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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 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

  2. 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.

  3. A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity

    PubMed Central

    Yasgar, Adam; Titus, Steven A.; Wang, Yuhong; Danchik, Carina; Yang, Shyh-Ming; Vasiliou, Vasilis; Jadhav, Ajit; Maloney, David J.; Simeonov, Anton

    2017-01-01

    Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules. PMID:28129349

  4. The beautiful cell: high-content screening in drug discovery.

    PubMed

    Bickle, Marc

    2010-09-01

    The term "high-content screening" has become synonymous with imaging screens using automated microscopes and automated image analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments and analyzing the data have come to maturity, so the full benefits of high-content screening can now be realized. Those benefits are the capability of carrying out phenotypic multiparametric cellular assays in an unbiased, fully automated, and quantitative fashion. Automated microscopes and automated image analysis are being applied at all stages of the drug discovery and development pipeline. All major pharmaceutical companies have adopted the technology and it is in the process of being embraced broadly by the academic community. This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.

  5. High Content Screening in Neurodegenerative Diseases

    PubMed Central

    Jain, Shushant; van Kesteren, Ronald E.; Heutink, Peter

    2012-01-01

    neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets. PMID:22257990

  6. Increasing the Content of High-Content Screening

    PubMed Central

    Singh, Shantanu; Genovesio, Auguste

    2014-01-01

    Target-based high-throughput screening (HTS) has recently been critiqued for its relatively poor yield compared to phenotypic screening approaches. One type of phenotypic screening, image-based high-content screening (HCS), has been seen as particularly promising. In this article, we assess whether HCS is as high content as it can be. We analyze HCS publications and find that although the number of HCS experiments published each year continues to grow steadily, the information content lags behind. We find that a majority of high-content screens published so far (60−80%) made use of only one or two image-based features measured from each sample and disregarded the distribution of those features among each cell population. We discuss several potential explanations, focusing on the hypothesis that data analysis traditions are to blame. This includes practical problems related to managing large and multidimensional HCS data sets as well as the adoption of assay quality statistics from HTS to HCS. Both may have led to the simplification or systematic rejection of assays carrying complex and valuable phenotypic information. We predict that advanced data analysis methods that enable full multiparametric data to be harvested for entire cell populations will enable HCS to finally reach its potential. PMID:24710339

  7. Functional genomic and high-content screening for target discovery and deconvolution

    PubMed Central

    Heynen-Genel, Susanne; Pache, Lars; Chanda, Sumit K

    2014-01-01

    Introduction Functional genomic screens apply knowledge gained from the sequencing of the human genome toward rapid methods of identifying genes involved in cellular function based on a specific phenotype. This approach has been made possible through the use of advances in both molecular biology and automation. The utility of this approach has been further enhanced through the application of image-based high content screening, an automated microscopy and quantitative image analysis platform. These approaches can significantly enhance acquisition of novel targets for drug discovery. Areas covered Both the utility and potential issues associated with functional genomic screening approaches are discussed along with examples that illustrate both. The considerations for high content screening applied to functional genomics are also presented. Expert opinion Functional genomic and high content screening are extremely useful in the identification of new drug targets. However, the technical, experimental, and computational parameters have an enormous influence on the results. Thus, although new targets are identified, caution should be applied toward interpretation of screening data in isolation. Genomic screens should be viewed as an integral component of a target identification campaign that requires both the acquisition of orthogonal data, as well as a rigorous validation strategy. PMID:22860749

  8. 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.

  9. Facilitating drug discovery: an automated high-content inflammation assay in zebrafish.

    PubMed

    Wittmann, Christine; Reischl, Markus; Shah, Asmi H; Mikut, Ralf; Liebel, Urban; Grabher, Clemens

    2012-07-16

    Zebrafish larvae are particularly amenable to whole animal small molecule screens due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos or tailfin amputation. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED2 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area

  10. 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.

  11. 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

  12. 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.

  13. High-content screening as a universal tool for fingerprinting of cytotoxicity of nanoparticles.

    PubMed

    Jan, Edward; Byrne, Stephen J; Cuddihy, Meghan; Davies, Anthony M; Volkov, Yuri; Gun'ko, Yurii K; Kotov, Nicholas A

    2008-05-01

    Recent advances and progress in nanobiotechnology have demonstrated many nanoparticles (NPs) as potential and novel drug delivery vehicles, therapeutic agents, and contrast agents and luminescent biological labels for bioimaging. The emergence of new biomedical applications based on NPs signifies the need to understand, compare, and manage their cytotoxicity. In this study, we demonstrated the use of high-content screening assay (HCA) as a universal tool to probe the cytotoxicity of NPs and specifically cadmium telluride quantum dots (CdTe QDs) and gold NPs (Au NPs) in NG108-15 murine neuroblastoma cells and HepG2 human hepatocellular carcinoma cells. Neural cells represent special interest for NP-induced cytotoxicity because the optical and electrical functionalities of materials necessary for neural imaging and interfacing are matched well with the properties of many NPs. In addition, the cellular morphology of neurons is particularly suitable for automated high content screening. HepG2 cells represent a good model for high content screening studies since they are commonly used as a surrogate for human hepatocytes in pharmaceutical studies. We found the CdTe QDs to induce primarily apoptotic response in a time- and dosage-dependent manner and produce different toxicological profiles and responses in undifferentiated and differentiated neural cells. Au NPs were found to inhibit the proliferation and intracellular calcium release of HepG2 cells.

  14. An oral multispecies biofilm model for high content screening applications

    PubMed Central

    Kommerein, Nadine; Stumpp, Sascha N.; Müsken, Mathias; Ehlert, Nina; Winkel, Andreas; Häussler, Susanne; Behrens, Peter; Buettner, Falk F. R.; Stiesch, Meike

    2017-01-01

    Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript. PMID:28296966

  15. Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

    2017-01-01

    We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

  16. High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

    PubMed Central

    Vaz, Gisela M. F.; Paszko, Edyta; Davies, Anthony M.; Senge, Mathias O.

    2013-01-01

    A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries. PMID:23923014

  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. A Multivariate Computational Method to Analyze High-Content RNAi Screening Data.

    PubMed

    Rameseder, Jonathan; Krismer, Konstantin; Dayma, Yogesh; Ehrenberger, Tobias; Hwang, Mun Kyung; Airoldi, Edoardo M; Floyd, Scott R; Yaffe, Michael B

    2015-09-01

    High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, multivariate robust analysis method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to an HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of hits in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A.

  19. A multivariate computational method to analyze high-content RNAi screening data

    PubMed Central

    Rameseder, Jonathan; Krismer, Konstantin; Dayma, Yogesh; Ehrenberger, Tobias; Hwang, Mun Kyung; Airoldi, Edoardo M.; Floyd, Scott R.; Yaffe, Michael B.

    2017-01-01

    High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, Multivariate Robust Analysis Method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to a HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of ‘hits’ in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A. PMID:25918037

  20. 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

  1. Rapid 3-D delineation of cell nuclei for high-content screening platforms.

    PubMed

    Gertych, Arkadiusz; Ma, Zhaoxuan; Tajbakhsh, Jian; Velásquez-Vacca, Adriana; Knudsen, Beatrice S

    2016-02-01

    High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery. However, cell confluency, complexity of nuclear staining as well as poor contrast between nuclei and background result in slow and unreliable 3-D image processing and therefore negatively affect the performance of studying a drug response. Here, we propose a new method, 3D-RSD, to delineate nuclei by means of 3-D radial symmetries and test it on high-resolution image data of human cancer cells treated by drugs. The nuclei detection performance was evaluated by means of manually generated ground truth from 2351 nuclei (27 confocal stacks). When compared to three other nuclei segmentation methods, 3D-RSD possessed a better true positive rate of 83.3% and F-score of 0.895±0.045 (p-value=0.047). Altogether, 3D-RSD is a method with a very good overall segmentation performance. Furthermore, implementation of radial symmetries offers good processing speed, and makes 3D-RSD less sensitive to staining patterns. In particular, the 3D-RSD method performs well in cell lines, which are often used in imaging-based HCS platforms and are afflicted by nuclear crowding and overlaps that hinder feature extraction.

  2. Rapid 3-D delineation of cell nuclei for high-content screening platforms

    PubMed Central

    Gertych, Arkadiusz; Ma, Zhaoxuan; Tajbakhsh, Jian; Velásquez-Vacca, Adriana; Knudsen, Beatrice S.

    2015-01-01

    High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery. However, cell confluency, complexity of nuclear staining as well as poor contrast between nuclei and background result in slow and unreliable 3-D image processing and therefore negatively affect the performance of studying a drug response. Here, we propose a new method, 3D-RSD, to delineate nuclei by means of 3-D radial symmetries and test it on high-resolution image data of human cancer cells treated by drugs. The nuclei detection performance was evaluated by means of manually generated ground truth from 2351 nuclei (27 confocal stacks). When compared to three other nuclei segmentation methods, 3D-RSD possessed a better true positive rate of 83.3% and F-score of 0.895+/-0.045 (p- value=0.047). Altogether, 3D-RSD is a method with a very good overall segmentation performance. Furthermore, implementation of radial symmetries offers good processing speed, and makes 3D-RSD less sensitive to staining patterns. In particular the 3D-RSG method performs well in cell lines, which are often used in imaging-based HCS platforms and are afflicted by nuclear crowding and overlaps that hinder feature extraction. PMID:25982066

  3. 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

  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.

  5. A morphology- and kinetics-based cascade for human neural cell high content screening.

    PubMed

    Richards, Gillian R; Smith, Alison J; Parry, Frances; Platts, Amy; Chan, Grace K Y; Leveridge, Mathew; Kerby, Julie E; Simpson, Peter B

    2006-04-01

    The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional high-throughput screening approaches.

  6. Graph cut and image intensity-based splitting improves nuclei segmentation in high-content screening

    NASA Astrophysics Data System (ADS)

    Farhan, Muhammad; Ruusuvuori, Pekka; Emmenlauer, Mario; Rämö, Pauli; Yli-Harja, Olli; Dehio, Christoph

    2013-02-01

    Quantification of phenotypes in high-content screening experiments depends on the accuracy of single cell analysis. In such analysis workflows, cell nuclei segmentation is typically the first step and is followed by cell body segmentation, feature extraction, and subsequent data analysis workflows. Therefore, it is of utmost importance that the first steps of high-content analysis are done accurately in order to guarantee correctness of the final analysis results. In this paper, we present a novel cell nuclei image segmentation framework which exploits robustness of graph cut to obtain initial segmentation for image intensity-based clump splitting method to deliver the accurate overall segmentation. By using quantitative benchmarks and qualitative comparison with real images from high-content screening experiments with complicated multinucleate cells, we show that our method outperforms other state-of-the-art nuclei segmentation methods. Moreover, we provide a modular and easy-to-use implementation of the method for a widely used platform.

  7. 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...

  8. 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....

  9. An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

    PubMed Central

    Yang, Gyongseon; Lee, Changbok; Moon, Hong Kee; Chatelain, Eric; Genovesio, Auguste; Cechetto, Jonathan; Freitas-Junior, Lucio H.

    2012-01-01

    Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania. PMID:22720099

  10. 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.

  11. Active Learning Strategies for Phenotypic Profiling of High-Content Screens.

    PubMed

    Smith, Kevin; Horvath, Peter

    2014-06-01

    High-content screening is a powerful method to discover new drugs and carry out basic biological research. Increasingly, high-content screens have come to rely on supervised machine learning (SML) to perform automatic phenotypic classification as an essential step of the analysis. However, this comes at a cost, namely, the labeled examples required to train the predictive model. Classification performance increases with the number of labeled examples, and because labeling examples demands time from an expert, the training process represents a significant time investment. Active learning strategies attempt to overcome this bottleneck by presenting the most relevant examples to the annotator, thereby achieving high accuracy while minimizing the cost of obtaining labeled data. In this article, we investigate the impact of active learning on single-cell-based phenotype recognition, using data from three large-scale RNA interference high-content screens representing diverse phenotypic profiling problems. We consider several combinations of active learning strategies and popular SML methods. Our results show that active learning significantly reduces the time cost and can be used to reveal the same phenotypic targets identified using SML. We also identify combinations of active learning strategies and SML methods which perform better than others on the phenotypic profiling problems we studied.

  12. High-throughput, high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.

    PubMed

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

    2014-02-01

    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 microphthalmia transcription factor (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 4000 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.

  13. 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

  14. Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

    PubMed

    Schembri, Laura; Zanese, Marion; Depierre-Plinet, Gaelle; Petit, Muriel; Elkaoukabi-Chaibi, Assia; Tauzin, Loic; Florean, Cristina; Lartigue, Lydia; Medina, Chantal; Rey, Christophe; Belloc, Francis; Reiffers, Josy; Ichas, François; De Giorgi, Francesca

    2009-12-01

    Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

  15. Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis.

    PubMed

    Schulz, Martin Michael Peter; Reisen, Felix; Zgraggen, Silvana; Fischer, Stephanie; Yuen, Don; Kang, Gyeong Jin; Chen, Lu; Schneider, Gisbert; Detmar, Michael

    2012-10-02

    Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.

  16. A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

    PubMed Central

    Matthews, Daniel R.; Fruhwirth, Gilbert O.; Weitsman, Gregory; Carlin, Leo M.; Ofo, Enyinnaya; Keppler, Melanie; Barber, Paul R.; Tullis, Iain D. C.; Vojnovic, Borivoj; Ng, Tony; Ameer-Beg, Simon M.

    2012-01-01

    Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing

  17. A high-content screening assay for small-molecule modulators of oncogene-induced senescence.

    PubMed

    Bitler, Benjamin G; Fink, Lauren S; Wei, Zhi; Peterson, Jeffrey R; Zhang, Rugang

    2013-10-01

    Cellular senescence is a state of stable cell growth arrest. Activation of oncogenes such as RAS in mammalian cells typically triggers cellular senescence. Oncogene-induced senescence (OIS) is an important tumor suppression mechanism, and suppression of OIS contributes to cell transformation. Oncogenes trigger senescence through a multitude of incompletely understood downstream signaling events that frequently involve protein kinases. To identify target proteins required for RAS-induced senescence, we developed a small-molecule screen in primary human fibroblasts undergoing senescence induced by oncogenic RAS (H-Ras(G12V)). Using a high-content imaging system to monitor two hallmarks of senescence, senescence-associated β-galactosidase activity expression and inhibition of proliferation, we screened a library of known small-molecule kinase inhibitors for those that suppressed OIS. Identified compounds were subsequently validated and confirmed using a third marker of senescence, senescence-associated heterochromatin foci. In summary, we have established a novel high-content screening platform that may be useful for elucidating signaling pathways mediating OIS by targeting critical pathway components.

  18. A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

    PubMed

    Smith, Lorey K; Thomas, Daniel W; Simpson, Kaylene J; Humbert, Patrick O

    2016-10-01

    Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

  19. 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

  20. 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

  1. High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.

    PubMed

    Bin Zhang; Xiubin Gu; Uppalapati, Uma; Ashwell, Mark A; Leggett, David S; Li, Chiang J

    2008-07-01

    Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

  2. Adapting human pluripotent stem cells to high-throughput and high-content screening.

    PubMed

    Desbordes, Sabrina C; Studer, Lorenz

    2013-01-01

    The increasing use of human pluripotent stem cells (hPSCs) as a source of cells for drug discovery, cytotoxicity assessment and disease modeling requires their adaptation to large-scale culture conditions and screening formats. Here, we describe a simple and robust protocol for the adaptation of human embryonic stem cells (hESCs) to high-throughput screening (HTS). This protocol can also be adapted to human induced pluripotent stem cells (hiPSCs) and high-content screening (HCS). We also describe a 7-d assay to identify compounds with an effect on hESC self-renewal and differentiation. This assay can be adapted to a variety of applications. The procedure involves the culture expansion of hESCs, their adaptation to 384-well plates, the addition of small molecules or other factors, and finally data acquisition and processing. In this protocol, the optimal number of hESCs plated in 384-well plates has been adapted to HTS/HCS assays of 7 d.

  3. High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor.

    PubMed

    Geldenhuys, Werner J; Bergeron, Sadie A; Mullins, Jackie E; Aljammal, Rowaa; Gaasch, Briah L; Chen, Wei-Chi; Yun, June; Hazlehurst, Lori A

    2017-02-28

    In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC50=4.078μM) and surprisingly a DAPK1 kinase agonist/activator (EC50=39.525μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.

  4. Normalizing for individual cell population context in the analysis of high-content cellular screens

    PubMed Central

    2011-01-01

    Background High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology. Results We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. Conclusions Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results. PMID:22185194

  5. HCS-Neurons: identifying phenotypic changes in multi-neuron images upon drug treatments of high-content screening

    PubMed Central

    2013-01-01

    Background High-content screening (HCS) has become a powerful tool for drug discovery. However, the discovery of drugs targeting neurons is still hampered by the inability to accurately identify and quantify the phenotypic changes of multiple neurons in a single image (named multi-neuron image) of a high-content screen. Therefore, it is desirable to develop an automated image analysis method for analyzing multi-neuron images. Results We propose an automated analysis method with novel descriptors of neuromorphology features for analyzing HCS-based multi-neuron images, called HCS-neurons. To observe multiple phenotypic changes of neurons, we propose two kinds of descriptors which are neuron feature descriptor (NFD) of 13 neuromorphology features, e.g., neurite length, and generic feature descriptors (GFDs), e.g., Haralick texture. HCS-neurons can 1) automatically extract all quantitative phenotype features in both NFD and GFDs, 2) identify statistically significant phenotypic changes upon drug treatments using ANOVA and regression analysis, and 3) generate an accurate classifier to group neurons treated by different drug concentrations using support vector machine and an intelligent feature selection method. To evaluate HCS-neurons, we treated P19 neurons with nocodazole (a microtubule depolymerizing drug which has been shown to impair neurite development) at six concentrations ranging from 0 to 1000 ng/mL. The experimental results show that all the 13 features of NFD have statistically significant difference with respect to changes in various levels of nocodazole drug concentrations (NDC) and the phenotypic changes of neurites were consistent to the known effect of nocodazole in promoting neurite retraction. Three identified features, total neurite length, average neurite length, and average neurite area were able to achieve an independent test accuracy of 90.28% for the six-dosage classification problem. This NFD module and neuron image datasets are provided as a

  6. High content screening for G protein-coupled receptors using cell-based protein translocation assays.

    PubMed

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne; Linde, Viggo; Pedersen, Hans-Christian; Krog-Jensen, Christian; Rosenkilde, Mette M; Pagliaro, Len

    2005-06-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

  7. 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.

  8. 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

  9. 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

    2011-01-07

    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.

  10. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation.

    PubMed

    Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E; Pertsemlidis, Alexander; Du, Liqin

    2014-05-15

    Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.

  11. Systematic high-content genome-wide RNAi screens of endothelial cell migration and morphology

    PubMed Central

    Williams, Steven P.; Gould, Cathryn M.; Nowell, Cameron J.; Karnezis, Tara; Achen, Marc G.; Simpson, Kaylene J.; Stacker, Steven A.

    2017-01-01

    Many cell types undergo migration during embryogenesis and disease. Endothelial cells line blood vessels and lymphatics, which migrate during development as part of angiogenesis, lymphangiogenesis and other types of vessel remodelling. These processes are also important in wound healing, cancer metastasis and cardiovascular conditions. However, the molecular control of endothelial cell migration is poorly understood. Here, we present a dataset containing siRNA screens that identify known and novel components of signalling pathways regulating migration of lymphatic endothelial cells. These components are compared to signalling in blood vascular endothelial cells. Further, using high-content microscopy, we captured a dataset of images of migrating cells following transfection with a genome-wide siRNA library. These datasets are suitable for the identification and analysis of genes involved in endothelial cell migration and morphology, and for computational approaches to identify signalling networks controlling the migratory response and integration of cell morphology, gene function and cell signaling. This may facilitate identification of protein targets for therapeutically modulating angiogenesis and lymphangiogenesis in the context of human disease. PMID:28248931

  12. 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...

