P.; Soloveva, Veronica; Wanner, Laura M.; Gomba, Glenn; Kiris, Erkan; Panchal, Rekha G.; Kane, Christopher D.; Bavari, Sina

    2014-01-01

    Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system. PMID:25489815

  5. A high content imaging assay for identification of Botulinum neurotoxin inhibitors.

    PubMed

    Kota, Krishna P; Soloveva, Veronica; Wanner, Laura M; Gomba, Glenn; Kiris, Erkan; Panchal, Rekha G; Kane, Christopher D; Bavari, Sina

    2014-01-01

    Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system. PMID:25489815

  6. iMSRC: converting a standard automated microscope into an intelligent screening platform

    PubMed Central

    Carro, Angel; Perez-Martinez, Manuel; Soriano, Joaquim; Pisano, David G.; Megias, Diego

    2015-01-01

    Microscopy in the context of biomedical research is demanding new tools to automatically detect and capture objects of interest. The few extant packages addressing this need, however, have enjoyed limited uptake due to complexity of use and installation. To overcome these drawbacks, we developed iMSRC, which combines ease of use and installation with high flexibility and enables applications such as rare event detection and high-resolution tissue sample screening, saving time and resources. PMID:26015081

  7. iMSRC: converting a standard automated microscope into an intelligent screening platform.

    PubMed

    Carro, Angel; Perez-Martinez, Manuel; Soriano, Joaquim; Pisano, David G; Megias, Diego

    2015-01-01

    Microscopy in the context of biomedical research is demanding new tools to automatically detect and capture objects of interest. The few extant packages addressing this need, however, have enjoyed limited uptake due to complexity of use and installation. To overcome these drawbacks, we developed iMSRC, which combines ease of use and installation with high flexibility and enables applications such as rare event detection and high-resolution tissue sample screening, saving time and resources. PMID:26015081

  8. Comparison of Clinical and Automated Breast Density Measurements: Implications for Risk Prediction and Supplemental Screening.

    PubMed

    Brandt, Kathleen R; Scott, Christopher G; Ma, Lin; Mahmoudzadeh, Amir P; Jensen, Matthew R; Whaley, Dana H; Wu, Fang Fang; Malkov, Serghei; Hruska, Carrie B; Norman, Aaron D; Heine, John; Shepherd, John; Pankratz, V Shane; Kerlikowske, Karla; Vachon, Celine M

    2016-06-01

    Purpose To compare the classification of breast density with two automated methods, Volpara (version 1.5.0; Matakina Technology, Wellington, New Zealand) and Quantra (version 2.0; Hologic, Bedford, Mass), with clinical Breast Imaging Reporting and Data System (BI-RADS) density classifications and to examine associations of these measures with breast cancer risk. Materials and Methods In this study, 1911 patients with breast cancer and 4170 control subjects matched for age, race, examination date, and mammography machine were evaluated. Participants underwent mammography at Mayo Clinic or one of four sites within the San Francisco Mammography Registry between 2006 and 2012 and provided informed consent or a waiver for research, in compliance with HIPAA regulations and institutional review board approval. Digital mammograms were retrieved a mean of 2.1 years (range, 6 months to 6 years) before cancer diagnosis, with the corresponding clinical BI-RADS density classifications, and Volpara and Quantra density estimates were generated. Agreement was assessed with weighted κ statistics among control subjects. Breast cancer associations were evaluated with conditional logistic regression, adjusted for age and body mass index. Odds ratios, C statistics, and 95% confidence intervals (CIs) were estimated. Results Agreement between clinical BI-RADS density classifications and Volpara and Quantra BI-RADS estimates was moderate, with κ values of 0.57 (95% CI: 0.55, 0.59) and 0.46 (95% CI: 0.44, 0.47), respectively. Differences of up to 14% in dense tissue classification were found, with Volpara classifying 51% of women as having dense breasts, Quantra classifying 37%, and clinical BI-RADS assessment used to classify 43%. Clinical and automated measures showed similar breast cancer associations; odds ratios for extremely dense breasts versus scattered fibroglandular densities were 1.8 (95% CI: 1.5, 2.2), 1.9 (95% CI: 1.5, 2.5), and 2.3 (95% CI: 1.9, 2.8) for Volpara, Quantra

  9. Automated eligibility screening and monitoring for genotype-driven precision oncology trials.

    PubMed

    Eubank, Michael H; Hyman, David M; Kanakamedala, Amritha D; Gardos, Stuart M; Wills, Jonathan M; Stetson, Peter D

    2016-07-01

    The Information Systems Department at Memorial Sloan Kettering Cancer Center developed the DARWIN Cohort Management System (DCMS). The DCMS identifies and tracks cohorts of patients based on genotypic and clinical data. It assists researchers and treating physicians in enrolling patients to genotype-matched IRB-approved clinical trials. The DCMS sends automated, actionable, and secure email notifications to users with information about eligible or enrolled patients before their upcoming appointments. The system also captures investigators input via annotations on patient eligibility and preferences on future status updates. As of August 2015, the DCMS is tracking 159,893 patients on both clinical operations and research cohorts. 134 research cohorts have been established and track 64,473 patients. 51,192 of these have had one or more genomic tests including MSK-IMPACT, comprising the pool eligible for genotype-matched studies. This paper describes the design and evolution of this Informatics solution. PMID:27016727

  10. Integrated chip-based physiometer for automated fish embryo toxicity biotests in pharmaceutical screening and ecotoxicology.

    PubMed

    Akagi, Jin; Zhu, Feng; Hall, Chris J; Crosier, Kathryn E; Crosier, Philip S; Wlodkowic, Donald

    2014-06-01

    Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micromechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo-trapping suction manifold, drug delivery manifold, and optically transparent indium tin oxide heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves, and embedded miniaturized fluorescent USB microscope. Our results showed that the innovative device has 100% embryo-trapping efficiency while supporting normal embryo development for up to 72 hr in a confined microfluidic environment. We also showed data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational antiangiogenic agents in transgenic zebrafish lines. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the lab-on-a-chip systems a step closer to realization of complete analytical automation. PMID:24664821

  11. Automated phenotype pattern recognition of zebrafish for high-throughput screening.

    PubMed

    Schutera, Mark; Dickmeis, Thomas; Mione, Marina; Peravali, Ravindra; Marcato, Daniel; Reischl, Markus; Mikut, Ralf; Pylatiuk, Christian

    2016-07-01

    Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented. PMID:27285638

  12. Sustainable synthesis and automated deposition: an accessible discovery screening library of fragment-like purines.

    PubMed

    Kamper, Christoph; Korpis, Katharina; Specker, Edgar; Anger, Lennart; Neuenschwander, Martin; Bednarski, Patrick J; Link, Andreas

    2012-08-01

    A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays. PMID:22890959

  13. Fully automated screening of immunocytochemically stained specimens for early cancer detection

    NASA Astrophysics Data System (ADS)

    Bell, André A.; Schneider, Timna E.; Müller-Frank, Dirk A. C.; Meyer-Ebrecht, Dietrich; Böcking, Alfred; Aach, Til

    2007-03-01

    Cytopathological cancer diagnoses can be obtained less invasive than histopathological investigations. Cells containing specimens can be obtained without pain or discomfort, bloody biopsies are avoided, and the diagnosis can, in some cases, even be made earlier. Since no tissue biopsies are necessary these methods can also be used in screening applications, e.g., for cervical cancer. Among the cytopathological methods a diagnosis based on the analysis of the amount of DNA in individual cells achieves high sensitivity and specificity. Yet this analysis is time consuming, which is prohibitive for a screening application. Hence, it will be advantageous to retain, by a preceding selection step, only a subset of suspicious specimens. This can be achieved using highly sensitive immunocytochemical markers like p16 ink4a for preselection of suspicious cells and specimens. We present a method to fully automatically acquire images at distinct positions at cytological specimens using a conventional computer controlled microscope and an autofocus algorithm. Based on the thus obtained images we automatically detect p16 ink4a-positive objects. This detection in turn is based on an analysis of the color distribution of the p16 ink4a marker in the Lab-colorspace. A Gaussian-mixture-model is used to describe this distribution and the method described in this paper so far achieves a sensitivity of up to 90%.

  14. Automated Zebrafish Chorion Removal and Single Embryo Placement: Optimizing Throughput of Zebrafish Developmental Toxicity Screens

    PubMed Central

    Mandrell, David; Truong, Lisa; Jephson, Caleb; Sarker, Mushfiqur R.; Moore, Aaron; Lang, Christopher; Simonich, Michael T.; Tanguay, Robert L.

    2012-01-01

    The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly characterized permeability enclosing the developing embryo. We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. The robotic embryo placement allocated 6-hpf embryos to 94.7% ± 4.2% of the wells in multiple 96-well trials. The rate of embryo mortality was 2.8% (43 of 1536) from robotic handling, the rate of missed wells was 1.2% (18 of 1536), and the frequency of multipicks was <0.1%. Embryo malformations observed at 24 hpf occurred nearly twice as frequently from robotic handling (16 of 864; 1.9%) as from manual pipetting (9 of 864; 1%). There was no statistical difference between the success of performing the embryo placement robotically or manually. PMID:22357610

  15. Keeping participants on board: increasing uptake by automated respondent reminders in an Internet-based Chlamydia Screening in the Netherlands