  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.

  14. High-Content FRET-FLIM Screening in Inhibitors of Oncogenic Transcription by c-myc in Breast Cancer

    DTIC Science & Technology

    2008-06-01

    need for novel anti-breast cancer therapeutics. Our hypothesis is that by identifying small molecules that target the Myc oncogene, we will develop an...To this end, we aim to 1) develop a novel high content screen to identify inhibitors that block Myc:TRRAP interaction; 2) determine the...resonance energy transfer (FRET) in vivo. We have identified FRET pairs that are functional and established methodology using novel instrumentation

  15. 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

  16. 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.

  17. Discovery of a novel ROCK2 inhibitor with anti-migration effects via docking and high-content drug screening.

    PubMed

    Chong, Cheong-Meng; Kou, Man-Teng; Pan, Peichen; Zhou, Hefeng; Ai, Nana; Li, Chuwen; Zhong, Hai-Jing; Leung, Chung-Hang; Hou, Tingjun; Lee, Simon Ming-Yuen

    2016-08-16

    Rho-associated protein kinase (ROCK) mediated the reorganization of the actin cytoskeleton and has been implicated in the spread and metastatic process of cancer. In this study, structure-based high-throughput virtual screening was used to identify candidate compounds targeting ROCK2 from a chemical library. Moreover, high-content screening based on neurite outgrowth of SH-SY5Y cells (a human neuroblastoma cell line) was used for accelerating the identification of compounds with characteristics of ROCK2 inhibitors. The effects of bioactive ROCK2 inhibitor candidates were further validated using other bioassays including cell migration and wound healing in SH-SY5Y cells. Through the combined virtual and high-content drug screening, the compound 1,3-benzodioxol-5-yl[1-(5-isoquinolinylmethyl)-3-piperidinyl]-methanone (BIPM) was identified as a novel and potent ROCK2 inhibitor. Exposure of SH-SY5Y cells to BIPM led to significant changes in neurite length, cell migration and actin stress fibers. Further experiments demonstrated that BIPM was able to significantly inhibit phosphorylation of cofilin, a regulatory protein of actin cytoskeleton. These results suggest that BIPM could be considered as a promising scaffold for the further development of ROCK2 inhibitors for anti-cancer metastasis.

  18. A system and methodology for high-content visual screening of individual intact living cells in suspension

    NASA Astrophysics Data System (ADS)

    Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

    2007-02-01

    Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

  19. 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...

  20. Toward high-content screening of mitochondrial morphology and membrane potential in living cells.

    PubMed

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

    2015-06-01

    Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These "mitochondrial dynamics" are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial "health" and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  1. 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

  2. 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.

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

    PubMed

    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

  4. 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

    2016-05-03

    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.

  5. Automated reticulocyte parameters for hereditary spherocytosis screening.

    PubMed

    Lazarova, Elena; Pradier, Olivier; Cotton, Frédéric; Gulbis, Béatrice

    2014-11-01

    The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories.

  6. A method for characterizing phenotypic changes in highly variable cell populations and its application to high content screening of Arabidopsis thaliana protoplasts.

    PubMed

    Johnson, Gregory R; Kangas, Joshua D; Dovzhenko, Alexander; Trojok, Rüdiger; Voigt, Karsten; Majarian, Timothy D; Palme, Klaus; Murphy, Robert F

    2017-02-28

    Quantitative image analysis procedures are necessary for the automated discovery of effects of drug treatment in large collections of fluorescent micrographs. When compared to their mammalian counterparts, the effects of drug conditions on protein localization in plant species are poorly understood and underexplored. To investigate this relationship, we generated a large collection of images of single plant cells after various drug treatments. For this, protoplasts were isolated from six transgenic lines of A. thaliana expressing fluorescently tagged proteins. Eight drugs at three concentrations were applied to protoplast cultures followed by automated image acquisition. For image analysis, we developed a cell segmentation protocol for detecting drug effects using a Hough transform-based region of interest detector and a novel cross-channel texture feature descriptor. In order to determine treatment effects, we summarized differences between treated and untreated experiments with an L1 Cramér-von Mises statistic. The distribution of these statistics across all pairs of treated and untreated replicates was compared to the variation within control replicates to determine the statistical significance of observed effects. Using this pipeline, we report the dose dependent drug effects in the first high-content Arabidopsis thaliana drug screen of its kind. These results can function as a baseline for comparison to other protein organization modeling approaches in plant cells. © 2017 International Society for Advancement of Cytometry.

  7. High-content screening of natural products reveals novel nuclear export inhibitors.

    PubMed

    Cautain, Bastien; de Pedro, Nuria; Murillo Garzón, Virginia; Muñoz de Escalona, María; González Menéndez, Victor; Tormo, José R; Martin, Jesús; El Aouad, Noureddine; Reyes, Fernando; Asensio, Francisco; Genilloud, Olga; Vicente, Francisca; Link, Wolfgang

    2014-01-01

    Natural products are considered an extremely valuable source for the discovery of new drugs against diverse pathologies. As yet, we have only explored a fraction of the diversity of bioactive compounds, and opportunities for discovering new natural products leading to new drugs are huge. In the present study, U2nesRELOC, a previously established cell-based imaging assay, was employed to screen a collection of extracts of microbial origin for nuclear export inhibition activity. The fluorescent signal of untreated U2nesRELOC cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B, the fluorescent-tagged reporter proteins appear as speckles in the nucleus. A proprietary collection of extracts from fungi, actinomycetes, and unicellular bacteria that covers an uncommonly broad chemical space was used to interrogate this nuclear export assay system. A two-step image-based analysis allowed us to identify 12 extracts with biological activities that are not associated with previously known active metabolites. The fractionation and structural elucidation of active compounds revealed several chemical structures with nuclear export inhibition activity. Here we show that substrates of the nuclear export receptor CRM1, such as Rev, FOXO3a and NF-κB, accumulate in the nucleus in the presence of the fungal metabolite MDN-0105 with an IC50 value of 3.4 µM. Many important processes in tumor formation and progression, as well as in many viral infections, critically depend on the nucleocytoplasmic trafficking of proteins and RNA molecules. Therefore, the disruption of nuclear export is emerging as a novel therapeutic approach with enormous clinical potential. Our work highlights the potential of applying high-throughput phenotypic imaging on natural product extracts to identify novel nuclear export inhibitors.

  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. Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag-Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression.

    PubMed

    Berges, Nina; Arens, Katharina; Kreusch, Verena; Fischer, Rainer; Di Fiore, Stefano

    2017-04-01

    Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z' factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

  10. Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures.

    PubMed

    Trumpi, Kari; Egan, David A; Vellinga, Thomas T; Borel Rinkes, Inne H M; Kranenburg, Onno

    2015-01-01

    Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.

  11. 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

    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 lysosome

  12. High content screening of a kinase-focused library reveals compounds broadly-active against dengue viruses.

    PubMed

    Cruz, Deu John M; Koishi, Andrea Cristine; Taniguchi, Juliana Bosso; Li, Xiaolan; Milan Bonotto, Rafaela; No, Joo Hwan; Kim, Keum Hyun; Baek, Sungmin; Kim, Hee Young; Windisch, Marc Peter; Pamplona Mosimann, Ana Luiza; de Borba, Luana; Liuzzi, Michel; Hansen, Michael Adsetts Edberg; Duarte dos Santos, Claudia Nunes; Freitas-Junior, Lucio Holanda

    2013-01-01

    Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

  13. Getting the whole picture: High content screening using three-dimensional cellular model systems and whole animal assays.

    PubMed

    Kriston-Vizi, Janos; Flotow, Horst

    2017-02-01

    Phenotypic or High Content Screening (HCS) is becoming more widely used for primary screening campaigns in drug discovery. Currently the vast majority of HCS campaigns are using cell lines grown in well-established monolayer cultures (2D tissue culture). There is widespread recognition that the more biologically relevant 3D tissue culture technologies such as spheroids and organoids and even whole animal assays will eventually be run as primary HCS. Upgrading the IT infrastructure to cope with the increase in data volumes requires investments in hardware (and software) and this will be manageable. However, the main bottleneck for the effective adoption and use of 3D tissue culture and whole animal assays in HCS is anticipated to be the development of software for the analysis of 3D images. In this review we summarize the current state of the available software and how they may be applied to analyzing 3D images obtained from a HCS campaign. © 2016 International Society for Advancement of Cytometry.

  14. 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

  15. 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

    2016-04-08

    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.

  16. High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death

    PubMed Central

    Wang, Huaishan; Sherbini, Omar; Ling-lin Pai, Emily; Kwon, Ji-Sun; He, Wei; Wang, Hong; Chi, Zhikai; Xu, Jinchong; Jiang, Haisong; Andrabi, Shaida A.

    2016-01-01

    Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N′-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation–induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death. PMID:27822494

  17. High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death.

    PubMed

    Zhang, Jianmin; Wang, Huaishan; Sherbini, Omar; Ling-Lin Pai, Emily; Kang, Sung-Ung; Kwon, Ji-Sun; Yang, Jia; He, Wei; Wang, Hong; Eacker, Stephen M; Chi, Zhikai; Mao, Xiaobo; Xu, Jinchong; Jiang, Haisong; Andrabi, Shaida A; Dawson, Ted M; Dawson, Valina L

    2016-01-01

    Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N'-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation-induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death.

  18. 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.

  19. 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

  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. Towards automated screening of two-dimensional crystals.

    PubMed

    Cheng, Anchi; Leung, Albert; Fellmann, Denis; Quispe, Joel; Suloway, Christian; Pulokas, James; Abeyrathne, Priyanka D; Lam, Joseph S; Carragher, Bridget; Potter, Clinton S

    2007-12-01

    Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible. We, and others, have developed working prototypes for several of these tools and tested and evaluated them in a simple screen of 24 crystallization conditions. While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach.

  2. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

    PubMed

    Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

    2016-10-01

    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.

  3. Development of a 3D Tissue Culture–Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases

    PubMed Central

    Booij, Tijmen H.; Klop, Maarten J. D.; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S.

    2016-01-01

    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. PMID:27412535

  4. 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

  5. High-content screening technology combined with a human granuloma model as a new approach to evaluate the activities of drugs against Mycobacterium tuberculosis.

    PubMed

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

    2015-01-01

    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.

  6. 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

  7. 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...

  8. 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...

  9. 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...

  10. 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.

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

    PubMed

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

    2015-09-29

    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.

  12. High-Content FRET-FLIM Screening in Inhibitors of Oncogenic Transcription by C-Myc in Breast Cancer

    DTIC Science & Technology

    2009-06-01

    the two together were not additive. See Figure 11. ~ 4 ~ d- 4P New Goal: Screen optimal donor:acceptor pair using cells expressing stable Myc...from four independent biological replicates, of asynchronously growing MCF-7 and MDA-MB-231 cells +/- induction of TRRAP(F8). PCR amplify Chip’d DNA...for each cell line (see schematic Figure 18). In addition, expression profiling of four biological replicates of each cell line was also conducted

  13. 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

  14. Development of a cell-based assay for measurement of c-Met phosphorylation using AlphaScreen technology and high-content imaging analysis.

    PubMed

    Smotrov, Nadya; Mathur, Anjili; Kariv, Ilona; Moxham, Christopher M; Bays, Nathan

    2009-04-01

    c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases.

  15. 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.

  16. 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

  17. Large-scale screening by the automated Wassermann reaction

    PubMed Central

    Wagstaff, W.; Firth, R.; Booth, J. R.; Bowley, C. C.

    1969-01-01

    In view of the drawbacks in the use of the Kahn test for large-scale screening of blood donors, mainly those of human error through work overload and fatiguability, an attempt was made to adapt an existing automated complement-fixation technique for this purpose. This paper reports the successful results of that adaptation. PMID:5776559

  18. 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…

  19. Automation to improve efficiency of field expedient injury prediction screening.

    PubMed

    Teyhen, Deydre S; Shaffer, Scott W; Umlauf, Jon A; Akerman, Raymond J; Canada, John B; Butler, Robert J; Goffar, Stephen L; Walker, Michael J; Kiesel, Kyle B; Plisky, Phillip J

    2012-07-01

    Musculoskeletal injuries are a primary source of disability in the U.S. Military. Physical training and sports-related activities account for up to 90% of all injuries, and 80% of these injuries are considered overuse in nature. As a result, there is a need to develop an evidence-based musculoskeletal screen that can assist with injury prevention. The purpose of this study was to assess the capability of an automated system to improve the efficiency of field expedient tests that may help predict injury risk and provide corrective strategies for deficits identified. The field expedient tests include survey questions and measures of movement quality, balance, trunk stability, power, mobility, and foot structure and mobility. Data entry for these tests was automated using handheld computers, barcode scanning, and netbook computers. An automated algorithm for injury risk stratification and mitigation techniques was run on a server computer. Without automation support, subjects were assessed in 84.5 ± 9.1 minutes per subject compared with 66.8 ± 6.1 minutes per subject with automation and 47.1 ± 5.2 minutes per subject with automation and process improvement measures (p < 0.001). The average time to manually enter the data was 22.2 ± 7.4 minutes per subject. An additional 11.5 ± 2.5 minutes per subject was required to manually assign an intervention strategy. Automation of this injury prevention screening protocol using handheld devices and netbook computers allowed for real-time data entry and enhanced the efficiency of injury screening, risk stratification, and prescription of a risk mitigation strategy.

  20. High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

    PubMed

    Mudhasani, Rajini; Kota, Krishna P; Retterer, Cary; Tran, Julie P; Whitehouse, Chris A; Bavari, Sina

    2014-08-01

    High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and

  1. From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

    PubMed Central

    Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

    2012-01-01

    The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

  2. High-Content Functional Screening of AEG-1 and AKR1C2 for the Promotion of Metastasis in Liver Cancer.

    PubMed

    Li, Cong; Wu, Xia; Zhang, Wei; Li, Jia; Liu, Huawei; Hao, Ming; Wang, Junsong; Zhang, Honghai; Yang, Gengxia; Hao, Meijun; Sheng, Shoupeng; Sun, Yu; Long, Jiang; Li, Juan; Zhuang, Fengfeng; Hu, Caixia; Li, Li; Zheng, Jiasheng

    2016-01-01

    Liver cancer is one of the most lethal cancer types in humans, but our understanding of the molecular mechanisms underlying this process remains insufficient. Here, we conducted high-content screening of the potential genes involved in liver cancer metastasis, which we selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, based on the SAMcell method and RNA interference technology. We identified two powerful genes in the liver cancer metastasis process, AEG-1 and AKR1C2, both of which proved to be positive regulators in promoting metastasis in liver cancer. Further clinical results verified their roles in liver cancer. In summary, these findings could provide new insight into the liver cancer mechanism and potentially therapeutic novel targets for liver cancer therapies in the future.

  3. Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.

    PubMed

    Song, Ok Ryul; Kim, Tae-Hee; Perrodon, Xavier; Lee, Changbok; Jeon, Hee Kyoung; Seghiri, Zahir; Kwon, Ho Jeong; Cechetto, Jonathan; Christophe, Thierry

    2010-02-01

    Rapid mixing in microplates is still an underappreciated challenge in screening assay development, particularly with the use of noncontact nanoliter liquid handlers. In high-content/throughput screening (HC/TS), fast and efficient mixing between compounds and cell culture medium is even more critical as biological kinetics dictates speed of mixing, usually within a few minutes. Moreover, mixing in HC/TS should be gentle enough to avoid any negative disruption in cell layer. Here the authors introduce a method to accurately quantify drop diffusion into a microplate well, independently of buffer, liquid handler, or dispensing protocol. This method was used to determine the effect of various mixing methods on the diffusion of a nanoliter drop of pure DMSO in aqueous buffer in 384-well plates. Rapid plate shaking and additional buffer addition were shown to be the most efficient and effective mixing methods for HC/TS. However, efficient mixing by plate shaking is limited by assay volume. Bulk addition shows fast and efficient mixing, without negative effects on cells. Moreover, this simple, fast, and inexpensive method can be easily adapted on any platform.

  4. Genotoxicity evaluation of individual cigarette smoke toxicants using the in vitro γH2AX assay by high content screening.

    PubMed

    Garcia-Canton, Carolina; Anadon, Arturo; Meredith, Clive

    2013-10-23

    Cigarette smoke is a complex mixture consisting of more than 5600 identified chemical constituents of which approximately 150 have been identified so far as "tobacco smoke toxicants". Proposals made by the World Health Organisation Framework Convention on Tobacco Control mandate the lowering of nine tobacco smoke priority toxicants, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), and benzo[a]pyrene (B[a]P) and monitoring the levels of a further nine including cadmium. Here, we evaluated the genotoxic potential in human bronchial epithelial BEAS-2B cells of four cigarette smoke toxicants; NNK, NNN, B[a]P and cadmium using the novel in vitro γH2AX assay by High Content Screening (HCS). We also examined the genotoxicity of binary mixtures of NNK and NNN reporting their relative contribution to the genotoxic end-point. The results of this preliminary assessment showed that the in vitro γH2AX assay by HCS could be used as a pre-screening tool to detect and quantify the genotoxicity effect of cigarette smoke toxicants individually and in binary mixture. Moreover, the data produced could contribute to the prioritisation of toxicant reduction research in modified tobacco products.

  5. Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

    PubMed Central

    Krueger, Chateen; Hoffmann, F. Michael

    2010-01-01

    Background Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1. Methodology/Principal Findings A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells. Conclusions/Significance Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for

  6. Omnisphero: a high-content image analysis (HCA) approach for phenotypic developmental neurotoxicity (DNT) screenings of organoid neurosphere cultures in vitro.

    PubMed

    Schmuck, Martin R; Temme, Thomas; Dach, Katharina; de Boer, Denise; Barenys, Marta; Bendt, Farina; Mosig, Axel; Fritsche, Ellen

    2017-04-01

    Current developmental neurotoxicity (DNT) testing in animals faces major limitations, such as high cost and time demands as well as uncertainties in their methodology, evaluation and regulation. Therefore, the use of human-based 3D in vitro systems in combination with high-content image analysis (HCA) might contribute to DNT testing with lower costs, increased throughput and enhanced predictivity for human hazard identification. Human neural progenitor cells (hNPCs) grown as 3D neurospheres mimic basic processes of brain development including hNPC migration and differentiation and are therefore useful for DNT hazard identification. HCA of migrated neurospheres creates new challenges for automated evaluations because it encompasses variable cell densities, inconsistent z-layers and heterogeneous cell populations. We tackle those challenges with our Omnisphero software, which assesses multiple endpoints of the 'Neurosphere Assay.' For neuronal identification, Omnisphero reaches a true positive rate (TPR) of 83.8 % and a false discovery rate (FDR) of 11.4 %, thus being comparable to the interindividual difference among two researchers (TPR = 94.3, FDR = 11.0 %) and largely improving the results obtained by an existing HCA approach, whose TPR does not exceed 50 % at a FDR above 50 %. The high FDR of existing methods results in incorrect measurements of neuronal morphological features accompanied by an overestimation of compound effects. Omnisphero additionally includes novel algorithms to assess 'neurosphere-specific' endpoints like radial migration and neuronal density distribution within the migration area. Furthermore, a user-assisted parameter optimization procedure makes Omnisphero accessible to non-expert end users.

  7. High-Content pSTAT3/1 Imaging Assays to Screen for Selective Inhibitors of STAT3 Pathway Activation in Head and Neck Cancer Cell Lines

    PubMed Central

    Sen, Malabika; Hua, Yun; Camarco, Daniel; Shun, Tong Ying; Lazo, John S.; Grandis, Jennifer R.

    2014-01-01

    Abstract The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is hyperactivated in most cancers and represents a plausible therapeutic target. In the absence of STAT3-selective small-molecule inhibitors, we sought to develop pSTAT3/1 high-content imaging (HCS) assays to screen for selective inhibitors of STAT3 pathway activation in head and neck squamous cell carcinomas (HNSCC) tumor cell lines. Based on the expression of the interleukin-6 (IL-6)Rα and gp130 subunits of the IL-6 receptor complex and STAT3, we selected the Cal33 HNSCC cell line as our model. After developing image acquisition and analysis procedures, we rigorously investigated the cytokine activation responses to optimize the dynamic ranges of both assays and demonstrated that the pan-Janus kinase inhibitor pyridone 6 nonselectively inhibited pSTAT3 and pSTAT1 activation with 50% inhibition concentrations of 7.19±4.08 and 16.38±8.45 nM, respectively. The optimized pSTAT3 HCS assay performed very well in a pilot screen of 1,726 compounds from the Library of Pharmacologically Active Compounds and the National Institutes of Health clinical collection sets, and we identified 51 inhibitors of IL-6-induced pSTAT3 activation. However, only three of the primary HCS actives selectively inhibited STAT3 compared with STAT1. Our follow-up studies indicated that the nonselective inhibition of cytokine induced pSTAT3 and pSTAT1 activation by G-alpha stimulatory subunit-coupled G-protein-coupled receptor agonists, and forskolin was likely due to cyclic adenosine monophosphate-mediated up-regulation of suppressors of cytokine signaling 3. Azelastine, an H1 receptor antagonist approved for the treatment of seasonal allergic rhinitis, nonallergic vasomotor rhinitis, and ocular conjunctivitis, was subsequently confirmed as a selective inhibitor of IL-6-induced pSTAT3 activation that also reduced the growth of HNSCC cell lines. These data illustrate the power of a chemical

  8. Development and implementation of industrialized, fully automated high throughput screening systems

    PubMed Central

    2003-01-01

    Automation has long been a resource for high-throughput screening at Bristol-Myers Squibb. However, with growing deck sizes and decreasing time lines, a new generation of more robust, supportable automated systems was necessary for accomplishing high-throughput screening goals. Implementation of this new generation of automated systems required numerous decisions concerning hardware, software and the value of in-house automation expertise. This project has resulted in fast, flexible, industrialized automation systems with a strong in-house support structure that we believe meets our current high-throughput screening requirements and will continue to meet them well into the future. PMID:18924614

  9. Automated microfluidic screening assay platform based on DropLab.

    PubMed

    Du, Wen-Bin; Sun, Meng; Gu, Shu-Qing; Zhu, Ying; Fang, Qun

    2010-12-01

    This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.

  10. A testing strategy to predict risk for drug-induced liver injury in humans using high-content screen assays and the 'rule-of-two' model.

    PubMed

    Chen, Minjun; Tung, Chun-Wei; Shi, Qiang; Guo, Lei; Shi, Leming; Fang, Hong; Borlak, Jürgen; Tong, Weida

    2014-07-01

    Drug-induced liver injury (DILI) is a major cause of drug failures in both the preclinical and clinical phase. Consequently, improving prediction of DILI at an early stage of drug discovery will reduce the potential failures in the subsequent drug development program. In this regard, high-content screening (HCS) assays are considered as a promising strategy for the study of DILI; however, the predictive performance of HCS assays is frequently insufficient. In the present study, a new testing strategy was developed to improve DILI prediction by employing in vitro assays that was combined with the RO2 model (i.e., 'rule-of-two' defined by daily dose ≥100 mg/day & logP ≥3). The RO2 model was derived from the observation that high daily doses and lipophilicity of an oral medication were associated with significant DILI risk in humans. In the developed testing strategy, the RO2 model was used for the rational selection of candidates for HCS assays, and only the negatives predicted by the RO2 model were further investigated by HCS. Subsequently, the effects of drug treatment on cell loss, nuclear size, DNA damage/fragmentation, apoptosis, lysosomal mass, mitochondrial membrane potential, and steatosis were studied in cultures of primary rat hepatocytes. Using a set of 70 drugs with clear evidence of clinically relevant DILI, the testing strategy improved the accuracies by 10 % and reduced the number of drugs requiring experimental assessment by approximately 20 %, as compared to the HCS assay alone. Moreover, the testing strategy was further validated by including published data (Cosgrove et al. in Toxicol Appl Pharmacol 237:317-330, 2009) on drug-cytokine-induced hepatotoxicity, which improved the accuracies by 7 %. Taken collectively, the proposed testing strategy can significantly improve the prediction of in vitro assays for detecting DILI liability in an early drug discovery phase.

  11. 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.

  12. High-throughput automated refolding screening of inclusion bodies.

    PubMed

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

    2004-10-01

    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.

  13. 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.

  14. 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

  15. Shedding Light on Filovirus Infection with High-Content Imaging

    PubMed Central

    Pegoraro, Gianluca; Bavari, Sina; Panchal, Rekha G.

    2012-01-01

    Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the population and at the single-cell level. High-Content Imaging (HCI) has been used to characterize host-virus interactions in genome-wide reverse genetic screens and to identify novel cellular factors implicated in the binding, entry, replication and egress of several pathogenic viruses. Here we present an overview of the most significant applications of HCI in the context of the cell biology of filovirus infection. HCI assays have been recently implemented to quantitatively study filoviruses in cell culture, employing either infectious viruses in a BSL-4 environment or surrogate genetic systems in a BSL-2 environment. These assays are becoming instrumental for small molecule and siRNA screens aimed at the discovery of both cellular therapeutic targets and of compounds with anti-viral properties. We discuss the current practical constraints limiting the implementation of high-throughput biology in a BSL-4 environment, and propose possible solutions to safely perform high-content, high-throughput filovirus infection assays. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection. PMID:23012631

  16. Shedding light on filovirus infection with high-content imaging.

    PubMed

    Pegoraro, Gianluca; Bavari, Sina; Panchal, Rekha G

    2012-08-01

    Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the population and at the single-cell level. High-Content Imaging (HCI) has been used to characterize host-virus interactions in genome-wide reverse genetic screens and to identify novel cellular factors implicated in the binding, entry, replication and egress of several pathogenic viruses. Here we present an overview of the most significant applications of HCI in the context of the cell biology of filovirus infection. HCI assays have been recently implemented to quantitatively study filoviruses in cell culture, employing either infectious viruses in a BSL-4 environment or surrogate genetic systems in a BSL-2 environment. These assays are becoming instrumental for small molecule and siRNA screens aimed at the discovery of both cellular therapeutic targets and of compounds with anti-viral properties. We discuss the current practical constraints limiting the implementation of high-throughput biology in a BSL-4 environment, and propose possible solutions to safely perform high-content, high-throughput filovirus infection assays. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection.

  17. Combinatorial parallel synthesis and automated screening of a novel class of liquid crystalline materials.

    PubMed

    Deeg, Oliver; Kirsch, Peer; Pauluth, Detlef; Bäuerle, Peter

    2002-12-07

    Combinatorial parallel synthesis has led to the rapid generation of a single-compound library of novel fluorinated quaterphenyls. Subsequent automated screening revealed liquid crystalline (LC) behaviour and gave qualitative relationships of molecular structures and solid state properties.

  18. Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

    PubMed

    Bolt, M J; Stossi, F; Callison, A M; Mancini, M G; Dandekar, R; Mancini, M A

    2015-01-08

    Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

  19. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  20. 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

  1. Automated recycling of chemistry for virtual screening and library design.

    PubMed

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

    2012-07-23

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

  2. 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

  3. 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.

  4. 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).

  5. Automated cell-based assay for screening of aquaporin inhibitors.

    PubMed

    Mola, Maria Grazia; Nicchia, Grazia Paola; Svelto, Maria; Spray, David C; Frigeri, Antonio

    2009-10-01

    Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 microM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders.

  6. Automated Cell-Based Assay for Screening of Aquaporin Inhibitors

    PubMed Central

    Mola, Maria Grazia; Nicchia, Grazia Paola; Svelto, Maria; Spray, David C.; Frigeri, Antonio

    2010-01-01

    Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 μM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders. PMID:19705854

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

    NASA Astrophysics Data System (ADS)

    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.

  8. 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.

  9. High-content single-cell analysis on-chip using a laser microarray scanner.

    PubMed

    Zhou, Jing; Wu, Yu; Lee, Sang-Kwon; Fan, Rong

    2012-12-07

    High-content cellomic analysis is a powerful tool for rapid screening of cellular responses to extracellular cues and examination of intracellular signal transduction pathways at the single-cell level. In conjunction with microfluidics technology that provides unique advantages in sample processing and precise control of fluid delivery, it holds great potential to transform lab-on-a-chip systems for high-throughput cellular analysis. However, high-content imaging instruments are expensive, sophisticated, and not readily accessible. Herein, we report on a laser scanning cytometry approach that exploits a bench-top microarray scanner as an end-point reader to perform rapid and automated fluorescence imaging of cells cultured on a chip. Using high-content imaging analysis algorithms, we demonstrated multiplexed measurements of morphometric and proteomic parameters from all single cells. Our approach shows the improvement of both sensitivity and dynamic range by two orders of magnitude as compared to conventional epifluorescence microscopy. We applied this technology to high-throughput analysis of mesenchymal stem cells on an extracellular matrix protein array and characterization of heterotypic cell populations. This work demonstrates the feasibility of a laser microarray scanner for high-content cellomic analysis and opens up new opportunities to conduct informative cellular analysis and cell-based screening in the lab-on-a-chip systems.

  10. 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.

  11. Evaluation of an automated connective tissue disease screening assay in Korean patients with systemic rheumatic diseases

    PubMed Central

    Jeong, Seri; Yang, Heeyoung; Hwang, Hyunyong

    2017-01-01

    This study aimed to evaluate the diagnostic utilities of the automated connective tissues disease screening assay, CTD screen, in patients with systemic rheumatic diseases. A total of 1093 serum samples were assayed using CTD screen and indirect immunofluorescent (IIF) methods. Among them, 162 were diagnosed with systemic rheumatic disease, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCT). The remaining 931 with non-systemic rheumatic disease were assigned to the control group. The median ratios of CTD screen tests were significantly higher in the systemic rheumatic disease group than in the control group. The positive likelihood ratios of the CTD screen were higher than those of IIF in patients with total rheumatic diseases (4.1 vs. 1.6), including SLE (24.3 vs. 10.7). The areas under the receiver operating characteristic curves (ROC-AUCs) of the CTD screen for discriminating total rheumatic diseases, RA, SLE, and MCT from controls were 0.68, 0.56, 0.92 and 0.80, respectively. The ROC-AUCs of the combinations with IIF were significantly higher in patients with total rheumatic diseases (0.72) and MCT (0.85) than in those of the CTD screen alone. Multivariate analysis indicated that both the CTD screen and IIF were independent variables for predicting systemic rheumatic disease. CTD screen alone and in combination with IIF were a valuable diagnostic tool for predicting systemic rheumatic diseases, particularly for SLE. PMID:28273146

  12. Evaluation of an automated connective tissue disease screening assay in Korean patients with systemic rheumatic diseases.

    PubMed

    Jeong, Seri; Yang, Heeyoung; Hwang, Hyunyong

    2017-01-01

    This study aimed to evaluate the diagnostic utilities of the automated connective tissues disease screening assay, CTD screen, in patients with systemic rheumatic diseases. A total of 1093 serum samples were assayed using CTD screen and indirect immunofluorescent (IIF) methods. Among them, 162 were diagnosed with systemic rheumatic disease, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCT). The remaining 931 with non-systemic rheumatic disease were assigned to the control group. The median ratios of CTD screen tests were significantly higher in the systemic rheumatic disease group than in the control group. The positive likelihood ratios of the CTD screen were higher than those of IIF in patients with total rheumatic diseases (4.1 vs. 1.6), including SLE (24.3 vs. 10.7). The areas under the receiver operating characteristic curves (ROC-AUCs) of the CTD screen for discriminating total rheumatic diseases, RA, SLE, and MCT from controls were 0.68, 0.56, 0.92 and 0.80, respectively. The ROC-AUCs of the combinations with IIF were significantly higher in patients with total rheumatic diseases (0.72) and MCT (0.85) than in those of the CTD screen alone. Multivariate analysis indicated that both the CTD screen and IIF were independent variables for predicting systemic rheumatic disease. CTD screen alone and in combination with IIF were a valuable diagnostic tool for predicting systemic rheumatic diseases, particularly for SLE.

  13. 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.

  14. High-content screening data management for drug discovery in a small- to medium-size laboratory: results of a collaborative pilot study focused on user expectations as indicators of effectiveness.

    PubMed

    Berlinicke, Cynthia A; Ackermann, Christopher F; Chen, Steve H; Schulze, Christoph; Shafranovich, Yakov; Myneni, Sahiti; Patel, Vimla L; Wang, Jian; Zack, Donald J; Lindvall, Mikael; Bova, G Steven

    2012-08-01

    High-content screening (HCS) technology provides a powerful vantage point to approach biological problems; it allows analysis of cell parameters, including changes in cell or protein movement, shape, or texture. As part of a collaborative pilot research project to improve bioscience research data integration, we identified HCS data management as an area ripe for advancement. A primary goal was to develop an integrated data management and analysis system suitable for small- to medium-size HCS programs that would improve research productivity and increase work satisfaction. A system was developed that uses Labmatrix, a Web-based research data management platform, to integrate and query data derived from a Cellomics STORE database. Focusing on user expectations, several barriers to HCS productivity were identified and reduced or eliminated. The impact of the project on HCS research productivity was tested through a series of 18 lab-requested integrated data queries, 7 of which were fully enabled, 7 partially enabled, and 4 enabled through data export to standalone data analysis tools. The results are limited to one laboratory, but this pilot suggests that through an "implementation research" approach, a network of small- to medium-size laboratories involved in HCS projects could achieve greater productivity and satisfaction in drug discovery research.

  15. Automated panning and screening procedure on microplates for antibody generation from phage display libraries.

    PubMed

    Turunen, Laura; Takkinen, Kristiina; Söderlund, Hans; Pulli, Timo

    2009-03-01

    Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.

  16. An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

    PubMed Central

    Linke, Pawel; Amaning, Kwame; Maschberger, Melanie; Vallee, Francois; Steier, Valerie; Baaske, Philipp; Duhr, Stefan; Breitsprecher, Dennis; Rak, Alexey

    2015-01-01

    Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns. PMID:26637553

  17. An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery.

    PubMed

    Linke, Pawel; Amaning, Kwame; Maschberger, Melanie; Vallee, Francois; Steier, Valerie; Baaske, Philipp; Duhr, Stefan; Breitsprecher, Dennis; Rak, Alexey

    2016-04-01

    Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

  18. 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.

  19. 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

    2016-07-01

    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.

  20. 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.

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

    SciTech Connect

    Li, Fenglei

    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

  2. Clinical evaluation of the vector algorithm for neonatal hearing screening using automated auditory brainstem response.

    PubMed

    Keohane, Bernie M; Mason, Steve M; Baguley, David M

    2004-02-01

    A novel auditory brainstem response (ABR) detection and scoring algorithm, entitled the Vector algorithm is described. An independent clinical evaluation of the algorithm using 464 tests (120 non-stimulated and 344 stimulated tests) on 60 infants, with a mean age of approximately 6.5 weeks, estimated test sensitivity greater than 0.99 and test specificity at 0.87 for one test. Specificity was estimated to be greater than 0.95 for a two stage screen. Test times were of the order of 1.5 minutes per ear for detection of an ABR and 4.5 minutes per ear in the absence of a clear response. The Vector algorithm is commercially available for both automated screening and threshold estimation in hearing screening devices.

  3. Fully automated cellular-resolution vertebrate screening platform with parallel animal processing

    PubMed Central

    Chang, Tsung-Yao; Pardo-Martin, Carlos; Allalou, Amin; Wählby, Carolina; Yanik, Mehmet Fatih

    2012-01-01

    The zebrafish larva is an optically-transparent vertebrate model with complex organs that is widely used to study genetics, developmental biology, and to model various human diseases. In this article, we present a set of novel technologies that significantly increase the throughput and capabilities of previously described vertebrate automated screening technology (VAST). We developed a robust multi-thread system that can simultaneously process multiple animals. System throughput is limited only by the image acquisition speed rather than by the fluidic or mechanical processes. We developed image recognition algorithms that fully automate manipulation of animals, including orienting and positioning regions of interest within the microscope’s field of view. We also identified the optimal capillary materials for high-resolution, distortion-free, low-background imaging of zebrafish larvae. PMID:22159032

  4. 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.

  5. 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.

  6. 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.

  7. New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

    PubMed Central

    Soltis, S. Michael; Cohen, Aina E.; Deacon, Ashley; Eriksson, Thomas; González, Ana; McPhillips, Scott; Chui, Hsui; Dunten, Pete; Hollenbeck, Michael; Mathews, Irimpan; Miller, Mitch; Moorhead, Penjit; Phizackerley, R. Paul; Smith, Clyde; Song, Jinhu; van dem Bedem, Henry; Ellis, Paul; Kuhn, Peter; McPhillips, Timothy; Sauter, Nicholas; Sharp, Kenneth; Tsyba, Irina; Wolf, Guenter

    2008-01-01

    Complete automation of the macromolecular crystallography experiment has been achieved at SSRL through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface. These highly reliable systems have enabled crystallography experiments to be carried out from the researchers’ home institutions and other remote locations while retaining complete control over even the most challenging systems. A breakthrough component of the system, the Stanford Auto-Mounter (SAM), has enabled the efficient mounting of cryocooled samples without human intervention. Taking advantage of this automation, researchers have successfully screened more than 200 000 samples to select the crystals with the best diffraction quality for data collection as well as to determine optimal crystallization and cryocooling conditions. These systems, which have been deployed on all SSRL macromolecular crystallography beamlines and several beamlines worldwide, are used by more than 80 research groups in remote locations, establishing a new paradigm for macromolecular crystallo­graphy experimentation. PMID:19018097

  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. Color features as an approach for the automated screening of Salmonella strain

    NASA Astrophysics Data System (ADS)

    Trujillo, Alejandra Serrano; González, Viridiana Contreras; Andrade Rincón, Saulo E.; Palafox, Luis E.

    2016-11-01

    We present the implementation of a feature extraction approach for the automated screening of Salmonella sp., a task visually carried out by a microbiologist, where the resulting color characteristics of the culture media plate indicate the presence of this strain. The screening of Salmonella sp. is based on the inoculation and incubation of a sample on an agar plate, allowing the isolation of this strain, if present. This process uses three media: Xylose lysine deoxycholate, Salmonella Shigella, and Brilliant Green agar plates, which exhibit specific color characteristics over the colonies and over the surrounding medium for a presumed positive interpretation. Under a controlled illumination environment, images of plates are captured and the characteristics found over each agar are processed separately. Each agar is analyzed using statistical descriptors for texture, to determine the presence of colonies, followed by the extraction of color features. A comparison among the color features seen over the three media, according to the FDA Bacteriological Analytical Manual, determines the presence of Salmonella sp. on a given sample. The implemented process proves that the task addressed can be accomplished under an image processing approach, leading to the future validation and automation of additional screening processes.

  10. Consistency of breast density categories in serial screening mammograms: A comparison between automated and human assessment.

    PubMed

    Holland, Katharina; van Zelst, Jan; den Heeten, Gerard J; Imhof-Tas, Mechli; Mann, Ritse M; van Gils, Carla H; Karssemeijer, Nico

    2016-10-01

    Reliable breast density measurement is needed to personalize screening by using density as a risk factor and offering supplemental screening to women with dense breasts. We investigated the categorization of pairs of subsequent screening mammograms into density classes by human readers and by an automated system. With software (VDG) and by four readers, including three specialized breast radiologists, 1000 mammograms belonging to 500 pairs of subsequent screening exams were categorized into either two or four density classes. We calculated percent agreement and the percentage of women that changed from dense to non-dense and vice versa. Inter-exam agreement (IEA) was calculated with kappa statistics. Results were computed for each reader individually and for the case that each mammogram was classified by one of the four readers by random assignment (group reading). Higher percent agreement was found with VDG (90.4%, CI 87.9-92.9%) than with readers (86.2-89.2%), while less plausible changes from non-dense to dense occur less often with VDG (2.8%, CI 1.4-4.2%) than with group reading (4.2%, CI 2.4-6.0%). We found an IEA of 0.68-0.77 for the readers using two classes and an IEA of 0.76-0.82 using four classes. IEA is significantly higher with VDG compared to group reading. The categorization of serial mammograms in density classes is more consistent with automated software than with a mixed group of human readers. When using breast density to personalize screening protocols, assessment with software may be preferred over assessment by radiologists.