    PubMed Central

    2012-01-01

    Background Effectiveness of Chlamydia screening programs is determined by an adequate level of participation and the capturing of high-risk groups. This study aimed to evaluate the contribution of automated reminders by letter, email and short message service (SMS) on package request and sample return in an Internet-based Chlamydia screening among people aged 16 to 29 years in the Netherlands. Methods Individuals not responding to the invitation letter received a reminder letter after 1 month. Email- and SMS-reminders were sent to persons who did not return their sample. It was examined to what extent reminders enhanced the response rate (% of package requests) and participation rate (% of sample return). Sociodemographic and behavioural correlates of providing a cell phone number and participation after the reminder(s) were studied by logistic regression models. Results Of all respondents (screening round 1: 52,628, round 2: 41,729), 99% provided an email address and 72% a cell phone number. Forty-two percent of all package requests were made after the reminder letter. The proportion of invitees returning a sample increased significantly from 10% to 14% after email/SMS reminders (round 2: from 7% to 10%). Determinants of providing a cell-phone number were younger age (OR in 25-29 year olds versus 16-19 year olds = 0.8, 95%CI 0.8-0.9), non-Dutch (OR in Surinam/Antillean versus Dutch = 1.3, 95%CI 1.2-1.4, Turkish/Moroccan: 1.1, 95%CI 1.0-1.2, Sub Sahara African: 1.5, 95%CI 1.3-1.8, non-Western other 1.1, 95%CI 1.1-1.2), lower educational level (OR in high educational level versus low level = 0.8, 95%CI 0.7-0.9), no condom use during the last contact with a casual partner (OR no condom use versus condom use 1.2, 95%CI 1.1-1.3), younger age at first sexual contact (OR 19 years or older versus younger than 16: 0.7, 95%CI 0.6-0.8). Determinants for requesting a test-package after the reminder letter were male gender (OR female versus male 0.9 95%CI 0.8-0.9), non

  16. Discriminative power of an assay for automated in vitro screening of teratogens.

    PubMed

    Walmod, Peter S; Gravemann, Ute; Nau, Heinz; Berezin, Vladimir; Bock, Elisabeth

    2004-08-01

    Screening for potential teratogenicity of 20 test compounds was performed using a computerised microscope workstation for determination of cytotoxicity, proliferation and morphology of fibroblastoid murine L929-cells. The test compounds, which were divided into four classes according to teratogenicity were: 5-bromo-2(')-deoxyuridine, 6-aminonicotinamide, acrylamide, boric acid, D-(+)-camphor, dimethadione, dimethyl phthalate, diphenhydramine, hydroxyurea, isobutyl-ethyl-valproic acid, lithium chloride, methyl mercury chloride, methotrexate, methoxyacetic acid, penicillin G, all-trans-retinoic acid, pentyl-4-yn-valproic acid, saccharin, salicylic acid and valproic acid. All compounds, with the exception of dimethadione inhibited proliferation in a linear dose-dependent manner, and there were statistically significant compound class-dependent differences between the IC(50)-values for the compounds (p<0.0374), the strongest teratogens being the most potent. Furthermore, the average efficacies (maximum relative change) for 10 parameters describing cell morphology exhibited statistically significant compound class-dependent differences (p<0.0001), the class I and II compounds exhibiting significantly lower efficacies than the class III and IV compounds (p<0.01). Thus, test compounds affected both the proliferation and morphology of L-cells in manner demonstrating a general relationship with the teratogenic potency of the compounds. However, the moderate teratogens dimethadione and lithium chloride only had minor effects on the morphology and proliferation of the cells whereas the non-teratogen diphenhydramine had effects on both proliferation and morphology comparable to the strong teratogens. PMID:15130609

  17. Automated scheme for measuring mandibular cortical thickness on dental panoramic radiographs for osteoporosis screening

    NASA Astrophysics Data System (ADS)

    Matsumoto, T.; Hayashi, T.; Hara, T.; Katsumata, A.; Muramatsu, C.; Zhou, X.; Iida, Y.; Matsuoka, M.; Katagi, Ki.; Fujita, H.

    2012-03-01

    Findings of dental panoramic radiographs (DPRs) have shown that the mandibular cortical thickness (MCT) was significantly correlated with osteoporosis. Identifying asymptomatic patients with osteoporosis through dental examinations may bring a supplemental benefit for the patients. However, most of the DPRs are used for only diagnosing dental conditions by dentists in their routine clinical work. The aim of this study was to develop a computeraided diagnosis scheme that automatically measures MCT to assist dentists in screening osteoporosis. First, the inferior border of mandibular bone was detected by use of an active contour method. Second, the locations of mental foramina were estimated on the basis of the inferior border of mandibular bone. Finally, MCT was measured on the basis of the grayscale profile analysis. One hundred DPRs were used to evaluate our proposed scheme. Experimental results showed that the sensitivity and specificity for identifying osteoporotic patients were 92.6 % and 100 %, respectively. We conducted multiclinic trials, in which 223 cases have been obtained and processed in about a month. Our scheme succeeded in detecting all cases of suspected osteoporosis. Therefore, our scheme may have a potential to identify osteoporotic patients at an early stage.

  18. Bilateral Image Subtraction and Multivariate Models for the Automated Triaging of Screening Mammograms

    PubMed Central

    Celaya-Padilla, José; Martinez-Torteya, Antonio; Rodriguez-Rojas, Juan; Galvan-Tejada, Jorge; Treviño, Victor; Tamez-Peña, José

    2015-01-01

    Mammography is the most common and effective breast cancer screening test. However, the rate of positive findings is very low, making the radiologic interpretation monotonous and biased toward errors. This work presents a computer-aided diagnosis (CADx) method aimed to automatically triage mammogram sets. The method coregisters the left and right mammograms, extracts image features, and classifies the subjects into risk of having malignant calcifications (CS), malignant masses (MS), and healthy subject (HS). In this study, 449 subjects (197 CS, 207 MS, and 45 HS) from a public database were used to train and evaluate the CADx. Percentile-rank (p-rank) and z-normalizations were used. For the p-rank, the CS versus HS model achieved a cross-validation accuracy of 0.797 with an area under the receiver operating characteristic curve (AUC) of 0.882; the MS versus HS model obtained an accuracy of 0.772 and an AUC of 0.842. For the z-normalization, the CS versus HS model achieved an accuracy of 0.825 with an AUC of 0.882 and the MS versus HS model obtained an accuracy of 0.698 and an AUC of 0.807. The proposed method has the potential to rank cases with high probability of malignant findings aiding in the prioritization of radiologists work list. PMID:26240818

  19. Automated screening of reversed-phase stationary phases for small-molecule separations using liquid chromatography with mass spectrometry.

    PubMed

    Appulage, Dananjaya K; Wang, Evelyn H; Carroll, Frances; Schug, Kevin A

    2016-05-01

    There are various reversed-phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed-phase stationary phases (aqueous stable C18 , biphenyl, pentafluorophenyl propyl, and polar-embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine-like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar-embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C18 phase. The electron-rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C18 with a methanolic phase, but it behaved very similarly to a C18 when an acetonitrile-based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation. PMID:26959840

  20. Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

    PubMed

    Riccardi Sirtori, Federico; Caronni, Dannica; Colombo, Maristella; Dalvit, Claudio; Paolucci, Mauro; Regazzoni, Luca; Visco, Carlo; Fogliatto, Gianpaolo

    2015-08-30

    ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with

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

    PubMed

    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. PMID:21717479

  2. The motion sensitivity screening test in clinical practice in abuja, Nigeria: affordable automated perimetry for the third world?

    PubMed

    Babalola, O E

    2005-06-01

    Perimetry is essential in the clinical management and evaluation of glaucoma patients and other patients with diseases impacting on visual fields, but automated equipment may be too expensive for many practitioners in the developing world. I have used the Wu-Jones automated motion sensitivity system in a medium sized practice in Nigeria, a developing country, and hereby present an audit of our experience with it. The Wu-Jones Motion Sensitivity screening test is a lap-top computer based test which integrates a number of components including a test program and reporting facility, a self organizing neural network, a database management mechanism, and a menu-mouse-windowing user interface. The test is available on the public domain and is small enough (194 mb) to fit into a diskette. This test has been used at the Rachel Eye Center in Abuja since 1998, and has been applied to 339 individuals, 298 of whom are included in this analysis. Patients tested fell into four main groups: those with clinical glaucoma (intraocular pressure > 20 mmHg on at least one occasion and optic cup/disc ratio of 0.5 or more), glaucoma suspects, (i.e. ocular hypertensives >20 mmHg or c/d ratio of 0.5 or more and first degree relatives of glaucoma patients) patients undergoing routine tests for pre-employment ('normals'), and 'others'. These 'normals' were used as controls. Records are available for 531 eyes. It took an average of two minutes to complete the test. Significant field defects (Motion sensitivity less than 50%) were detected overall in 15.6% of tested eyes, 7.2% of normals but in 32.6% of glaucoma eyes. Using the 'normals' as controls, the sensitivity of the test in our hands varied from 33% to 72% and specificity from 57% to 93% at motion sensitivity cut off points from 50% to 97%. At the 83% cut off point, positive and negative predictive values were 86.0% and 47.5% respectively. Reliability averaged 70%. I find the test easy to administer and understand by patients. Results

  3. Using high-content imaging data from ToxCast to analyze toxicological tipping points (TDS)

    EPA Science Inventory

    Translating results obtained from high-throughput screening to risk assessment is vital for reducing dependence on animal testing. We studied the effects of 976 chemicals (ToxCast Phase I and II) in HepG2 cells using high-content imaging (HCI) to measure dose and time-depende...

  4. Evaluation of the SediMax automated microscopy sediment analyzer and the Sysmex UF-1000i flow cytometer as screening tools to rule out negative urinary tract infections.

    PubMed

    Íñigo, Melania; Coello, Andreu; Fernández-Rivas, Gema; Carrasco, María; Marcó, Clara; Fernández, Anabel; Casamajor, Teresa; Ausina, Vicente

    2016-05-01

    Urinary tract infections (UTI) are highly prevalent in nosocomial and community settings, and their diagnosis is costly and time-consuming. Screening methods represent an important advance towards the final UTI diagnosis, diminishing inappropriate treatment or clinical complications. Automated analyzers have been developed and commercialized to screen and rule out negative urine samples. The aim of this study was to evaluate two of these automated analyzers (SediMax, an automatic sediment analyzer and UF-1000i a flow cytometer) to predict negative urine cultures. A total of 1934 urine samples were analyzed. A very strong correlation for white blood cells (WBC) (rs: 0.928) and a strong correlation for bacteria (BAC) (rs: 0.693) were obtained. We also calculated optimal cut-off points for both autoanalyzers: 18WBC/μL and 97BAC/μL for SediMax (sensitivity=96.25%, specificity=63.04%, negative predictive value=97.97%), and 40WBC/μL and 460BAC/μL for UF-1000i (sensitivity=98.13%, specificity=79.16%, negative predictive value=99.18%). The use of SediMax and UF-1000i resulted in a 46.33% and 57.19% reduction of all samples cultured, respectively. In conclusion, both analyzers are good UTI screening tools in our setting. PMID:26921459

  5. [Non-target screening of organic pollutants in sediments and sludges using gas chromatography-mass spectrometry and automated mass spectral deconvolution].