  11. 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

  12. Added value of a mandible movement automated analysis in the screening of obstructive sleep apnea.

    PubMed

    Maury, Gisele; Cambron, Laurent; Jamart, Jacques; Marchand, Eric; Senny, Frédéric; Poirrier, Robert

    2013-02-01

    In-laboratory polysomnography is the 'gold standard' for diagnosing obstructive sleep apnea syndrome, but is time consuming and costly, with long waiting lists in many sleep laboratories. Therefore, the search for alternative methods to detect respiratory events is growing. In this prospective study, we compared attended polysomnography with two other methods, with or without mandible movement automated analysis provided by a distance-meter and added to airflow and oxygen saturation analysis for the detection of respiratory events. The mandible movement automated analysis allows for the detection of salient mandible movement, which is a surrogate for arousal. All parameters were recorded simultaneously in 570 consecutive patients (M/F: 381/189; age: 50±14 years; body mass index: 29±7 kg m(-2) ) visiting a sleep laboratory. The most frequent main diagnoses were: obstructive sleep apnea (344; 60%); insomnia/anxiety/depression (75; 13%); and upper airway resistance syndrome (25; 4%). The correlation between polysomnography and the method with mandible movement automated analysis was excellent (r: 0.95; P<0.001). Accuracy characteristics of the methods showed a statistical improvement in sensitivity and negative predictive value with the addition of mandible movement automated analysis. This was true for different diagnostic thresholds of obstructive sleep severity, with an excellent efficiency for moderate to severe index (apnea-hypopnea index ≥15h(-1) ). A Bland & Altman plot corroborated the analysis. The addition of mandible movement automated analysis significantly improves the respiratory index calculation accuracy compared with an airflow and oxygen saturation analysis. This is an attractive method for the screening of obstructive sleep apnea syndrome, increasing the ability to detect hypopnea thanks to the salient mandible movement as a marker of arousals.

  13. 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

  14. 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.

  15. 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

  16. The use of the PAPNET automated cytological screening system for the diagnosis of oral squamous carcinoma.

    PubMed

    Levine, T S; Njemenze, V; Cowpe, J G; Coleman, D V

    1998-12-01

    The automated PAPNET screening system has been developed to recognize abnormal cells in cervical smears. Given that the oral mucosa sheds cells resembling superficial and intermediate cells of the cervix, the aim of this study was to assess whether the PAPNET system could be used to detect dysplastic cells in oral mucosal smears. Sixty-two oral smears from 27 patients were examined by both light microscopy and using the PAPNET system from clinically abnormal and normal areas by two pathologists. The clinically abnormal sites were also biopsied for histological analysis. There was 100% correlation between the manual and PAPNET screening results. Cytological interpretation of oral smears by both manual and PAPNET screening methods correctly diagnosed squamous cell carcinoma in 14/23 (61%) of patients who had all been confirmed by biopsy. The nine patients with false-negative cases could be attributed to poor smear technique and preparation. The PAPNET system can be used to identify abnormal cells in oral smears and, as such, may have an application for screening those populations at high risk of oral cancer--provided that adequate tuition is given in smear technique.

  17. Automated toxicological screening reports of modified Agilent MSD Chemstation combined with Microsoft Visual Basic application programs.

    PubMed

    Choe, Sanggil; Kim, Suncheun; Choi, Hyeyoung; Choi, Hwakyoung; Chung, Heesun; Hwang, Bangyeon

    2010-06-15

    Agilent GC-MS MSD Chemstation offers automated library search report for toxicological screening using total ion chromatogram (TIC) and mass spectroscopy in normal mode. Numerous peaks appear in the chromatogram of biological specimen such as blood or urine and often large migrating peaks obscure small target peaks, in addition, any target peaks of low abundance regularly give wrong library search result or low matching score. As a result, retention time and mass spectrum of all the peaks in the chromatogram have to be checked to see if they are relevant. These repeated actions are very tedious and time-consuming to toxicologists. MSD Chemstation software operates using a number of macro files which give commands and instructions on how to work on and extract data from the chromatogram and spectroscopy. These macro files are developed by the own compiler of the software. All the original macro files can be modified and new macro files can be added to the original software by users. To get more accurate results with more convenient method and to save time for data analysis, we developed new macro files for reports generation and inserted new menus in the Enhanced Data Analysis program. Toxicological screening reports generated by these new macro files are in text mode or graphic mode and these reports can be generated with three different automated subtraction options. Text reports have Brief mode and Full mode and graphic reports have the option with or without mass spectrum mode. Matched mass spectrum and matching score for detected compounds are printed in reports by modified library searching modules. We have also developed an independent application program named DrugMan. This program manages drug groups, lists and parameters that are in use in MSD Chemstation. The incorporation of DrugMan with modified macro modules provides a powerful tool for toxicological screening and save a lot of valuable time on toxicological work.

  18. 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.

  19. 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

  20. Optoelectronic parallel processing with smart pixel arrays for automated screening of cervical smear imagery

    NASA Astrophysics Data System (ADS)

    Metz, John Langdon

    2000-10-01

    This thesis investigates the use of optoelectronic parallel processing systems with smart photosensor arrays (SPAs) to examine cervical smear images. The automation of cervical smear screening seeks to reduce human workload and improve the accuracy of detecting pre- cancerous and cancerous conditions. Increasing the parallelism of image processing improves the speed and accuracy of locating regions-of-interest (ROI) from images of the cervical smear for the first stage of a two-stage screening system. The two-stage approach first detects ROI optoelectronically before classifying them using more time consuming electronic algorithms. The optoelectronic hit/miss transform (HMT) is computed using gray scale modulation spatial light modulators in an optical correlator. To further the parallelism of this system, a novel CMOS SPA computes the post processing steps required by the HMT algorithm. The SPA reduces the subsequent bandwidth passed into the second, electronic image processing stage classifying the detected ROI. Limitations in the miss operation of the HMT suggest using only the hit operation for detecting ROI. This makes possible a single SPA chip approach using only the hit operation for ROI detection which may replace the optoelectronic correlator in the screening system. Both the HMT SPA postprocessor and the SPA ROI detector design provide compact, efficient, and low-cost optoelectronic solutions to performing ROI detection on cervical smears. Analysis of optoelectronic ROI detection with electronic ROI classification shows these systems have the potential to perform at, or above, the current error rates for manual classification of cervical smears.

  1. Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

    PubMed Central

    Sowa, Mandy; Grossmann, Kai; Knütter, Ilka; Hiemann, Rico; Röber, Nadja; Anderer, Ursula; Csernok, Elena; Bogdanos, Dimitrios P.; Borghi, Maria Orietta; Meroni, Pier Luigi; Schierack, Peter; Reinhold, Dirk; Conrad, Karsten; Roggenbuck, Dirk

    2014-01-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by Cyto

  2. Autonomy Versus Automation: Perceptions of Nonmydriatic Camera Choice for Teleretinal Screening in an Urban Safety Net Clinic

    PubMed Central

    Moran, Erin; Patty Daskivich, Lauren; George, Sheba; Teklehaimanot, Senait; Ilapakurthi, Ramarao; Lopez, Kevin; Norris, Keith

    2013-01-01

    Abstract Objective: Teleretinal screening with nonmydriatic cameras has been presented as a means of increasing the number of patients assessed for diabetic retinopathy in urban safety net clinics. It has been hypothesized that automated nonmydriatic cameras may improve screening rates by reducing the learning curve for camera use. In this article, we examine the impact of introducing automated nonmydriatic cameras to urban safety net clinics whose photographers had previously used manual cameras. Materials and Methods: We evaluated the impact of manual and automated digital nonmydriatic cameras on teleretinal screening using a quantitative analysis of readers' image quality ratings as well as a qualitative analysis, through in-depth interviews, of photographers' experiences. Results: With the manual camera, 68% of images were rated “adequate” or better, including 24% rated “good” and 20% rated “excellent.” With the automated camera, 61% were rated “adequate” or better, including 9% rated “good” and 0% rated “excellent.” Photographers expressed frustration with their inability to control image-taking settings with the automated camera, which led to unexpected delays. Conclusions: For safety net clinics in which medical assistants are already trained to take photographs for diabetic retinopathy screening with a manual camera, the introduction of automated cameras may lead to frustration and paradoxically contribute to increased patient wait times. When photographers have achieved a high degree of aptitude with manual cameras and value the control they have over camera features, the introduction of automated cameras should be approached with caution and may require extensive training to increase user acceptability. PMID:23763609

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts

    PubMed Central

    Vandenburgh, Herman; Shansky, Janet; Benesch-Lee, Frank; Skelly, Kirsten; Spinazzola, Janelle M.; Saponjian, Yero; Tseng, Brian S.

    2009-01-01

    Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, β-hydroxy-β-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC50 of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.—Vandenburgh, H., Shansky, J., Benesch-Lee, F., Skelly, K., Spinazzola, J.M., Saponjian, Y., Tseng, B.S. Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts. PMID:19487307

  9. 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

    2014-02-26

    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

  10. 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

  11. High-content analysis to leverage a robust phenotypic profiling approach to vascular modulation.

    PubMed

    Isherwood, Beverley J; Walls, Rebecca E; Roberts, Mark E; Houslay, Thomas M; Brave, Sandra R; Barry, Simon T; Carragher, Neil O

    2013-12-01

    Phenotypic screening seeks to identify substances that modulate phenotypes in a desired manner with the aim of progressing first-in-class agents. Successful campaigns require physiological relevance, robust screening, and an ability to deconvolute perturbed pathways. High-content analysis (HCA) is increasingly used in cell biology and offers one approach to prosecution of phenotypic screens, but challenges exist in exploitation where data generated are high volume and complex. We combine development of an organotypic model with novel HCA tools to map phenotypic responses to pharmacological perturbations. We describe implementation for angiogenesis, a process that has long been a focus for therapeutic intervention but has lacked robust models that recapitulate more completely mechanisms involved. The study used human primary endothelial cells in co-culture with stromal fibroblasts to model multiple aspects of angiogenic signaling: cell interactions, proliferation, migration, and differentiation. Multiple quantitative descriptors were derived from automated microscopy using custom-designed algorithms. Data were extracted using a bespoke informatics platform that integrates processing, statistics, and feature display into a streamlined workflow for building and interrogating fingerprints. Ninety compounds were characterized, defining mode of action by phenotype. Our approach for assessing phenotypic outcomes in complex assay models is robust and capable of supporting a range of phenotypic screens at scale.

  12. Process automation toward ultra-high-throughput screening of combinatorial one-bead-one-compound (OBOC) peptide libraries.

    PubMed

    Cha, Junhoe; Lim, Jaehong; Zheng, Yiran; Tan, Sylvia; Ang, Yi Li; Oon, Jessica; Ang, Mei Wei; Ling, Jingjing; Bode, Marcus; Lee, Su Seong

    2012-06-01

    With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.

  13. The Gray Institute 'open' high-content, fluorescence lifetime microscopes.

    PubMed

    Barber, P R; Tullis, I D C; Pierce, G P; Newman, R G; Prentice, J; Rowley, M I; Matthews, D R; Ameer-Beg, S M; Vojnovic, B

    2013-08-01

    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.

  14. From Sample Changer to the Robotic Rheometer: Automation and High Throughput Screening in Rotational Rheometry

    NASA Astrophysics Data System (ADS)

    Läuger, Jörg; Krenn, Michael

    2008-07-01

    A fully automated, robotically operated rheometer was developed. The full functionality, modularity and accuracy of the rotational rheometer are available, which means the modern principles of high-throughput screening are brought to full function on the rheometer. The basic rheometer setup remains as modular as before including the ability to run all test modes the rheometer offers with the difference that the high-throughput rheometer now performs all measuring steps automatically. In addition, the standard and proven environmental chambers of the rheometer are available. The rheometer itself runs by the standard rheometer software and the measurement data and analysis results can be transferred to a monitoring database. The sample loading and the cleaning of the geometries is assisted by a sample preparation unit and a cleaning station, respectively. The sample throughput is further maximized by the use of multiple geometries allowing the simultaneous rheological measurement by the rheometer and the cleaning of the geometries at the cleaning station by the robot. The High-Throughput Rheometer (HTR) and its special adaptation to different applications like dispersions and polymer melts are described.

  15. Toxicological screening in urine: comparison of two automated HPLC screening systems, toxicological identification system (TOX.I.S.*) versus REMEDI-HS.

    PubMed

    Schönberg, Lena; Grobosch, Thomas; Lampe, Dagmar; Kloft, Charlotte

    2007-01-01

    In this paper, the comparison of two automated high-performance liquid chromatography (HPLC) screening systems, a newly developed toxicological identification system (TOX.I.S.) versus the commercially available Remedi-HS (Bio-Rad), is presented. Urine samples from 405 cases screened positive for amphetamines, cocaine, and opiates by immunological assays and confirmed by GC-MS were analyzed with both systems. In more than 80% (TOX.I.S.) and 78% (Remedi-HS) of the cases (except for cocaine), the results obtained by both HPLC methods showed agreement with the earlier obtained results by immunoassay prescreening and gas chromatography-mass spectrometry (GC-MS). The evaluation showed that both automated HPLC methods led to comparable results and can be used alternatively. As the confirmation results for cocaine were rather poor (45% TOX.I.S., 54% Remedi-HS) in comparison to GC-MS, the TOX.I.S. was further optimized for the detection of the cocaine metabolite benzoylecgonine (BEC). The BEC method improved the detectability of BEC from 45% to 80%. Besides confirmation screening, the use of both systems in cases of acute intoxications was investigated. Information about basic compounds was obtained from urine screening by both systems, which therefore were useful as complementary techniques in the toxicological laboratory. The TOX.I.S. offers advantages such as common equipment, modern software, and higher versatility with the opportunity to establish additional methods in the system.

  16. Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.

    PubMed

    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.

  17. 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

  18. Automation-assisted cervical cancer screening in manual liquid-based cytology with hematoxylin and eosin staining.

    PubMed

    Zhang, Ling; Kong, Hui; Ting Chin, Chien; Liu, Shaoxiong; Fan, Xinmin; Wang, Tianfu; Chen, Siping

    2014-03-01

    Current automation-assisted technologies for screening cervical cancer mainly rely on automated liquid-based cytology slides with proprietary stain. This is not a cost-efficient approach to be utilized in developing countries. In this article, we propose the first automation-assisted system to screen cervical cancer in manual liquid-based cytology (MLBC) slides with hematoxylin and eosin (H&E) stain, which is inexpensive and more applicable in developing countries. This system consists of three main modules: image acquisition, cell segmentation, and cell classification. First, an autofocusing scheme is proposed to find the global maximum of the focus curve by iteratively comparing image qualities of specific locations. On the autofocused images, the multiway graph cut (GC) is performed globally on the a* channel enhanced image to obtain cytoplasm segmentation. The nuclei, especially abnormal nuclei, are robustly segmented by using GC adaptively and locally. Two concave-based approaches are integrated to split the touching nuclei. To classify the segmented cells, features are selected and preprocessed to improve the sensitivity, and contextual and cytoplasm information are introduced to improve the specificity. Experiments on 26 consecutive image stacks demonstrated that the dynamic autofocusing accuracy was 2.06 μm. On 21 cervical cell images with nonideal imaging condition and pathology, our segmentation method achieved a 93% accuracy for cytoplasm, and a 87.3% F-measure for nuclei, both outperformed state of the art works in terms of accuracy. Additional clinical trials showed that both the sensitivity (88.1%) and the specificity (100%) of our system are satisfyingly high. These results proved the feasibility of automation-assisted cervical cancer screening in MLBC slides with H&E stain, which is highly desirable in community health centers and small hospitals.

  19. 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.

  20. 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.

  1. Reagent strip screening for sediment abnormalities identified by automated microscopy in urine from patients suspected to have urinary tract disease.

    PubMed

    Bartlett, R C; Zern, D A; Ratkiewicz, I; Tetreault, J Z

    1994-11-01

    We assessed the ability of reagent strip screening to predict the finding of blood cells and bacteria using an automated urinalysis workstation (The Yellow IRIS, International Remote Imaging Systems, Chatsworth, Calif) in 427 specimens submitted for urine culture. The sensitivities of leukocyte esterase, hemoglobin, and nitrite detection on reagent strips were 71.9%, 70.8%, and 56.7%, respectively, at 5 or more white blood cells per high-power field, at 3 or more red blood cells per high-power field and bacteria observed using the IRIS. Screening results for leukocyte esterase associated with negative results using the IRIS for white blood cells represented mostly false-positive screening test results based on chart review. Positive screening test results for hemoglobin associated with negative results obtained with the IRIS for red blood cells consisted of equal numbers of false-positive screening test results and IRIS test results based on chart review. A common screening algorithm using a combination of these three reagent strip variables exhibited a false-negative rate of 30.1%: review of medical records found clinical evidence of urinary tract infection in 14 patients and genitourinary or renal disease or hypertension in another 13 patients. Adding more variables to the algorithm increases the sensitivity and decreases the specificity. Both microscopic examination and reagent strip testing of urine are necessary for the detection of abnormalities associated with disease.

  2. High content kinetic assays of neuronal signaling implemented on BD pathway HT.

    PubMed

    Chan, Grace K Y; Richards, Gillian R; Peters, Marco; Simpson, Peter B

    2005-12-01

    A great deal of information can be gained from kinetic fluorescence-based measurement of cellular responses; however, until recently the use of such approaches has been limited by the manual nature of the instrumentation available. Higher-throughput kinetic studies of signaling pathways are greatly facilitated by new confocal, liquid handling-enabled, high content screening (HCS) platforms. In the present work, we have implemented one such instrument, the BD(TM) Pathway HT bioimager (BD Biosciences, Rockville, MD), for studying regulation of neuronal signaling pathways. We have established a neuronal calcium oscillation model, whereby rate of oscillation, amplitude of oscillation, and level of synchronicity across the culture can be measured. We have implemented membrane potential measurement using fluorescence resonance energy transfer-based dyes, for single cell characterization on this platform, showing the benefits of a truly flexible excitation and recording system; this dye combination cannot be readily implemented on all HCS platforms because of constraints of excitation wavelengths. We have validated long-term intracellular calcium imaging experiments, using innovative dyes and BD Pathway HT's spinning disk-based confocal excitation. To maximize both throughput and reproducibility, walk-away automation integration of this bioimaging technology has been implemented, producing an affordable, compact platform for fully automated kinetic HCS.

  3. High content live cell imaging for the discovery of new antimalarial marine natural products

    PubMed Central

    2012-01-01

    Background The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. Methods A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Results Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Conclusion Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. PMID:22214291

  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.