    PubMed

    Wang, Gang; Ma, Huilian; Wang, Longxing; Chen, Jiping; Hou, Xiaohong

    2015-12-01

    A screening method in the combination of ultrasonic extraction, gas chromatography-mass spectrometry detection and automated mass spectrometry deconvolution technique was developed for non-target screening of non-polar and weak polar pollutants in sediments and sludges. The samples were extracted by ultrasonication for 20 min using dichloromethane for three times. The extraction solutions were cleaned-up by gel permeation chromatography and a silica gel column, and then 3 g of copper powder was used to remove the sulfur by ultrasonication for 10 min. Parallel experiments were carried out for 5 times and the RSDs were ranged from 5.8% to 14.9%. Automated mass spectral deconvolution & identification system (AMDIS) would improve the resolution of overlapping peaks, and identify the pure mass spectrum of the analytes in the cases of stronger background interference and co-extracted substances covering. Standard spectrum databases, such as NISTDRUG, NISTEPA, NISTFDA, Mass Spectral Library, etc, would qualitatively identify the organic pollutants in the samples. As a result, a total of 290 organic pollutants were identified, of which 190 and 153 pollutants were found in sediments and sludges, respectively. The identified pollutants included the Environmental Protection Agency (EPA) priority pollutants, pharmaceuticals, herbicides, antioxidants, intermediates, organic solvents and chemical raw materials. The proposed method is proved to be a promising one for non-target screening of complex matrix samples with the advantages of higher sensitivity and better repeatability. PMID:27097463

  6. Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay.

    PubMed

    Wille, T; Thiermann, H; Worek, F

    2010-04-01

    Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system. PMID:19961920

  7. DG-AMMOS: A New tool to generate 3D conformation of small molecules using Distance Geometry and Automated Molecular Mechanics Optimization for in silico Screening

    PubMed Central

    2009-01-01

    Background Discovery of new bioactive molecules that could enter drug discovery programs or that could serve as chemical probes is a very complex and costly endeavor. Structure-based and ligand-based in silico screening approaches are nowadays extensively used to complement experimental screening approaches in order to increase the effectiveness of the process and facilitating the screening of thousands or millions of small molecules against a biomolecular target. Both in silico screening methods require as input a suitable chemical compound collection and most often the 3D structure of the small molecules has to be generated since compounds are usually delivered in 1D SMILES, CANSMILES or in 2D SDF formats. Results Here, we describe the new open source program DG-AMMOS which allows the generation of the 3D conformation of small molecules using Distance Geometry and their energy minimization via Automated Molecular Mechanics Optimization. The program is validated on the Astex dataset, the ChemBridge Diversity database and on a number of small molecules with known crystal structures extracted from the Cambridge Structural Database. A comparison with the free program Balloon and the well-known commercial program Omega generating the 3D of small molecules is carried out. The results show that the new free program DG-AMMOS is a very efficient 3D structure generator engine. Conclusion DG-AMMOS provides fast, automated and reliable access to the generation of 3D conformation of small molecules and facilitates the preparation of a compound collection prior to high-throughput virtual screening computations. The validation of DG-AMMOS on several different datasets proves that generated structures are generally of equal quality or sometimes better than structures obtained by other tested methods. PMID:19912625

  8. The Gray Institute ‘open’ high-content, fluorescence lifetime microscopes

    PubMed Central

    BARBER, PR; TULLIS, IDC; PIERCE, GP; NEWMAN, RG; PRENTICE, J; ROWLEY, MI; MATTHEWS, DR; AMEER-BEG, SM; VOJNOVIC, B

    2013-01-01

    Summary We describe a microscopy design methodology and details of microscopes built to this ‘open’ design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam ‘end-stations’ at Oxford and Surrey Universities, showing the versatility and extendibility of this approach. PMID:23772985

  9. Automated Fast Screening Method for Cocaine Identification in Seized Drug Samples Using a Portable Fourier Transform Infrared (FT-IR) Instrument.

    PubMed

    Mainali, Dipak; Seelenbinder, John

    2016-05-01

    Quick and presumptive identification of seized drug samples without destroying evidence is necessary for law enforcement officials to control the trafficking and abuse of drugs. This work reports an automated screening method to detect the presence of cocaine in seized samples using portable Fourier transform infrared (FT-IR) spectrometers. The method is based on the identification of well-defined characteristic vibrational frequencies related to the functional group of the cocaine molecule and is fully automated through the use of an expert system. Traditionally, analysts look for key functional group bands in the infrared spectra and characterization of the molecules present is dependent on user interpretation. This implies the need for user expertise, especially in samples that likely are mixtures. As such, this approach is biased and also not suitable for non-experts. The method proposed in this work uses the well-established "center of gravity" peak picking mathematical algorithm and combines it with the conditional reporting feature in MicroLab software to provide an automated method that can be successfully employed by users with varied experience levels. The method reports the confidence level of cocaine present only when a certain number of cocaine related peaks are identified by the automated method. Unlike library search and chemometric methods that are dependent on the library database or the training set samples used to build the calibration model, the proposed method is relatively independent of adulterants and diluents present in the seized mixture. This automated method in combination with a portable FT-IR spectrometer provides law enforcement officials, criminal investigators, or forensic experts a quick field-based prescreening capability for the presence of cocaine in seized drug samples. PMID:27006022

  10. Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.

    PubMed

    Jensen, Nickels A; Gerth, Klaus; Grotjohann, Tim; Kapp, Dieter; Keck, Matthias; Niehaus, Karsten

    2009-03-10

    High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (<10%) and apoptotic (<5%) cells. Treatment with the reference substances blasticidin, colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts. PMID:19111838

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

    PubMed Central

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

    2016-01-01

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

  12. Combining High-Content Imaging and Phenotypic Classification Analysis of Senescence-Associated Beta-Galactosidase Staining to Identify Regulators of Oncogene-Induced Senescence.

    PubMed

    Chan, Keefe T; Paavolainen, Lassi; Hannan, Katherine M; George, Amee J; Hannan, Ross D; Simpson, Kaylene J; Horvath, Peter; Pearson, Richard B

    2016-09-01

    Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets. High-throughput screening provides a powerful platform for target discovery. Here, we describe an approach to use RNAi transfection of a pre-established AKT-induced senescent cell population and subsequent high-content imaging to screen for senescence regulators. We have incorporated multiparametric readouts, including cell number, proliferation, and senescence-associated beta-galactosidase (SA-βGal) staining. Using machine learning and automated image analysis, we also describe methods to classify distinct phenotypes of cells with SA-βGal staining. These methods can be readily adaptable to high-throughput functional screens interrogating the mechanisms that maintain and prevent senescence in various contexts. PMID:27552145

  13. High-Throughput Screening of NaV1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument.

    PubMed

    Chambers, Chris; Witton, Ian; Adams, Cathryn; Marrington, Luke; Kammonen, Juha

    2016-03-01

    Voltage-gated sodium (NaV) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, NaV1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel NaV1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube(®) is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a NaV1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC50 assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC50 potencies generated on the Qube correlate with data generated on the more established Sophion QPatch(®) APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study NaV1.7 in a robust and reliable APC assay for compound screening. PMID:26991360

  14. Dexterous robotic manipulation of alert adult Drosophila for high-content experimentation

    PubMed Central

    Huang, Cheng; Maxey, Jessica R.; Schnitzer, Mark J.

    2015-01-01

    We present a robot that enables high-content studies of alert adult Drosophila by combining operations including gentle picking, translations and rotations, characterizations of fly phenotypes and behaviors, micro-dissection or release. To illustrate, we assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies’ complex attributes and behavioral patterns. PMID:26005812

  15. Dexterous robotic manipulation of alert adult Drosophila for high-content experimentation.

    PubMed

    Savall, Joan; Ho, Eric Tatt Wei; Huang, Cheng; Maxey, Jessica R; Schnitzer, Mark J

    2015-07-01

    We present a robot that enables high-content studies of alert adult Drosophila by combining operations including gentle picking; translations and rotations; characterizations of fly phenotypes and behaviors; microdissection; or release. To illustrate, we assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies' complex attributes and behavioral patterns. PMID:26005812

  16. High-content analysis of Rab protein function at the ER-Golgi interface

    PubMed Central

    Galea, George; Simpson, Jeremy C

    2015-01-01

    ABSTRACT The Rab family of small GTPases play fundamental roles in the regulation of trafficking pathways between intracellular membranes in eukaryotic cells. In this short commentary we highlight a recent high-content screening study that investigates the roles of Rab proteins in retrograde trafficking from the Golgi complex to the endoplasmic reticulum, and we discuss how the findings of this work and other literature might influence our thoughts on how the architecture of the Golgi complex is regulated. PMID:26693811

  17. Comparative evaluation of two fully-automated real-time PCR methods for MRSA admission screening in a tertiary-care hospital.