  5. High-Content Analysis of Sequential Events during the Early Phase of Influenza A Virus Infection

    PubMed Central

    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

  6. Toward a Trustworthy Voice: Increasing the Effectiveness of Automated Outreach Calls to Promote Colorectal Cancer Screening among African Americans

    PubMed Central

    Albright, Karen; Richardson, Terri; Kempe, Karin L; Wallace, Kristin

    2014-01-01

    Introduction: Colorectal cancer screening rates are lower among African-American members of Kaiser Permanente Colorado (KPCO) than among members of other races and ethnicities. This study evaluated use of a linguistically congruent voice in interactive voice response outreach calls about colorectal cancer screening as a strategy to increase call completion and response. Methods: After an initial discussion group to assess cultural acceptability of the project, 6 focus groups were conducted with 33 KPCO African-American members. Participants heard and discussed recordings of 5 female voices reading the same segment of the standard-practice colorectal cancer message using interactive voice response. The linguistic palette included the voices of a white woman, a lightly accented Latina, and 3 African-American women. Results: Participants strongly preferred the African-American voices, particularly two voices. Participants considered these voices the most trustworthy and reported that they would be the most effective at increasing motivation to complete an automated call. Participants supported the use of African-American voices when designing outgoing automated calls for African Americans because the sense of familiarity engendered trust among listeners. Participants also indicated that effective automated messages should provide immediate clarity of purpose; explain why the issue is relevant to African Americans; avoid sounding scripted; emphasize that the call is for the listener’s benefit only; sound personable, warm, and positive; and not create fear among listeners. Discussion: Establishing linguistic congruence between African Americans and the voices used in automated calls designed to reach them may increase the effectiveness of outreach efforts. PMID:24867548

  7. 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

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

    PubMed Central

    Ihlow, Alexander; Schweizer, Patrick; Seiffert, Udo

    2008-01-01

    Background To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps. Results We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects. Conclusion Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically. PMID:18215267

  9. Automated grid handling and image acquisition for two-dimensional crystal screening.

    PubMed

    Cheng, Anchi

    2013-01-01

    Routine large-scale two-dimensional (2D) crystallization trials are good candidates for automation. For imaging the large number of grids prepared from 2D crystallization trials, a two-pass imaging protocol using Leginon and a grid handling robot is described. A manual target selection bridges the two imaging passes. The two passes can be combined into one if objects of interests at different stages of trials can be reliably found using automated methods.

  10. Automated Confocal Microscope Bias Correction

    NASA Astrophysics Data System (ADS)

    Dorval, Thierry; Genovesio, Auguste

    2006-10-01

    Illumination artifacts systematically occur in 2D cross-section confocal microscopy imaging . These bias can strongly corrupt an higher level image processing such as a segmentation, a fluorescence evaluation or even a pattern extraction/recognition. This paper presents a new fully automated bias correction methodology based on large image database preprocessing. This method is very appropriate to the High Content Screening (HCS), method dedicated to drugs discovery. Our method assumes that the amount of pictures available is large enough to allow a reliable statistical computation of an average bias image. A relevant segmentation evaluation protocol and experimental results validate our correction algorithm by outperforming object extraction on non corrupted images.

  11. 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.

  12. Automated Assessment of Patients' Self-Narratives for Posttraumatic Stress Disorder Screening Using Natural Language Processing and Text Mining.

    PubMed

    He, Qiwei; Veldkamp, Bernard P; Glas, Cees A W; de Vries, Theo

    2017-03-01

    Patients' narratives about traumatic experiences and symptoms are useful in clinical screening and diagnostic procedures. In this study, we presented an automated assessment system to screen patients for posttraumatic stress disorder via a natural language processing and text-mining approach. Four machine-learning algorithms-including decision tree, naive Bayes, support vector machine, and an alternative classification approach called the product score model-were used in combination with n-gram representation models to identify patterns between verbal features in self-narratives and psychiatric diagnoses. With our sample, the product score model with unigrams attained the highest prediction accuracy when compared with practitioners' diagnoses. The addition of multigrams contributed most to balancing the metrics of sensitivity and specificity. This article also demonstrates that text mining is a promising approach for analyzing patients' self-expression behavior, thus helping clinicians identify potential patients from an early stage.

  13. A time-table organizer for the planning and implementation of screenings in manual or semi-automation mode

    PubMed Central

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

    2013-01-01

    We have designed a software to facilitate the planning and execution of screenings performed manually or in semi-automation mode, which follow a sequential sequence 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 in order to achieve reliable outcomes. The Excel Macro-Enabled Workbook presented in this study analyzes and breaks down the timings for all tasks, calculates the maximum 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 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

  14. Design and implementation of high-content imaging platforms: lessons learned from end user-developer collaboration.

    PubMed

    Adams, Cynthia L; Sjaastad, Michael D

    2009-11-01

    Automated high-content screening and analysis (HCS/HCA) technology solutions have become indispensable in expediting the pace of drug discovery. Because of the complexity involved in designing, building, and validating HCS/HCA platforms, it is important to design, build, and validate a HCS/HCA platform before it is actually needed. Managed properly, collaboration between technology providers and end users in research is essential in accelerating development of the hardware and software of new HCS/HCA platforms before they become commercially available. Such a collaboration results in the cost effective creation of new technologies that meet specific and customized industrial requirements. This review outlines the history of, and considerations relevant to, the development of the Cytometrix Profiling System by Cytokinetics, Inc. and the "Complete Imaging Solution" for high-content screening, developed by Molecular Devices Corporation (MDC) (now MDS Analytical Technologies), from original conception and testing of various components, to multiple development cycles from 1998 to the present, and finally to market consolidation.

  15. Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation

    PubMed Central

    Ketteler, Robin; Freeman, Jamie; Ferraro, Francesco; Bata, Nicole; Cutler, Dan F.; Kriston-Vizi, Janos

    2017-01-01

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology. PMID:28248923

  16. Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

    PubMed

    Ketteler, Robin; Freeman, Jamie; Ferraro, Francesco; Bata, Nicole; Cutler, Dan F; Kriston-Vizi, Janos

    2017-03-01

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

  17. Automated analysis of behavior: a computer-controlled system for drug screening and the investigation of learning.

    PubMed

    Hicks, Caitlin; Sorocco, Debra; Levin, Michael

    2006-08-01

    Efforts to understand cognition will be greatly facilitated by computerized systems that enable the automated analysis of animal behavior. A number of controversies in the invertebrate learning field have resulted from difficulties inherent in manual experiments. Driven by the necessity to overcome these problems during investigation of neural function in planarian flatworms and frog larvae, we designed and developed a prototype for an inexpensive, flexible system that enables automated control and analysis of behavior and learning. Applicable to a variety of small animals such as flatworms and zebrafish, this system allows automated analysis of innate behavior, as well as of learning and memory in a plethora of conditioning paradigms. We present here the schematics of a basic prototype, which overcomes experimenter effects and operator tedium, enabling a large number of animals to be analyzed with transparent on-line access to primary data. A scaled-up version of this technology represents an efficient methodology to screen pharmacological and genetic libraries for novel neuroactive reagents of basic and biomedical relevance.

  18. 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.

  19. Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS.

    PubMed

    Andersen, David W; Linnet, Kristian

    2014-01-01

    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids.

  20. 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

  1. A Parallel Microfluidic Flow Cytometer for High Content Screening

    PubMed Central

    McKenna, Brian K.; Evans, James G.; Cheung, Man Ching; Ehrlich, Daniel J.

    2011-01-01

    A parallel microfluidic cytometer (PMC) uses a high-speed scanning photomultiplier-based detector to combine low-pixel-count 1-D imaging with flow cytometry. The 384 parallel flow channels of the PMC decouple count rate from signal-to-noise ratio. Using 6-pixel 1-D images, we investigated protein-localization in a yeast model for a human protein-misfolding disease and demonstrated the feasibility of a nuclear-translocation assay in Chinese-hamster-ovary (CHO) cells expressing a NFκB-GFP reporter. PMID:21478861

  2. Screening urine samples for the absence of urinary tract infection using the sediMAX automated microscopy analyser.

    PubMed

    Sterry-Blunt, Rosanne E; S Randall, Karen; J Doughton, Michael; H Aliyu, Sani; Enoch, David A

    2015-06-01

    Urinalysis culminates in a workload skew within the clinical microbiology laboratory. Routine processing involves screening via manual microscopy or biochemical dipstick measurement, followed by culture for each sample. Despite this, as many as 80% of specimens are reported as negative; thus, there is vast wastage of resources and time, as well as delayed turnaround time of results as numerous negative cultures fulfil their required incubation time. Automation provides the potential for streamlining sample screening by efficiently (>30% sample exclusion) and reliably [negative predictive value (NPV) ≥ 95%] ruling out those likely to be negative, whilst also reducing resource usage and hands-on time. The present study explored this idea by using the sediMAX automated microscopy urinalysis platform. We prospectively collected and processed 1411 non-selected samples directly after routine laboratory processing. The results from this study showed multiple optimum cut-off values for microscopy. However, although optimum cut-off values permitted rule-out of 40.1% of specimens, an associated 87.5% NPV was lower than the acceptable limit of 95%. Sensitivity and specificity of leukocytes and bacteria in determining urinary tract infection was assessed by receiver operator characteristic curves with area under the curve values found to be 0.697 [95% confidence interval (CI): 0.665-0.729] and 0.587 (95% CI: 0.551-0.623), respectively. We suggested that the sediMAX was not suitable for use as a rule-out screen prior to culture and further validation work must be carried out before routine use of the analyser.

  3. Microfluidic microscopy-assisted label-free approach for cancer screening: automated microfluidic cytology for cancer screening.

    PubMed

    Jagannadh, Veerendra Kalyan; Gopakumar, G; Subrahmanyam, Gorthi R K Sai; Gorthi, Sai Siva

    2016-07-22

    Each year, about 7-8 million deaths occur due to cancer around the world. More than half of the cancer-related deaths occur in the less-developed parts of the world. Cancer mortality rate can be reduced with early detection and subsequent treatment of the disease. In this paper, we introduce a microfluidic microscopy-based cost-effective and label-free approach for identification of cancerous cells. We outline a diagnostic framework for the same and detail an instrumentation layout. We have employed classical computer vision techniques such as 2D principal component analysis-based cell type representation followed by support vector machine-based classification. Analogous to criminal face recognition systems implemented with help of surveillance cameras, a signature-based approach for cancerous cell identification using microfluidic microscopy surveillance is demonstrated. Such a platform would facilitate affordable mass screening camps in the developing countries and therefore help decrease cancer mortality rate.

  4. Feasibility of implementing an automated culture system for bacteria screening in platelets in the blood bank routine.

    PubMed

    Castro, E; Bueno, J L; Barea, L; González, R

    2005-06-01

    Bacterial contamination of blood components is the principal infectious complication linked to transfusion. The aim of the study was to evaluate the applicability of an automated culture system for platelets. 10 141 platelet concentrates were cultured individually and in pools of five on storage days 1 and 7 using Bact/Alert system aerobic bottles. A modified collection bag was used for improved sampling. Five-millilitre samples were cultured at 37 degrees C for 7 days. Only those samples where the same bacteria were identified in reculture were considered true positives (TP). Homogeneity of proportions was tested by Fisher's exact test. The rate of TP was 30 per 100 000 (95% CI, 6.1-86.4) sampling on day 1; 33 per 100 000 (95% CI, 7-96) on day 7; and 40 per 100 000 (95% CI, 1.28-122.4) if the screening was based on taking both samples (day 1 and 7). Only one TP was detected in the pool testing. The time for detection among TPs on day 1 ranged between 30 and 134 h. The system is not considered practical for use as a routine screening method, as the time for detection is too long. Pool testing is insensitive. Faster screening methods or pathogen-inactivation systems are needed.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    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.

  6. 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...

  7. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.

    PubMed

    Quaglia, Daniela; Ebert, Maximilian C C J C; Mugford, Paul F; Pelletier, Joelle N

    2017-01-01

    The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

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

    2015-01-01

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

  10. 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

  11. Automation and validation of the Transflour technology: a universal screening assay for G protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Hudson, Christine C.; Oakley, Robert H.; Cruickshank, Rachael D.; Rhem, Shay M.; Loomis, Carson R.

    2002-06-01

    G protein-coupled receptors (GPCRs) are historically the richest targets for drug discovery, accounting for nearly 60 percent of prescription drugs. The ligands and functions of only 200 out of possibly 1000 GPCRs are known. Screening methods that directly and accurately measure GPCR activation and inhibition are required to identify ligands for orphan receptors and cultivate superior drugs for known GPCRs. Norak Biosciences utilizes the redistribution of a fluorescently-labeled protein, arrestin, as a novel screen for monitoring GPCR activation. In contrast to the present methods of analyzing GPCR function, the power of the Transfluor technology is in its simplicity, large signal to noise ratio, and applicability to all GPCRs. Here, we demonstrate that the Transfluor technology can be automated and quantitated on high throughput image analysis systems. Cells transfected with an arrestin-green fluorescent protein conjugate and the neurokinin-1 GPCR were seeded on 96-well plates. Activation of the NK-1 receptor with Substance P induced translocation of arrestin-GFP from the cytosol to the receptor. Image quantitation of the arrestin-GFP translocation was used to generate dose dependent curves. These results reveal that the Transfluor technology combined with an image analysis system forms a universal platform capable of measuring ligand-receptor interactions for all GPCRs.

  12. 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.

  13. 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

  14. Development of an Automated Modality-Independent Elastographic Image Analysis System for Tumor Screening

    DTIC Science & Technology

    2006-02-01

    increased computational complexity, developing and testing metrics for the evaluation of reconstructions, and the fabrication of a compression chamber... tested on a tissue-like polymer phantom. elastography, breast cancer screening, image processing 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...design and a share of 100 CPUs on the Vanderbilt University Advanced Computing Center for Research and 5 Education cluster, this has been tested to be

  15. 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.

  16. Detection and classification of threat agents via high-content assays of mammalian cells.

    PubMed

    Tencza, Sarah B; Sipe, Michael A

    2004-01-01

    One property common to all chemical or biological threat agents is that they damage mammalian cells. A threat detection and classification method based on the effects of compounds on cells has been developed. This method employs high-content screening (HCS), a concept in drug discovery that enables those who practice cell-based assays to generate deeper biological information about the compounds they are testing. A commercial image-based cell screening platform comprising fluorescent reagents, automated image acquisition hardware, image analysis algorithms, data management and informatics was used to develop assays and detection/classification methods for threat agents. These assays measure a cell's response to a compound, which may include activation or inhibition of signal transduction pathways, morphological changes or cytotoxic effects. Data on cell responses to a library of compounds was collected and used as a training set. At the EILATox-Oregon Workshop, cellular responses following exposure to unknown samples were measured by conducting assays of p38 MAP kinase, NF-kappaB, extracellular-signal related kinase (ERK) MAP kinase, cyclic AMP-response element binding protein (CREB), cell permeability, lysosomal mass and nuclear morphology. Although the assays appeared to perform well, only four of the nine toxic samples were detected. However the system was specific, because no false positives were detected. Opportunities for improvement to the system were identified during the course of this enlightening workshop. Some of these improvements were applied in subsequent tests in the Cellomics laboratories, resulting in a higher level of detection. Thus, an HCS approach was shown to have potential in detecting threat agents, but additional work is necessary to make this a comprehensive detection and classification system.

  17. SleepAp: an automated obstructive sleep apnoea screening application for smartphones.

    PubMed

    Behar, Joachim; Roebuck, Aoife; Shahid, Mohammed; Daly, Jonathan; Hallack, Andre; Palmius, Niclas; Stradling, John; Clifford, Gari D

    2015-01-01

    Obstructive sleep apnoea (OSA) is a sleep disorder with long-term consequences. Long-term effects include sleep-related issues and cardiovascular diseases. OSA is often diagnosed with an overnight sleep test called a polysomnogram. Monitoring can be costly with long wait times for diagnosis. In this paper, a novel OSA screening framework and prototype phone application are introduced. A database of 856 patients that underwent at-home polygraphy was collected. Features were derived from audio, actigraphy, photoplethysmography (PPG), and demographics, and used as the inputs of a support vector machine (SVM) classifier. The SVM was trained on 735 patients and tested on 121 patients. Classification on the test set had an accuracy of up to 92.2% when classifying subjects as having moderate or severe OSA versus being healthy or a snorer based on the clinicians' diagnoses. The signal processing and machine learning algorithms were ported to Java and integrated into the phone application-SleepAp. SleepAp records the body position, audio, actigraphy and PPG signals, and implements the clinically validated STOP-BANG questionnaire. It derives features from the signals and classifies the user as having OSA or not using the SVM trained on the clinical database. The resulting software could provide a new, easy-to-use, low-cost, and widely available modality for OSA screening.

  18. 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.

  19. 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%.

  20. Automated, high-throughput platform for protein solubility screening using a split-GFP system.

    PubMed

    Listwan, Pawel; Terwilliger, Thomas C; Waldo, Geoffrey S

    2009-03-01

    Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are "difficult to handle" as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216-228) can be detected in vivo and in vitro using the engineered GFP 1-10 "detector" fragment (amino acids 1-215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.

  1. 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

  2. Automated image-based phenotypic analysis in zebrafish embryos

    PubMed Central

    Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

    2009-01-01

    Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

  3. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

    PubMed Central

    Ebert, Maximilian C. C. J. C.; Mugford, Paul F.; Pelletier, Joelle N.

    2017-01-01

    The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells. PMID:28178357

  4. 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.

  5. An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation.

    PubMed

    Bursey, Evan H; Kim, Chang-Yub; Yu, Minmin; Terwilliger, Thomas C; Hung, Li-Wei

    2006-12-01

    A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.

  6. 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

  7. 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...

  8. 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

  9. A micropatterned hepatocyte coculture model for assessment of liver toxicity using high-content imaging analysis.

    PubMed

    Trask, O Joseph; Moore, Amanda; LeCluyse, Edward L

    2014-01-01

    The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time.

  10. 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

  11. High content image cytometry in the context of subnuclear organization.

    PubMed

    De Vos, W H; Van Neste, L; Dieriks, B; Joss, G H; Van Oostveldt, P

    2010-01-01

    The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high-throughput applications, such as functional protein assays or drug compound screening.

  12. High-throughput pharmacokinetics screen of VLA-4 antagonists by LC/MS/MS coupled with automated solid-phase extraction sample preparation.

    PubMed

    Tong, Xinchun S; Wang, Junying; Zheng, Song; Pivnichny, James V

    2004-06-29

    Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors.

  13. 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: 18 WBC/μL and 97 BAC/μL for SediMax (sensitivity=96.25%, specificity=63.04%, negative predictive value=97.97%), and 40 WBC/μL and 460 BAC/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.

  14. Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis.

    PubMed

    Coudray, Nicolas; Hermann, Gilles; Caujolle-Bert, Daniel; Karathanou, Argyro; Erne-Brand, Françoise; Buessler, Jean-Luc; Daum, Pamela; Plitzko, Juergen M; Chami, Mohamed; Mueller, Urs; Kihl, Hubert; Urban, Jean-Philippe; Engel, Andreas; Rémigy, Hervé-W

    2011-02-01

    We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.

  15. High-Throughput Screening of Na(V)1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument.

    PubMed

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

    2016-03-01

    Voltage-gated sodium (Na(V)) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na(V)1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na(V)1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube(®) is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na(V)1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC50 assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC50 potencies generated on the Qube correlate with data generated on the more established Sophion QPatch(®) APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na(V)1.7 in a robust and reliable APC assay for compound screening.

  16. A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity.

    PubMed

    Anderl, Janet L; Redpath, Stella; Ball, Andrew J

    2009-05-05

    High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with betaIII-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co

  17. Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish.

    PubMed

    Sanker, Subramaniam; Cirio, Maria Cecilia; Vollmer, Laura L; Goldberg, Natasha D; McDermott, Lee A; Hukriede, Neil A; Vogt, Andreas

    2013-12-01

    Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

  18. Developing a UHPLC-QTOF-MS and Automated Library Search Method for Screening Drugs and Toxic Compounds in Postmortem Specimens.

    PubMed

    Liu, Hsiu-Chuan; Yang, Chu-An; Liu, Ray H; Lin, Dong-Liang

    2017-03-24

    Screening and confirming the presence of drugs and toxic compounds in various matrices are important and challenging tasks routinely faced by forensic and clinical laboratories. Recent advances in the liquid chromatographic and mass spectrometric technologies have provided an opportunity for the development of more specific and effective approaches to achieve the "screening" and "confirmation" goals in a single analytical step. The objectives of this study are: (i) the establishment of an ultra-high performance liquid chromatographic, quadrupole time-of-flight mass spectrometric mass spectrometric and MS-MS spectral database, including 1,200 compounds of interest; and (ii) the development of an effective protocol, using this database and three searching algorithms, for general unknown screening of these compounds. The established database and protocol were evaluated through the analysis of 30 external proficiency test and 100 postmortem samples and found to be significantly more effective than the LC-IT-MS and GC-MS approaches previously established in our laboratory.