    PubMed

    Hos, N J; Wiegel, P; Fischer, J; Plum, G

    2016-09-01

    We evaluated two fully-automated real-time PCR systems, the novel QIAGEN artus MRSA/SA QS-RGQ and the widely used BD MAX MRSA assay, for their diagnostic performance in MRSA admission screening in a tertiary-care university hospital. Two hundred sixteen clinical swabs were analyzed for MRSA DNA using the BD MAX MRSA assay. In parallel, the same specimens were tested with the QIAGEN artus MRSA/SA QS-RGQ. Automated steps included lysis of bacteria, DNA extraction, real-time PCR and interpretation of results. MRSA culture was additionally performed as a reference method for MRSA detection. Sensitivity values were similar for both assays (80 %), while the QIAGEN artus MRSA/SA QS-RGQ reached a slightly higher specificity (95.8 % versus 90.0 %). Positive (PPVs) and negative predictive values (NPVs) were 17.4 % and 99.4 % for the BD MAX MRSA assay and 33.3 % and 99.5 % for the QIAGEN artus MRSA/SA QS-RGQ, respectively. Total turn-around time (TAT) for 24 samples was 3.5 hours for both assays. In conclusion, both assays represent reliable diagnostic tools due to their high negative predictive values, especially for the rapid identification of MRSA negative patients in a low prevalence MRSA area. PMID:27259711

  18. Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media: implementation of automated Quanterix SIMOA instrumentation.

    PubMed

    Pleil, Joachim D; Angrish, Michelle M; Madden, Michael C

    2015-12-01

    Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1β and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2)  >  0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels. PMID:26658359

  19. Automated pancreatic cyst screening using natural language processing: a new tool in the early detection of pancreatic cancer

    PubMed Central

    Roch, Alexandra M; Mehrabi, Saeed; Krishnan, Anand; Schmidt, Heidi E; Kesterson, Joseph; Beesley, Chris; Dexter, Paul R; Palakal, Mathew; Schmidt, C Max

    2015-01-01

    Introduction As many as 3% of computed tomography (CT) scans detect pancreatic cysts. Because pancreatic cysts are incidental, ubiquitous and poorly understood, follow-up is often not performed. Pancreatic cysts may have a significant malignant potential and their identification represents a ‘window of opportunity’ for the early detection of pancreatic cancer. The purpose of this study was to implement an automated Natural Language Processing (NLP)-based pancreatic cyst identification system. Method A multidisciplinary team was assembled. NLP-based identification algorithms were developed based on key words commonly used by physicians to describe pancreatic cysts and programmed for automated search of electronic medical records. A pilot study was conducted prospectively in a single institution. Results From March to September 2013, 566 233 reports belonging to 50 669 patients were analysed. The mean number of patients reported with a pancreatic cyst was 88/month (range 78–98). The mean sensitivity and specificity were 99.9% and 98.8%, respectively. Conclusion NLP is an effective tool to automatically identify patients with pancreatic cysts based on electronic medical records (EMR). This highly accurate system can help capture patients ‘at-risk’ of pancreatic cancer in a registry. PMID:25537257

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

    PubMed

    Wang, Hongzhi; Liu, Jin; 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. Application of Fragment Ion Information as Further Evidence in Probabilistic Compound Screening Using Bayesian Statistics and Machine Learning: A Leap Toward Automation.

    PubMed

    Woldegebriel, Michael; Zomer, Paul; Mol, Hans G J; Vivó-Truyols, Gabriel

    2016-08-01

    In this work, we introduce an automated, efficient, and elegant model to combine all pieces of evidence (e.g., expected retention times, peak shapes, isotope distributions, fragment-to-parent ratio) obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS) data for screening purposes. Combining all these pieces of evidence requires a careful assessment of the uncertainties in the analytical system as well as all possible outcomes. To-date, the majority of the existing algorithms are highly dependent on user input parameters. Additionally, the screening process is tackled as a deterministic problem. In this work we present a Bayesian framework to deal with the combination of all these pieces of evidence. Contrary to conventional algorithms, the information is treated in a probabilistic way, and a final probability assessment of the presence/absence of a compound feature is computed. Additionally, all the necessary parameters except the chromatographic band broadening for the method are learned from the data in training and learning phase of the algorithm, avoiding the introduction of a large number of user-defined parameters. The proposed method was validated with a large data set and has shown improved sensitivity and specificity in comparison to a threshold-based commercial software package. PMID:27391247

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

    PubMed Central

    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

  3. Automated storage of gas chromatography-mass spectrometry data in a relational database to facilitate compound screening and identification.

    PubMed

    Staeb, J A; Epema, O J; van Duijn, P; Steevens, J; Klap, V A; Freriks, I L

    2002-10-18

    This paper describes a database containing massspectra from gas chromatography-mass spectrometry(GC-MS) measurements as a tool for easy screening for multiple compounds. In this way additional compounds can be reported from the same run together with routine pesticide monitoring with little effort. The relevant analytical data from the GC-MS measurements are transferred automatically to a database. Search algorithms in the database, containing the US EPA and Dutch NEN GC-MS identification criteria as standard settings, are used to identify compounds in the data. Screening of samples analysed in our laboratory show the ubiquitous presence of--up until now in monitoring largely overlooked--compounds in surface waters in The Netherlands. Most frequently found compounds include TAED (complexing agent), 2-methyl quinoline (industrial solvent), atrazin and desethylatrazin (pesticide and degradation product), caffeine (human consumption), surfinol-104 (anti foaming agent), HHCB (Galaxolide) and AHTN (Tonalide; fragrances). The database can also be used to quickly search a large number of datafiles for rare contaminants. This way, some interesting compounds such as pentoxifilin (a pharmaceutical) and Irgarol 1051 (an antifouling compound) were found. PMID:12458939