  19. Quantification of bacteria on abiotic surfaces by laser scanning cytometry: an automated approach to screen the antifouling properties of new surface coatings.

    PubMed

    Regina, Viduthalai Rasheedkhan; Poulsen, Morten; Søhoel, Helmer; Bischoff, Claus; Meyer, Rikke Louise

    2012-08-01

    Bacterial biofilms are a persistent source of contamination, and much effort has been invested in developing antifouling surfaces or coatings. A bottleneck in developing such coatings is often the time-consuming task of screening and evaluating a large number of surface materials. An automated high-throughput assay is therefore needed. In this study, we present a promising technique, laser scanning cytometry (LSC), for automated quantification of bacteria on surfaces. The method was evaluated by quantifying young Staphylococcus xylosus biofilms on glass surfaces using LSC and comparing the results with cell counts obtained by fluorescence microscopy. As an example of application, we quantified bacterial adhesion to seven different sol-gel-based coatings on stainless steel. The surface structure and hydrophobicity of the coatings were analyzed using atomic force microscopy and water contact angle measurements. Among the coatings tested, a significant reduction in adhesion of S. xylosus was observed only for one coating, which also had a unique surface microstructure. LSC was particularly sensitive for quantification at low cell densities, and the adhered bacteria could be quantified both as cell number and as area coverage. The method proved to be an excellent alternative to microscopy for fast and reproducible quantification of microbial colonization on abiotic surfaces.

  20. Automated decision stations

    NASA Technical Reports Server (NTRS)

    Tischendorf, Mark

    1990-01-01

    This paper discusses the combination of software robots and expert systems to automate everyday business tasks. Tasks which require people to repetitively interact with multiple systems screens as well as multiple systems.

  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

    2016-08-02

    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.

  2. Multiplexed assays by high-content imaging for assessment of GPCR activity.

    PubMed

    Ross, D A; Lee, S; Reiser, V; Xue, J; Alves, K; Vaidya, S; Kreamer, A; Mull, R; Hudak, E; Hare, T; Detmers, P A; Lingham, R; Ferrer, M; Strulovici, B; Santini, F

    2008-07-01

    G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor alone has limitations: relying on a single mechanistic step of beta-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multiplexed the Transfluor assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization.

  3. 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,...

  4. HC StratoMineR: A Web-Based Tool for the Rapid Analysis of High-Content Datasets.

    PubMed

    Omta, Wienand A; van Heesbeen, Roy G; Pagliero, Romina J; van der Velden, Lieke M; Lelieveld, Daphne; Nellen, Mehdi; Kramer, Maik; Yeong, Marley; Saeidi, Amir M; Medema, Rene H; Spruit, Marco; Brinkkemper, Sjaak; Klumperman, Judith; Egan, David A

    2016-10-01

    High-content screening (HCS) can generate large multidimensional datasets and when aligned with the appropriate data mining tools, it can yield valuable insights into the mechanism of action of bioactive molecules. However, easy-to-use data mining tools are not widely available, with the result that these datasets are frequently underutilized. Here, we present HC StratoMineR, a web-based tool for high-content data analysis. It is a decision-supportive platform that guides even non-expert users through a high-content data analysis workflow. HC StratoMineR is built by using My Structured Query Language for storage and querying, PHP: Hypertext Preprocessor as the main programming language, and jQuery for additional user interface functionality. R is used for statistical calculations, logic and data visualizations. Furthermore, C++ and graphical processor unit power is diffusely embedded in R by using the rcpp and rpud libraries for operations that are computationally highly intensive. We show that we can use HC StratoMineR for the analysis of multivariate data from a high-content siRNA knock-down screen and a small-molecule screen. It can be used to rapidly filter out undesirable data; to select relevant data; and to perform quality control, data reduction, data exploration, morphological hit picking, and data clustering. Our results demonstrate that HC StratoMineR can be used to functionally categorize HCS hits and, thus, provide valuable information for hit prioritization.

  5. Comparison of the Accuracy of Noninvasive Hemoglobin Sensor (NBM-200) and Portable Hemoglobinometer (HemoCue) with an Automated Hematology Analyzer (LH500) in Blood Donor Screening

    PubMed Central

    Kim, Moon Jung; Park, Quehn; Kim, Myung Hee; Shin, Jeong Won

    2013-01-01

    Background The Hb levels of prospective blood donors are usually determined using a finger prick test. A new noninvasive Hb device has the advantage of not causing any sampling pain. The purpose of this study was to evaluate the accuracy of the noninvasive Hb sensor and to compare its measurements with those of a currently used portable hemoglobinometer. Methods Hb was measured using a noninvasive Hb sensor (NBM-200; OrSense, Israel), a portable hemoglobinometer (HemoCue; HemoCue AB, Sweden), and an automated hematology analyzer (LH500; Beckman Coulter, USA). The correlations between Hb measurements taken by the NBM-200 and HemoCue with those by an automated hematology analyzer were assessed using intraclass correlation coefficients (ICCs). Hb measurements were compared among 3 different Hb level groups. Results The mean Hb values of 506 blood donors were 14.1 g/dL by the NBM-200, 14.0 g/dL by the LH500, and 14.3 g/dL by the HemoCue. The correlation between the LH500 and the NBM-200 was substantial (ICC=0.69), while that between the LH500 and the HemoCue agreed almost perfectly (ICC=0.86). Conclusions The possibility to judge to be eligible for donors who are ineligible to donate was substantial when using NBM-200. Even though the NBM-200 has the apparent advantage of noninvasiveness, its use in pre-screening should be given meticulous attention. Since pre-donation testing is crucial to protecting donors' health, complete evaluation of the instrument should be performed prior to use. PMID:23826562

  6. Automated high-performance cIMT measurement techniques using patented AtheroEdge™: a screening and home monitoring system.

    PubMed

    Molinari, Filippo; Meiburger, Kristen M; Suri, Jasjit

    2011-01-01

    The evaluation of the carotid artery wall is fundamental for the assessment of cardiovascular risk. This paper presents the general architecture of an automatic strategy, which segments the lumen-intima and media-adventitia borders, classified under a class of Patented AtheroEdge™ systems (Global Biomedical Technologies, Inc, CA, USA). Guidelines to produce accurate and repeatable measurements of the intima-media thickness are provided and the problem of the different distance metrics one can adopt is confronted. We compared the results of a completely automatic algorithm that we developed with those of a semi-automatic algorithm, and showed final segmentation results for both techniques. The overall rationale is to provide user-independent high-performance techniques suitable for screening and remote monitoring.

  7. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    PubMed Central

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-01

    Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. PMID:26771356

  8. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    PubMed

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  9. 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

  10. High-Content Assay to Identify Inhibitors of Dengue Virus Infection

    PubMed Central

    Shum, David; Smith, Jessica L.; Hirsch, Alec J.; Bhinder, Bhavneet; Radu, Constantin; Stein, David A.; Nelson, Jay A.; Früh, Klaus

    2010-01-01

    Abstract Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration–approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC50 potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections. PMID:20973722

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

    PubMed

    Hoh, Eunha; Lehotay, Steven J; Mastovska, Katerina; Huwe, Janice K

    2008-08-01

    An automated direct sample introduction technique coupled to comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (DSI-GC x GC/TOF-MS) was applied for the development of a relatively fast and easy analytical screening method for 17 polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and 4 non-ortho polychlorinated biphenyls (PCBs) in fish oil. Comparison of instrumental performance between DSI-GC x GC/TOF-MS and the traditional gas chromatographic high resolution mass spectrometric (GC-HRMS) method showed good agreement of results for standard solutions analyzed in blind fashion. Relatively high tolerance of the DSI technique for lipids in the final extracts enabled a streamlined sample preparation procedure that only required gel permeation chromatography (GPC) and solid-phase extraction (SPE) cleanup with graphitized carbon black. The sample size for the method was 2g of cod liver oil, which achieved limits of quantitation (LOQs) of 0.019-7.8 pg/g toxic equivalent quotients for the individual PCDD/Fs. Lower detection limits can be achieved by using larger sample size and scaling up the sample preparation procedure, but this adds to the labor, time, solvent consumption, and expense of the approach. However, the streamlined method yielded 0.94 pg/g and 2.3 pg/g LOQs for 2,3,7,8-tetrachloro dibenzofuran (TCDF) and 3,3',4,4',5-pentachloro biphenyl (CB126), which were sufficiently low for regulatory monitoring of 2g samples. Therefore, instead of congener specific analysis, this streamlined analytical screening method for TCDF and CB126 has the potential to monitor fish oil contaminated with dioxin and dioxin-like PCBs at or above current food safety limits. Acceptable recoveries for nearly all analytes at three different spiking levels in fish oil samples were achieved with good repeatability.

  12. Screening of drugs in equine plasma using automated on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry.

    PubMed

    Kwok, W H; Leung, David K K; Leung, Gary N W; Wan, Terence S M; Wong, Colton H F; Wong, Jenny K Y

    2010-05-07

    A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC-MS-MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and chromatographic separation of the targeted drugs were performed using respectively a polymeric extraction column (2 cm L x 2.1mm ID, 25 microm particle size) and a reversed-phase C18 LC column (3 cm L x 2.1mm ID, 3 microm particle size) with gradient elution to provide fast analysis time. The overall instrument turnaround time was 9.5 min, inclusive of post-run and equilibration time. Plasma samples fortified with 19 targeted drugs including narcotic analgesics, local anaesthetics, antipsychotics, bronchodilators, mucolytics, corticosteroids, sedative and tranquillisers at sub-parts per billion (ppb) to low parts per trillion (ppt) levels could be consistently detected. No significant matrix interference was observed at the expected retention times of the targeted ion transitions. Over 70% of the drugs studied gave detection limits at or below 100 pg/mL, with some detection limits reaching down to 19 pg/mL. The method had been validated for extraction recovery, precision and sensitivity, and a blockage study had also been carried out. This method is used regularly in the authors' laboratory to screen for the presence of targeted drugs in pre-race plasma samples from racehorses.

  13. 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.

  14. High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells.

    PubMed

    Liang, Shenxuan; Yin, Lei; Shengyang Yu, Kevin; Hofmann, Marie-Claude; Yu, Xiaozhong

    2017-01-01

    Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.

  15. 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.

  16. Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

    PubMed Central

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

    2013-01-01

    Abstract 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

  17. A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

    PubMed Central

    Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

    2012-01-01

    The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

  18. Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.

    PubMed

    Grove, Linnette E; Ghosh, Richik N

    2006-08-01

    A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.

  19. Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots.

    PubMed

    Williams, Y; Byrne, S; Bashir, M; Davies, A; Whelan, A; Gun'ko, Y; Kelleher, D; Volkov, Y

    2008-10-01

    Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots. Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence. This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.

  20. High-content analysis of tumour cell invasion in three-dimensional spheroid assays

    PubMed Central

    Cheng, Vinton; Esteves, Filomena; Chakrabarty, Aruna; Cockle, Julia; Short, Susan; Brüning-Richardson, Anke

    2015-01-01

    Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time. PMID:26244167

  1. Mechanism of action-based classification of antibiotics using high-content bacterial image analysis.

    PubMed

    Peach, Kelly C; Bray, Walter M; Winslow, Dustin; Linington, Peter F; Linington, Roger G

    2013-07-01

    Image-based screening has become a mature field over the past decade, largely due to the detailed information that can be obtained about compound mode of action by considering the phenotypic effects of test compounds on cellular morphology. However, very few examples exist of extensions of this approach to bacterial targets. We now report the first high-throughput, high-content platform for the prediction of antibiotic modes of action using image-based screening. This approach employs a unique feature segmentation and extraction protocol to quantify key size and shape metrics of bacterial cells over a range of compound concentrations, and matches the trajectories of these metrics to those of training set compounds of known molecular target to predict the test compound's mode of action. This approach has been used to successfully predict the modes of action of a panel of known antibiotics, and has been extended to the evaluation of natural products libraries for the de novo prediction of compound function directly from primary screening data.

  2. Cermet anode compositions with high content alloy phase

    DOEpatents

    Marschman, Steven C.; Davis, Norman C.

    1989-01-01

    Cermet electrode compositions comprising NiO-NiFe.sub.2 O.sub.4 -Cu-Ni, and methods for making, are disclosed. Addition of nickel metal prior to formation and densification of a base mixture into the cermet allows for an increase in the total amount of copper and nickel that can be contained in the NiO-NiFe.sub.2 O.sub.4 oxide system. Nickel is present in a base mixture weight concentration of from 0.1% to 10%. Copper is present in the alloy phase in a weight concentration of from 10% to 30% of the densified composition. Such cermet electrodes can be formed to have electrical conductivities well in excess of 100 ohm.sup.-1 cm.sup.-1. Other alloy and oxide system cermets having high content metal phases are also expected to be manufacturable in accordance with the invention.

  3. High-content analysis for drug delivery and nanoparticle applications.

    PubMed

    Brayden, David J; Cryan, Sally-Ann; Dawson, Kenneth A; O'Brien, Peter J; Simpson, Jeremy C

    2015-08-01

    High-content analysis (HCA) provides quantitative multiparametric cellular fluorescence data. From its origins in discovery toxicology, it is now addressing fundamental questions in drug delivery. Nanoparticles (NPs), polymers, and intestinal permeation enhancers are being harnessed in drug delivery systems to modulate plasma membrane properties and the intracellular environment. Identifying comparative mechanistic cytotoxicity on sublethal events is crucial to expedite the development of such systems. NP uptake and intracellular routing pathways are also being dissected using chemical and genetic perturbations, with the potential to assess the intracellular fate of targeted and untargeted particles in vitro. As we discuss here, HCA is set to make a major impact in preclinical delivery research by elucidating the intracellular pathways of NPs and the in vitro mechanistic-based toxicology of formulation constituents.

  4. Selective-plane illumination microscopy for high-content volumetric biological imaging

    NASA Astrophysics Data System (ADS)

    McGorty, Ryan; Huang, Bo

    2016-03-01

    Light-sheet microscopy, also named selective-plane illumination microscopy, enables optical sectioning with minimal light delivered to the sample. Therefore, it allows one to gather volumetric datasets of developing embryos and other light-sensitive samples over extended times. We have configured a light-sheet microscope that, unlike most previous designs, can image samples in formats compatible with high-content imaging. Our microscope can be used with multi-well plates or with microfluidic devices. In designing our optical system to accommodate these types of sample holders we encounter large optical aberrations. We counter these aberrations with both static optical components in the imaging path and with adaptive optics. Potential applications of this microscope include studying the development of a large number of embryos in parallel and over long times with subcellular resolution and doing high-throughput screens on organisms or cells where volumetric data is necessary.

  5. Automated Processing of Zebrafish Imaging Data: A Survey

    PubMed Central

    Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-01-01

    Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

  6. High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies*

    PubMed Central

    Ha, Kevin D.; Bidlingmaier, Scott M.; Zhang, Yafeng; Su, Yang; Liu, Bin

    2014-01-01

    Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly

  7. HIGH-CONTENT ANALYSIS OF PRIMARY RAT NEURAL CORTICALCULTURES FOR DEVELOPMENTAL NEUROTOXICITY SCREENING

    EPA Science Inventory

    Development of the vertebrate nervous system proceeds through a number of critical processes, ultimately concluding with the extension of neurites and establishment of synaptic networks. Early-life exposure to toxicants that perturb these critical developmental processes can po...

  8. Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening

    EPA Science Inventory

    Identification of chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health risks to infants and children. In response to the need for more efficient methods to identify potential developmental neurotoxicants,...

  9. CellH5: a format for data exchange in high-content screening

    PubMed Central

    Sommer, Christoph; Held, Michael; Fischer, Bernd; Huber, Wolfgang; Gerlich, Daniel W.

    2013-01-01

    Summary: High-throughput microscopy data require a diversity of analytical approaches. However, the construction of workflows that use algorithms from different software packages is difficult owing to a lack of interoperability. To overcome this limitation, we present CellH5, an HDF5 data format for cell-based assays in high-throughput microscopy, which stores high-dimensional image data along with inter-object relations in graphs. CellH5Browser, an interactive gallery image browser, demonstrates the versatility and performance of the file format on live imaging data of dividing human cells. CellH5 provides new opportunities for integrated data analysis by multiple software platforms. Availability: Source code is freely available at www.github.com/cellh5 under the GPL license and at www.bioconductor.org/packages/release/bioc/html/rhdf5.html under the Artistic-2.0 license. Demo datasets and the CellH5Browser are available at www.cellh5.org. A Fiji importer for cellh5 will be released soon. Contact: daniel.gerlich@imba.oeaw.ac.at or christoph.sommer@imba.oeaw.ac.at Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23595665

  10. Investigation of cell morphology for disease diagnostics via high content screening

    NASA Astrophysics Data System (ADS)

    Khatau, Shyam

    2013-03-01

    Ninety percent of all cancer-related deaths are caused by metastatic disease, i.e. the spreading of a subset of cells from a primary tumor in an organ to distal sites in other organs. Understanding this progression from localized to metastatic disease is essential for further developing effective therapeutic and treatment strategies. However, despite research efforts, no distinct genetic, epigenetic, or proteomic signature of cancer metastasis has been identified so far. Metastasis is a physical event: through invasion and migration through the dense, tortuous stromal matrix, intravasation, shear forces of blood flow, successful re-attachment to blood vessel walls, migration, the colonization of a distal site, and, finally, reactivation following dormancy, metastatic cells may share precise physical properties. Cell morphology is the most direct physical property that can be measured. In this work, we develop a high throughput cell phenotyping process and investigate the morphological signature of primary tumor cells and liver metastatic pancreatic cancer cells.

  11. ASSESSMENT OF CHEMICAL EFFECTS ON NEURONAL DIFFERENTIATION USING THE ARRAYSCAN HIGH CONTENT SCREENING SYSTEM

    EPA Science Inventory

    The development of alternative methods for toxicity testing is driven by the need for scientifically valid data that can be obtained in a rapid and cost-efficient manner. In vitro systems provide a model in which chemical effects on cellular events can be examined using technique...

  12. IN VITRO SCREENING OF DEVELOPMENTAL NEUROTOXICANTS IN RAT PRIMARY CORTICAL NEURONS USING HIGH CONTENT IMAGE

    EPA Science Inventory

    There is a need for more efficient and cost-effective methods for identifying, characterizing and prioritizing chemicals which may result in developmental neurotoxicity. One approach is to utilize in vitro test systems which recapitulate the critical processes of nervous system d...

  13. Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

    PubMed Central

    Garvis, Steven; Munder, Antje; Ball, Geneviève; de Bentzmann, Sophie; Wiehlmann, Lutz; Ewbank, Jonathan J.; Tümmler, Burkhard; Filloux, Alain

    2009-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity. PMID:19662168

  14. Fast and accurate automated cell boundary determination for fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

    2013-07-01

    Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

  15. Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra-high-performance liquid chromatography with TOF-MS with data-independent acquisition.

    PubMed

    Pedersen, Anders Just; Dalsgaard, Petur Weihe; Rode, Andrej Jaroslav; Rasmussen, Brian Schou; Müller, Irene Breum; Johansen, Sys Stybe; Linnet, Kristian

    2013-07-01

    A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.

  16. Detecting the Senescence-Associated Secretory Phenotype (SASP) by High Content Microscopy Analysis.

    PubMed

    Hari, Priya; Acosta, Juan Carlos

    2017-01-01

    The diverse arrays of proteins secreted by senescent cells have been described to influence aging and to have both pro-tumorigenic and anti-tumorigenic influences on the surrounding microenvironment. Further characterization of these proteins, known as the senescence-associated secretory phenotype (SASP), and their regulators is required to understand and further manipulate such activities. The use of high-throughput technology allows us to obtain visual and quantitative data on a large number of samples quickly and easily. Not only is this an invaluable tool for conducting large-scale RNAi or compound screenings, but also allows rapid validation of candidates of interest. Here, we describe how we use the Widefield High-Content Analysis Systems to characterize the phenotypes of cells following modulation by potential regulators of Oncogene-Induced Senescence (OIS) by measuring numerous markers of senescence, including the SASP. This approach can be also used to screen for siRNA able to perturb the expression of SASP components during OIS.