  4. Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

    EPA Science Inventory

    Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

  5. Toxicology screen

    MedlinePlus

    Barbiturates - screen; Benzodiazepines - screen; Amphetamines - screen; Analgesics - screen; Antidepressants - screen; Narcotics - screen; Phenothiazines - screen; Drug abuse screen; Blood alcohol test

  6. Label-free cytotoxicity screening assay by digital holographic microscopy.

    PubMed

    Kühn, Jonas; Shaffer, Etienne; Mena, Julien; Breton, Billy; Parent, Jérôme; Rappaz, Benjamin; Chambon, Marc; Emery, Yves; Magistretti, Pierre; Depeursinge, Christian; Marquet, Pierre; Turcatti, Gerardo

    2013-03-01

    We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy. PMID:23062077

  7. Effectiveness of a Web-Based Screening and Fully Automated Brief Motivational Intervention for Adolescent Substance Use: A Randomized Controlled Trial

    PubMed Central

    Elgán, Tobias H; De Paepe, Nina; Tønnesen, Hanne; Csémy, Ladislav; Thomasius, Rainer

    2016-01-01

    Background Mid-to-late adolescence is a critical period for initiation of alcohol and drug problems, which can be reduced by targeted brief motivational interventions. Web-based brief interventions have advantages in terms of acceptability and accessibility and have shown significant reductions of substance use among college students. However, the evidence is sparse among adolescents with at-risk use of alcohol and other drugs. Objective This study evaluated the effectiveness of a targeted and fully automated Web-based brief motivational intervention with no face-to-face components on substance use among adolescents screened for at-risk substance use in four European countries. Methods In an open-access, purely Web-based randomized controlled trial, a convenience sample of adolescents aged 16-18 years from Sweden, Germany, Belgium, and the Czech Republic was recruited using online and offline methods and screened online for at-risk substance use using the CRAFFT (Car, Relax, Alone, Forget, Friends, Trouble) screening instrument. Participants were randomized to a single session brief motivational intervention group or an assessment-only control group but not blinded. Primary outcome was differences in past month drinking measured by a self-reported AUDIT-C-based index score for drinking frequency, quantity, and frequency of binge drinking with measures collected online at baseline and after 3 months. Secondary outcomes were the AUDIT-C-based separate drinking indicators, illegal drug use, and polydrug use. All outcome analyses were conducted with and without Expectation Maximization (EM) imputation of missing follow-up data. Results In total, 2673 adolescents were screened and 1449 (54.2%) participants were randomized to the intervention or control group. After 3 months, 211 adolescents (14.5%) provided follow-up data. Compared to the control group, results from linear mixed models revealed significant reductions in self-reported past-month drinking in favor of the

  8. Energy efficiency by use of automated energy-saving windows with heat-reflective screens and solar battery for power supply systems of European and Russian buildings

    NASA Astrophysics Data System (ADS)

    Zakharov, V. M.; Smirnov, N. N.; Tyutikov, V. V.; Flament, B.

    2015-10-01

    The new energy saving windows with heat-reflecting shields have been developed, and for their practical use they need to be integrated into the automated system for controlling heat supply in buildings and the efficiency of their use together with the existing energy-saving measures must be determined. The study was based on the results of field tests of windows with heat-reflective shields in a certified climate chamber. The method to determine the minimum indoor air temperature under standby heating using heat-reflective shields in the windows and multifunctional energy-efficient shutter with solar battery have been developed. Annual energy saving for the conditions of different regions of Russia and France was determined. Using windows with heat-reflecting screens and a solar battery results in a triple power effect: reduced heat losses during the heating season due to increased window resistance; lower cost of heating buildings due to lowering of indoor ambient temperature; also electric power generation.

  9. A touch screen-automated cognitive test battery reveals impaired attention, memory abnormalities, and increased response inhibition in the TgCRND8 mouse model of Alzheimer's disease

    PubMed Central

    Romberg, Carola; Horner, Alexa E.; Bussey, Timothy J.; Saksida, Lisa M.

    2013-01-01

    Transgenic mouse models of Alzheimer's disease (AD) with abundant β-amyloid develop memory impairments. However, multiple nonmnemonic cognitive domains such as attention and executive control are also compromised early in AD individuals, but have not been routinely assessed in animal models. Here, we assessed the cognitive abilities of TgCRND8 mice—a widely used model of β-amyloid pathology—with a touch screen-based automated test battery. The test battery comprises highly translatable tests of multiple cognitive constructs impaired in human AD, such as memory, attention, and response control, as well as appropriate control tasks. We found that familial AD mutations affect not only memory, but also cause significant alterations of sustained attention and behavioral flexibility. Because changes in attention and response inhibition may affect performance on tests of other cognitive abilities including memory, our findings have important consequences for the assessment of disease mechanisms and therapeutics in animal models of AD. A more comprehensive phenotyping with specialized, multicomponent cognitive test batteries for mice might significantly advance translation from preclinical mouse studies to the clinic. PMID:22959727

  10. Sub-population analysis based on temporal features of high content images

    PubMed Central

    2009-01-01

    Background High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems. PMID:19958514