  17. High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

    PubMed Central

    Joshi, Pranav; Lee, Moo-Yeal

    2015-01-01

    High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology. PMID:26694477

  18. A high-content analysis toolbox permits dissection of diverse signaling pathways for T lymphocyte polarization.

    PubMed

    Freeley, Michael; Bakos, Gabor; Davies, Anthony; Kelleher, Dermot; Long, Aideen; Dunican, Dara J

    2010-06-01

    RNA interfering (RNAi) screening strategies offer the potential to elucidate the signaling pathways that regulate integrin and adhesion receptor-mediated changes in T lymphocyte morphology. Of crucial importance, however, is the definition of key sets of parameters that will provide accurate, quantitative, and nonredundant information to flag relevant hits in such assays. In this study, the authors have used an image-based high-content analysis (HCA) technology platform and a panel of 24 pharmacological inhibitors, at a range of concentrations, to define key sets of parameters that enables sensitive and quantitative effects on integrin (LFA-1)-mediated lymphocyte morphology to be evaluated. In particular, multiparametric analysis of lymphocyte morphology that was based on intracellular staining of both the F-actin and alpha-tubulin cytoskeleton resulted in improved ability to discriminate morphological behavior compared to F-actin staining alone. Morphological and fluorescence intensity/distribution profiling of pharmacologically treated lymphocytes stimulated with integrin (LFA-1) and adhesion receptors (CD44) also revealed notable differences in their sensitivity to inhibitors. The assay described here may be used in HCA strategies such as RNAi screening assays to elucidate the signaling pathways and molecules that regulate integrin/adhesion receptor-mediated T lymphocyte polarization.

  19. Automating Small Libraries.

    ERIC Educational Resources Information Center

    Swan, James

    1996-01-01

    Presents a four-phase plan for small libraries strategizing for automation: inventory and weeding, data conversion, implementation, and enhancements. Other topics include selecting a system, MARC records, compatibility, ease of use, industry standards, searching capabilities, support services, system security, screen displays, circulation modules,…

  20. Phaedra, a protocol-driven system for analysis and validation of high-content imaging and flow cytometry.

    PubMed

    Cornelissen, Frans; Cik, Miroslav; Gustin, Emmanuel

    2012-04-01

    High-content screening has brought new dimensions to cellular assays by generating rich data sets that characterize cell populations in great detail and detect subtle phenotypes. To derive relevant, reliable conclusions from these complex data, it is crucial to have informatics tools supporting quality control, data reduction, and data mining. These tools must reconcile the complexity of advanced analysis methods with the user-friendliness demanded by the user community. After review of existing applications, we realized the possibility of adding innovative new analysis options. Phaedra was developed to support workflows for drug screening and target discovery, interact with several laboratory information management systems, and process data generated by a range of techniques including high-content imaging, multicolor flow cytometry, and traditional high-throughput screening assays. The application is modular and flexible, with an interface that can be tuned to specific user roles. It offers user-friendly data visualization and reduction tools for HCS but also integrates Matlab for custom image analysis and the Konstanz Information Miner (KNIME) framework for data mining. Phaedra features efficient JPEG2000 compression and full drill-down functionality from dose-response curves down to individual cells, with exclusion and annotation options, cell classification, statistical quality controls, and reporting.

  1. Drug Discovery Goes Three-Dimensional: Goodbye to Flat High-Throughput Screening?

    PubMed

    Eglen, Richard M; Randle, David H

    2015-06-01

    Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.

  2. High content analysis of an in vitro model for metabolic toxicity: results with the model toxicants 4-aminophenol and cyclophosphamide.

    PubMed

    Cole, Stephanie D; Madren-Whalley, Janna S; Li, Albert P; Dorsey, Russell; Salem, Harry

    2014-12-01

    In vitro models that accurately and rapidly assess hepatotoxicity and the effects of hepatic metabolism on nonliver cell types are needed by the U.S. Department of Defense and the pharmaceutical industry to screen compound libraries. Here, we report the first use of high content analysis on the Integrated Discrete Multiple Organ Co-Culture (IdMOC) system, a high-throughput method for such studies. We cultured 3T3-L1 cells in the presence and absence of primary human hepatocytes, and exposed the cultures to 4-aminophenol and cyclophosphamide, model toxicants that are respectively detoxified and activated by the liver. Following staining with calcein-AM, ethidium homodimer-1, and Hoechst 33342, high content analysis of the cultures revealed four cytotoxic endpoints: fluorescence intensities of calcein-AM and ethidium homodimer-1, nuclear area, and cell density. Using these endpoints, we observed that the cytotoxicity of 4-aminophenol in 3T3-L1 cells in co-culture was less than that observed for 3T3-L1 monocultures, consistent with the known detoxification of 4-aminophenol by hepatocytes. Conversely, cyclophosphamide cytotoxicity for 3T3-L1 cells was enhanced by co-culturing with hepatocytes, consistent with the known metabolic activation of this toxicant. The use of IdMOC plates combined with high content analysis is therefore a multi-endpoint, high-throughput capability for measuring the effects of metabolism on toxicity.

  3. High Content Analysis of Human Pluripotent Stem Cell Derived Hepatocytes Reveals Drug Induced Steatosis and Phospholipidosis

    PubMed Central

    Pradip, Arvind; Steel, Daniella; Jacobsson, Susanna; Holmgren, Gustav; Ingelman-Sundberg, Magnus; Sartipy, Peter; Björquist, Petter; Johansson, Inger; Edsbagge, Josefina

    2016-01-01

    Hepatotoxicity is one of the most cited reasons for withdrawal of approved drugs from the market. The use of nonclinically relevant in vitro and in vivo testing systems contributes to the high attrition rates. Recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into pure cultures of hepatocyte-like cells expressing functional drug metabolizing enzymes open up possibilities for novel, more relevant human cell based toxicity models. The present study aimed to investigate the use of hiPSC derived hepatocytes for conducting mechanistic toxicity testing by image based high content analysis (HCA). The hiPSC derived hepatocytes were exposed to drugs known to cause hepatotoxicity through steatosis and phospholipidosis, measuring several endpoints representing different mechanisms involved in drug induced hepatotoxicity. The hiPSC derived hepatocytes were benchmarked to the HepG2 cell line and generated robust HCA data with low imprecision between plates and batches. The different parameters measured were detected at subcytotoxic concentrations and the order of which the compounds were categorized (as severe, moderate, mild, or nontoxic) based on the degree of injury at isomolar concentration corresponded to previously published data. Taken together, the present study shows how hiPSC derived hepatocytes can be used as a platform for screening drug induced hepatotoxicity by HCA. PMID:26880940

  4. A High-Content Larval Zebrafish Brain Imaging Method for Small Molecule Drug Discovery.

    PubMed

    Liu, Harrison; Chen, Steven; Huang, Kevin; Kim, Jeffrey; Mo, Han; Iovine, Raffael; Gendre, Julie; Pascal, Pauline; Li, Qiang; Sun, Yaping; Dong, Zhiqiang; Arkin, Michelle; Guo, Su; Huang, Bo

    2016-01-01

    Drug discovery in whole-organisms such as zebrafish is a promising approach for identifying biologically-relevant lead compounds. However, high content imaging of zebrafish at cellular resolution is challenging due to the difficulty in orienting larvae en masse such that the cell type of interest is in clear view. We report the development of the multi-pose imaging method, which uses 96-well round bottom plates combined with a standard liquid handler to repose the larvae within each well multiple times, such that an image in a specific orientation can be acquired. We have validated this method in a chemo-genetic zebrafish model of dopaminergic neuron degeneration. For this purpose, we have developed an analysis pipeline that identifies the larval brain in each image and then quantifies neuronal health in CellProfiler. Our method achieves a SSMD* score of 6.96 (robust Z'-factor of 0.56) and is suitable for screening libraries up to 105 compounds in size.

  5. A High-Content Larval Zebrafish Brain Imaging Method for Small Molecule Drug Discovery

    PubMed Central

    Liu, Harrison; Chen, Steven; Huang, Kevin; Kim, Jeffrey; Mo, Han; Iovine, Raffael; Gendre, Julie; Pascal, Pauline; Li, Qiang; Sun, Yaping; Dong, Zhiqiang; Arkin, Michelle; Guo, Su

    2016-01-01

    Drug discovery in whole-organisms such as zebrafish is a promising approach for identifying biologically-relevant lead compounds. However, high content imaging of zebrafish at cellular resolution is challenging due to the difficulty in orienting larvae en masse such that the cell type of interest is in clear view. We report the development of the multi-pose imaging method, which uses 96-well round bottom plates combined with a standard liquid handler to repose the larvae within each well multiple times, such that an image in a specific orientation can be acquired. We have validated this method in a chemo-genetic zebrafish model of dopaminergic neuron degeneration. For this purpose, we have developed an analysis pipeline that identifies the larval brain in each image and then quantifies neuronal health in CellProfiler. Our method achieves a SSMD* score of 6.96 (robust Z’-factor of 0.56) and is suitable for screening libraries up to 105 compounds in size. PMID:27732643

  6. Comparative studies on the immunoregulatory effects of three polysaccharides using high content imaging system.

    PubMed

    Lv, Xiaocheng; Chen, Dandan; Yang, Liecheng; Zhu, Ning; Li, Jingling; Zhao, Jian; Hu, Zhibi; Wang, Fu-Jun; Zhang, Leshuai W

    2016-05-01

    In this study, polysaccharides were isolated from Astragalus membranaceus, Ganoderma lucidum and Radix ophiopogonis and named APSII, GLPII and OGPII for comparison of their immunoactivities. MTT assay indicated that these polysaccharides increased the metabolic activity of Raw264.7 macrophages and induced cell differentiation to dendritic like cells. High content screening and mathematical modeling were used to quantify the cell irregularity, a hallmark of cell differentiation by polysaccharides. The results showed that GLPII increased cell irregularity, but APSII and OGPII had slightly less effects. Imaging analysis also revealed that polysaccharides inhibited cell proliferation while inducing the cell differentiation. In addition, APSII and GLPII but not OGPII induced NO production and enhanced cell phagocytic ability. Interestingly, inducible nitric oxide synthase inhibitor blocked polysaccharide-enhanced phagocytosis, indicating NO production is crucial for macrophages to acquire phagocytic ability, which was further confirmed by correlation studies. APSII and GLPII significantly promoted the maturation of macrophages by the increase in the expression of MHCII, CD40, CD80 and CD86, while OGPII had less effects. In summary, we have suggested a practical and economical method to quantify macrophage differentiation (irregularity) induced by polysaccharides for quality assurance and have found the role of NO production on macrophage phagocytic ability.

  7. Evaluation of the Droplet-Microarray Platform for High-Throughput Screening of Suspension Cells.

    PubMed

    Popova, Anna A; Depew, Claire; Permana, Katya Manuella; Trubitsyn, Alexander; Peravali, Ravindra; Ordiano, Jorge Ángel González; Reischl, Markus; Levkin, Pavel A

    2017-04-01

    Phenotypic cell-based high-throughput screenings play a central role in drug discovery and toxicology. The main tendency in cell screenings is the increase of the throughput and decrease of reaction volume in order to accelerate the experiments, reduce the costs, and enable screenings of rare cells. Conventionally, cell-based assays are performed in microtiter plates, which exist in 96- to 1536-wells formats and cannot be further miniaturized. In addition, performing screenings of suspension cells is associated with risk of losing cell content during the staining procedures and incompatibility with high-content microscopy. Here, we evaluate the Droplet-Microarray screening platform for culturing, screening, and imaging of suspension cells. We demonstrate pipetting-free cell seeding and proliferation of cells in individual droplets of 3-80 nL in volume. We developed a methodology to perform parallel treatment, staining, and fixation of suspension cells in individual droplets. Automated imaging of live suspension cells directly in the droplets combined with algorithms for pattern recognition for image analysis is demonstrated. We evaluated the developed methodology by performing a dose-response study with antineoplastic drugs. We believe that the DMA screening platform carries great potential to be adopted for broad spectrum of screenings of suspension cells.

  8. Use of a Machine Learning-Based High Content Analysis Approach to Identify Photoreceptor Neurite Promoting Molecules.

    PubMed

    Fuller, John A; Berlinicke, Cynthia A; Inglese, James; Zack, Donald J

    2016-01-01

    High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635-637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.

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

    PubMed

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

    2004-01-01

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

  10. Internet-Based Cervical Cytology Screening Program

    DTIC Science & Technology

    2005-04-01

    cancer is theoretically completely preventable by effective screening using cervical cytology methods (the Pap test). The process of preparing and...preparation and computerized primary screening make automated approaches to cervical cancer screening possible. In addition, advances in information technology... cervical cancer screening results - completed f) Adapt commercial software (Wellogic) to integrate screening results reporting with medical decision

  11. LC-HR-MS/MS standard urine screening approach: Pros and cons of automated on-line extraction by turbulent flow chromatography versus dilute-and-shoot and comparison with established urine precipitation.

    PubMed

    Helfer, Andreas G; Michely, Julian A; Weber, Armin A; Meyer, Markus R; Maurer, Hans H

    2017-02-01

    Comprehensive urine screening for drugs and metabolites by LC-HR-MS/MS using Orbitrap technology has been described with precipitation as simple workup. In order to fasten, automate, and/or simplify the workup, on-line extraction by turbulent flow chromatography and a dilute-and-shoot approach were developed and compared. After chromatographic separation within 10min, the Q-Exactive mass spectrometer was run in full scan mode with positive/negative switching and subsequent data dependent acquisition mode. The workup approaches were validated concerning selectivity, recovery, matrix effects, process efficiency, and limits of identification and detection for typical drug representatives and metabolites. The total workup time for on-line extraction was 6min, for the dilution approach 3min. For comparison, the established urine precipitation and evaporation lasted 10min. The validation results were acceptable. The limits for on-line extraction were comparable with those described for precipitation, but lower than for dilution. Thanks to the high sensitivity of the LC-HR-MS/MS system, all three workup approaches were sufficient for comprehensive urine screening and allowed fast, reliable, and reproducible detection of cardiovascular drugs, drugs of abuse, and other CNS acting drugs after common doses.

  12. Accelerated throughput metabolic route screening in early drug discovery using high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry and automated data analysis.

    PubMed

    Mortishire-Smith, Russell J; O'Connor, Desmond; Castro-Perez, Jose M; Kirby, Jane

    2005-01-01

    The resource investment required to characterise the metabolic fate of a compound is relatively large, meaning that within a drug discovery environment relatively few compounds are characterised in depth. Rate-limiting steps include the setting up of a complex array of mass spectrometry experiments and the subsequent analysis of the large data sets produced. We describe here a strategy for the evaluation of metabolic routes using full-scan high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QToFMS) with automated data analysis using Metabolynx, a commercially available software package. Data from several structurally diverse compounds taken from the literature illustrate that, with careful setting of key parameters, this approach is able to indicate the presence of a wide range of metabolites with only a limited requirement for manual intervention.

  13. Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

    NASA Astrophysics Data System (ADS)

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

    2008-03-01

    We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

  14. Automation of an interferon-γ release assay and comparison to the tuberculin skin test for screening basic military trainees for Mycobacterium tuberculosis infection.

    PubMed

    Goodwin, Donald J; Mazurek, Gerald H; Campbell, Brandon H; Bohanon, Jamaria; West, Kevin B; Bell, James J; Powell, Richard; Toney, Sean; Morris, John A; Yamane, Grover K; Sjoberg, Paul A

    2014-03-01

    We automated portions of the QuantiFERON-TB Gold In-Tube test (QFT-GIT) and assessed its quality when performed concurrently with the tuberculin skin test (TST) among U.S. Air Force basic military trainees (BMTs). The volume of blood collected for QFT-GIT was monitored. At least one of the three tubes required for QFT-GIT had blood volume outside the recommended 0.8- to 1.2-mL range for 688 (29.0%) of 2,373 subjects who had their blood collected. Of the 2,124 subjects who had TST and QFT-GIT completed, TST was positive for 0.6%; QFT-GIT was positive for 0.3% and indeterminate for 2.0%. Among 2,081 subjects with completed TST and determinate QFT-GIT results, overall agreement was 99.5% but positive agreement was 5.6%. Specificity among the 1,546 low-risk BMTs was identical (99.7%). Indeterminate QFT-GIT results were 2.7 times more likely when mitogen tubes contained >1.2 mL blood than when containing 0.8- to 1.2-mL blood. Automation can facilitate QFT-GIT completion, especially if the recommended volume of blood is collected. Mycobacterium tuberculosis infection prevalence among BMTs based on TST and QFT-GIT is similar and low. Selectively testing those with significant risk may be more appropriate than universal testing of all recruits.

  15. Evaluation and validation of an accurate mass screening method for the analysis of pesticides in fruits and vegetables using liquid chromatography-quadrupole-time of flight-mass spectrometry with automated detection.

    PubMed

    López, Mónica García; Fussell, Richard J; Stead, Sara L; Roberts, Dominic; McCullagh, Mike; Rao, Ramesh

    2014-12-19

    This study reports the development and validation of a screening method for the detection of pesticides in 11 different fruit and vegetable commodities. The method was based on ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-QTOF-MS). The objective was to validate the method in accordance with the SANCO guidance document (12571/2013) on analytical quality control and validation procedures for pesticide residues analysis in food and feed. Samples were spiked with 199 pesticides, each at two different concentrations (0.01 and 0.05 mg kg(-1)) and extracted using the QuEChERS approach. Extracts were analysed by UPLC-QTOF-MS using generic acquisition parameters. Automated detection and data filtering were performed using the UNIFI™ software and the peaks detected evaluated against a proprietary scientific library containing information for 504 pesticides. The results obtained using different data processing parameters were evaluated for 4378 pesticide/commodities combinations at 0.01 and 0.05 mg kg(-1). Using mass accuracy (± 5 ppm) with retention time (± 0.2 min) and a low response threshold (100 counts) the validated Screening Detection Limits (SDLs) were 0.01 mg kg(-1) and 0.05 mg kg(-1) for 57% and 79% of the compounds tested, respectively, with an average of 10 false detects per sample analysis. Excluding the most complex matrices (onion and leek) the detection rates increased to 69% and 87%, respectively. The use of additional parameters such as isotopic pattern and fragmentation information further reduced the number of false detects but compromised the detection rates, particularly at lower residue concentrations. The challenges associated with the validation and subsequent implementation of a pesticide multi-residue screening method are also discussed.

  16. Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans.

    PubMed

    Abraham, Vivek C; Towne, Danli L; Waring, Jeffrey F; Warrior, Usha; Burns, David J

    2008-07-01

    Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. The authors report the validation and implementation of a high-content multiparametric cytotoxicity assay based on simultaneous measurement of 8 key cell health indicators associated with nuclear morphology, plasma membrane integrity, mitochondrial function, and cell proliferation. Compounds are prioritized by (a) computing an in vitro safety margin using the minimum cytotoxic concentration (IC(20)) across all 8 indicators and cell-based efficacy data and (b) using the minimal cytotoxic concentration alone to take into account concentration of drug in tissues. Feasibility data using selected compounds, including quinolone antibiotics, thiazolidinediones, and statins, suggest the viability of this approach. To increase overall throughput of compound prioritization, the authors have identified the higher throughput, plate reader-based CyQUANT assay that is similar to the high-content screening (HCS) assay in sensitivity of measuring inhibition of cell proliferation. It is expected that the phenotypic output from the multiparametric HCS assay in combination with other highly sensitive approaches, such as microarray-based expression analysis of toxic signatures, will contribute to a better understanding and predictivity of human hepatotoxicity potential.

  17. In Vitro Testing of Biomaterials for Neural Repair: Focus on Cellular Systems and High-Content Analysis.

    PubMed

    Baldassarro, Vito Antonio; Dolci, Luisa Stella; Mangano, Chiara; Giardino, Luciana; Gualandi, Chiara; Focarete, Maria Letizia; Calzà, Laura

    2016-01-01

    Biomimetic materials are designed to stimulate specific cellular responses at the molecular level. To improve the soundness of in vitro testing of the biological impact of new materials, appropriate cell systems and technologies must be standardized also taking regulatory issues into consideration. In this study, the biological and molecular effects of different scaffolds on three neural systems, that is, the neural cell line SH-SY5Y, primary cortical neurons, and neural stem cells, were compared. The effect of poly(L-lactic acid) scaffolds having different surface geometry (conventional two-dimensional seeding flat surface, random or aligned fibers as semi3D structure) and chemical functionalization (laminin or ECM extract) were studied. The endpoints were defined for efficacy (i.e., neural differentiation and neurite elongation) and for safety (i.e., cell death/survival) using high-content analysis. It is demonstrated that (i) the definition of the biological properties of biomaterials is profoundly influenced by the test system used; (ii) the definition of the in vitro safety profile of biomaterials for neural repair is also influenced by the test system; (iii) cell-based high-content screening may well be successfully used to characterize both the efficacy and safety of novel biomaterials, thus speeding up and improving the soundness of this critical step in material science having medical applications.

  18. In Vitro Testing of Biomaterials for Neural Repair: Focus on Cellular Systems and High-Content Analysis

    PubMed Central

    Baldassarro, Vito Antonio; Dolci, Luisa Stella; Mangano, Chiara; Giardino, Luciana; Gualandi, Chiara; Focarete, Maria Letizia; Calzà, Laura

    2016-01-01

    Abstract Biomimetic materials are designed to stimulate specific cellular responses at the molecular level. To improve the soundness of in vitro testing of the biological impact of new materials, appropriate cell systems and technologies must be standardized also taking regulatory issues into consideration. In this study, the biological and molecular effects of different scaffolds on three neural systems, that is, the neural cell line SH-SY5Y, primary cortical neurons, and neural stem cells, were compared. The effect of poly(L-lactic acid) scaffolds having different surface geometry (conventional two-dimensional seeding flat surface, random or aligned fibers as semi3D structure) and chemical functionalization (laminin or ECM extract) were studied. The endpoints were defined for efficacy (i.e., neural differentiation and neurite elongation) and for safety (i.e., cell death/survival) using high-content analysis. It is demonstrated that (i) the definition of the biological properties of biomaterials is profoundly influenced by the test system used; (ii) the definition of the in vitro safety profile of biomaterials for neural repair is also influenced by the test system; (iii) cell-based high-content screening may well be successfully used to characterize both the efficacy and safety of novel biomaterials, thus speeding up and improving the soundness of this critical step in material science having medical applications. PMID:27588220

  19. A simple and predictive phenotypic High Content Imaging assay for Plasmodium falciparum mature gametocytes to identify malaria transmission blocking compounds

    PubMed Central

    Lucantoni, Leonardo; Silvestrini, Francesco; Signore, Michele; Siciliano, Giulia; Eldering, Maarten; Dechering, Koen J.; Avery, Vicky M.; Alano, Pietro

    2015-01-01

    Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining “classic” gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays. PMID:26553647

  20. An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models

    NASA Astrophysics Data System (ADS)

    Celli, Jonathan P.; Rizvi, Imran; Blanden, Adam R.; Massodi, Iqbal; Glidden, Michael D.; Pogue, Brian W.; Hasan, Tayyaba

    2014-01-01

    While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.

  1. Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

    PubMed Central

    Gelles, Jesse D; Edward Chipuk, Jerry

    2016-01-01

    Quantitative and kinetic analyses of apoptotic cell death are integral components of exploring cell biology, measuring cellular stress responses, and performing high-throughput genomic/RNAi/drug screens. Here, we present a detailed method that integrates robust kinetic real-time high-content imaging with Annexin V labelling to provide a highly sensitive, accurate, simple and zero-handling approach to quantify extrinsic and intrinsic inducers of apoptosis. The sensitivity of this non-toxic method outperforms previous high-throughput methodologies using viability dyes or caspase-activation reporters. This method also incorporates a multiplex adaptation to integrate variability in cell number due to treatment-induced proliferation changes and the detachment of dying cells. Compared to Annexin V detection by flow cytometry, this method is 10-fold more sensitive, eliminates extensive sample handling and processing, and provides real-time kinetics of apoptosis at both single-cell and population-level resolutions. PMID:27906190

  2. The application of high-content analysis in the study of targeted particulate delivery systems for intracellular drug delivery to alveolar macrophages.

    PubMed

    Lawlor, Ciaran; O'Sullivan, Mary P; Sivadas, Neera; O'Leary, Seonadh; Gallagher, Paul J; Keane, Joseph; Cryan, Sally-Ann

    2011-08-01

    With an ever increasing number of particulate drug delivery systems being developed for the intracellular delivery of therapeutics a robust high-throughput method for studying particle-cell interactions is urgently required. Current methods used for analyzing particle-cell interaction include spectrofluorimetry, flow cytometry, and fluorescence/confocal microscopy, but these methods are not high throughput and provide only limited data on the specific number of particles delivered intracellularly to the target cell. The work herein presents an automated high-throughput method to analyze microparticulate drug delivery system (DDS) uptake byalveolar macrophages. Poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared in a range of sizes using a solvent evaporation method. A human monocyte cell line (THP-1) was differentiated into macrophage like cells using phorbol 12-myristate 13-acetate (PMA), and cells were treated with microparticles for 1 h and studied using confocal laser scanning microscopy (CLSM), spectrofluorimetry and a high-content analysis (HCA). PLGA microparticles within the size range of 0.8-2.1 μm were found to be optimal for macrophage targeting (p < 0.05). Uptake studies carried out at 37 °C and 4 °C indicated that microparticles were internalized in an energy dependent manner. To improve particle uptake, a range of opsonic coatings were assessed. Coating PLGA particles with gelatin and ovalbumin was found to significantly increase particle uptake from 2.75 ± 0.98 particles per cell for particles coated with gelatin. Opsonic coating also significantly increased particle internalization into primary human alveolar macrophages (p < 0.01) with a 1.7-fold increase in uptake from 4.19 ± 0.48 for uncoated to 7.53 ± 0.88 particles per cell for coated particles. In comparison to techniques such as spectrofluorimetry and CLSM, HCA provides both qualitative and quantitative data on the influence of carrier design on cell targeting that can be

  3. Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

    EPA Science Inventory

    During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

  4. Automation or De-automation

    NASA Astrophysics Data System (ADS)

    Gorlach, Igor; Wessel, Oliver

    2008-09-01

    In the global automotive industry, for decades, vehicle manufacturers have continually increased the level of automation of production systems in order to be competitive. However, there is a new trend to decrease the level of automation, especially in final car assembly, for reasons of economy and flexibility. In this research, the final car assembly lines at three production sites of Volkswagen are analysed in order to determine the best level of automation for each, in terms of manufacturing costs, productivity, quality and flexibility. The case study is based on the methodology proposed by the Fraunhofer Institute. The results of the analysis indicate that fully automated assembly systems are not necessarily the best option in terms of cost, productivity and quality combined, which is attributed to high complexity of final car assembly systems; some de-automation is therefore recommended. On the other hand, the analysis shows that low automation can result in poor product quality due to reasons related to plant location, such as inadequate workers' skills, motivation, etc. Hence, the automation strategy should be formulated on the basis of analysis of all relevant aspects of the manufacturing process, such as costs, quality, productivity and flexibility in relation to the local context. A more balanced combination of automated and manual assembly operations provides better utilisation of equipment, reduces production costs and improves throughput.

  5. The international experience of bacterial screen testing of platelet components with an automated microbial detection system: a need for consensus testing and reporting guidelines.

    PubMed

    Benjamin, Richard J; McDonald, Carl P

    2014-04-01

    The BacT/ALERT microbial detection system (bioMerieux, Inc, Durham, NC) is in routine use in many blood centers as a prerelease test for platelet collections. Published reports document wide variation in practices and outcomes. A systematic review of the English literature was performed to describe publications assessing the use of the BacT/ALERT culture system on platelet collections as a routine screen test of more than 10000 platelet components. Sixteen publications report the use of confirmatory testing to substantiate initial positive culture results but use varying nomenclature to classify the results. Preanalytical and analytical variables that may affect the outcomes differ widely between centers. Incomplete description of protocol details complicates comparison between sites. Initial positive culture results range from 539 to 10606 per million (0.054%-1.061%) and confirmed positive from 127 to 1035 per million (0.013%-0.104%) donations. False-negative results determined by outdate culture range from 662 to 2173 per million (0.066%-0.217%) and by septic reactions from 0 to 66 per million (0%-0.007%) collections. Current culture protocols represent pragmatic compromises between optimizing analytical sensitivity and ensuring the timely availability of platelets for clinical needs. Insights into the effect of protocol variations on outcomes are generally restricted to individual sites that implement limited changes to their protocols over time. Platelet manufacturers should reassess the adequacy of their BacT/ALERT screening protocols in light of the growing international experience and provide detailed documentation of all variables that may affect culture outcomes when reporting results. We propose a framework for a standardized nomenclature for reporting of the results of BacT/ALERT screening.

  6. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

    PubMed

    Leonard, Anthony P; Cameron, Robert B; Speiser, Jaime L; Wolf, Bethany J; Peterson, Yuri K; Schnellmann, Rick G; Beeson, Craig C; Rohrer, Bärbel

    2015-02-01

    Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological

  7. Wide-scope screening of pesticides in fruits and vegetables using information-dependent acquisition employing UHPLC-QTOF-MS and automated MS/MS library searching.

    PubMed

    Wang, Zhibin; Cao, Yanzhong; Ge, Na; Liu, Xiaomao; Chang, Qiaoying; Fan, Chunlin; Pang, Guo-Fang

    2016-11-01

    This paper presents an application of ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for simultaneous screening and identification of 427 pesticides in fresh fruit and vegetable samples. Both full MS scan mode for quantification, and an artificial-intelligence-based product ion scan mode information-dependent acquisition (IDA) providing automatic MS to MS/MS switching of product ion spectra for identification, were conducted by one injection. A home-in collision-induced-dissociation all product ions accurate mass spectra library containing more than 1700 spectra was developed prior to actual application. Both qualitative and quantitative validations of the method were carried out. The result showed that 97.4 % of the pesticides had the screening detection limit (SDL) less than 50 μg kg(-1) and more than 86.7 % could be confirmed by accurate MS/MS spectra embodied in the home-made library. Meanwhile, calibration curves covering two orders of magnitude were performed, and they were linear over the concentration range studied for the selected matrices (from 5 to 500 μg kg(-1) for most of the pesticides). Recoveries between 80 and 110 % in four matrices (apple, orange, tomato, and spinach) at two spiked levels, 10 and 100 μg kg(-1), was 88.7 or 86.8 %. Furthermore, the overall relative standard deviation (RSD, n = 12) for 94.3 % of the pesticides in 10 μg kg(-1) and 98.1 % of the pesticides in 100 μg kg(-1) spiked levels was less than 20 %. In order to validate the suitability for routine analysis, the method was applied to 448 fruit and vegetable samples purchased in different local markets. The results show 83.3 % of the analyzed samples have positive findings (higher than the limits of identification and quantification), and 412 commodity-pesticide combinations are identified in our scope. The approach proved to be a cost-effective, time-saving and powerful strategy for routine large

  8. Automation pilot

    NASA Technical Reports Server (NTRS)

    1983-01-01

    An important concept of the Action Information Management System (AIMS) approach is to evaluate office automation technology in the context of hands on use by technical program managers in the conduct of human acceptance difficulties which may accompany the transition to a significantly changing work environment. The improved productivity and communications which result from application of office automation technology are already well established for general office environments, but benefits unique to NASA are anticipated and these will be explored in detail.

  9. Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library search.

    PubMed

    Liu, Hsiu-Chuan; Liu, Ray H; Lin, Dong-Liang; Ho, Hsiu-O

    2010-01-01

    Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the 'screen' and 'confirmation' goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI-MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid-liquid and solid-phase extraction protocols were coupled to LC/ESI-MS/MS using a 1.8-microm particle size analytical column operated at 50 degrees C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive-ion ESI-MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1-10 microg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens - based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two-step (immunoassay and GC/MS) approach in the past.

  10. Simultaneous screening of targeted and non-targeted contaminants using an LC-QTOF-MS system and automated MS/MS library searching.

    PubMed

    Herrera-Lopez, S; Hernando, M D; García-Calvo, E; Fernández-Alba, A R; Ulaszewska, M M

    2014-09-01

    Simultaneous high-resolution full-scan and tandem mass spectrometry (MS/MS) analysis using time of flight mass spectrometry brings an answer for increasing demand of retrospective and non-targeted data analysis. Such analysis combined with spectral library searching is a promising tool for targeted and untargeted screening of small molecules. Despite considerable extension of the panel of compounds of tandem mass spectral libraries, the heterogeneity of spectral data poses a major challenge against the effective usage of spectral libraries. Performance evaluation of available LC-MS/MS libraries will significantly increase credibility in the search results. The present work was aimed to evaluate fluctuation of MS/MS pattern, in the peak intensities distribution together with mass accuracy measurements, and in consequence, performance compliant with ion ratio and mass error criteria as principles in identification processes for targeted and untargeted contaminants at trace levels. Matrix effect and ultra-trace levels of concentration (from 50 ng l(-1) to 1000 ng l(-1) were evaluated as potential source of inaccuracy in the performance of spectral matching. Matrix-matched samples and real samples were screened for proof of applicability. By manual review of data and application of ion ratio and ppm error criteria, false negatives were obtained; this number diminished when in-house library was used, while with on-line MS/MS databases 100% of positive samples were found. In our experience, intensity of peaks across spectra was highly correlated to the concentration effect and matrix complexity. In turn, analysis of spectra acquired at trace concentrations and in different matrices results in better performance in providing correct and reliable identification.

  11. Rapid screening of mycotoxins in liquid milk and milk powder by automated size-exclusion SPE-UPLC-MS/MS and quantification of matrix effects over the whole chromatographic run.

    PubMed

    Wang, Xiupin; Li, Peiwu

    2015-04-15

    An automated, size-exclusion solid phase extraction (SPE)-UPLC-MS/MS protocol without pre-treatment of samples was developed to screen for four mycotoxins (OTA, ZEN, AFB1, and AFM1) in liquid milk and milk powder. Firstly, a mixed macropore-silica gel cartridge was established as a size-exclusion SPE column. The proposed methodology could be a candidate in green analytical chemistry because it saves on manpower and organic solvent. Permanent post-column infusion of mycotoxin standards was used to quantify matrix effects throughout the chromatographic run. Matrix-matched calibration could effectively compensate for matrix effects, which may be caused by liquid milk or milk powder matrix. Recovery of the four mycotoxins in fortified liquid milk was in the range 89-120% and RSD 2-9%. The LOD for the four mycotoxins in liquid milk and milk powder were 0.05-2 ng L(-1) and 0.25-10 ng kg(-1), respectively. The LOQ for the four mycotoxins in liquid milk and milk powder were 0.1-5 ng L(-1) and 0.5-25 ng kg(-1), respectively.

  12. VSDMIP 1.5: an automated structure- and ligand-based virtual screening platform with a PyMOL graphical user interface

    NASA Astrophysics Data System (ADS)

    Cabrera, Álvaro Cortés; Gil-Redondo, Rubén; Perona, Almudena; Gago, Federico; Morreale, Antonio

    2011-09-01

    A graphical user interface (GUI) for our previously published virtual screening (VS) and data management platform VSDMIP (Gil-Redondo et al. J Comput Aided Mol Design, 23:171-184, 2009) that has been developed as a plugin for the popular molecular visualization program PyMOL is presented. In addition, a ligand-based VS module (LBVS) has been implemented that complements the already existing structure-based VS (SBVS) module and can be used in those cases where the receptor's 3D structure is not known or for pre-filtering purposes. This updated version of VSDMIP is placed in the context of similar available software and its LBVS and SBVS capabilities are tested here on a reduced set of the Directory of Useful Decoys database. Comparison of results from both approaches confirms the trend found in previous studies that LBVS outperforms SBVS. We also show that by combining LBVS and SBVS, and using a cluster of 100 modern processors, it is possible to perform complete VS studies of several million molecules in less than a month. As the main processes in VSDMIP are 100% scalable, more powerful processors and larger clusters would notably decrease this time span. The plugin is distributed under an academic license upon request from the authors.

  13. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  14. Office automation.

    PubMed

    Arenson, R L

    1986-03-01

    By now, the term "office automation" should have more meaning for those readers who are not intimately familiar with the subject. Not all of the preceding material pertains to every department or practice, but certainly, word processing and simple telephone management are key items. The size and complexity of the organization will dictate the usefulness of electronic mail and calendar management, and the individual radiologist's personal needs and habits will determine the usefulness of the home computer. Perhaps the most important ingredient for success in the office automation arena relates to the ability to integrate information from various systems in a simple and flexible manner. Unfortunately, this is perhaps the one area that most office automation systems have ignored or handled poorly. In the personal computer world, there has been much emphasis recently on integration of packages such as spreadsheet, database management, word processing, graphics, time management, and communications. This same philosophy of integration has been applied to a few office automation systems, but these are generally vendor-specific and do not allow for a mixture of foreign subsystems. During the next few years, it is likely that a few vendors will emerge as dominant in this integrated office automation field and will stress simplicity and flexibility as major components.

  15. Habitat automation

    NASA Technical Reports Server (NTRS)

    Swab, Rodney E.

    1992-01-01

    A habitat, on either the surface of the Moon or Mars, will be designed and built with the proven technologies of that day. These technologies will be mature and readily available to the habitat designer. We believe an acceleration of the normal pace of automation would allow a habitat to be safer and more easily maintained than would be the case otherwise. This document examines the operation of a habitat and describes elements of that operation which may benefit from an increased use of automation. Research topics within the automation realm are then defined and discussed with respect to the role they can have in the design of the habitat. Problems associated with the integration of advanced technologies into real-world projects at NASA are also addressed.

  16. High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

    PubMed Central

    Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

    2016-01-01

    Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24 hours after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. PMID:26747759

  17. Automating Finance

    ERIC Educational Resources Information Center

    Moore, John

    2007-01-01

    In past years, higher education's financial management side has been riddled with manual processes and aging mainframe applications. This article discusses schools which had taken advantage of an array of technologies that automate billing, payment processing, and refund processing in the case of overpayment. The investments are well worth it:…

  18. Automation in biological crystallization.

    PubMed

    Stewart, Patrick Shaw; Mueller-Dieckmann, Jochen

    2014-06-01

    Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given.

  19. Automation in biological crystallization

    PubMed Central

    Shaw Stewart, Patrick; Mueller-Dieckmann, Jochen

    2014-01-01

    Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given. PMID:24915074

  20. CONCENTRATION-RESPONSE ASSESSMENT OF CHEMICAL EFFECTS ON SYNAPTOGENESIS USING A HIGH CONTENT IMAGE ANALYSIS-BASED SCREENING ASSAY

    EPA Science Inventory

    Functional connectivity of the nervous system is dependent upon the development of synapses: i.e. specialized cell-cell contacts which facilitate the unidirectional flow of fast neurotransmission. Prenatal and/or early postnatal exposure to chemicals which disrupt synaptogenesis ...

  1. PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

    EPA Science Inventory

    New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

  2. Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

    NASA Astrophysics Data System (ADS)

    Solmesky, Leonardo J.; Shuman, Michal; Goldsmith, Meir; Weil, Miguel; Peer, Dan

    2011-12-01

    Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

  3. ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells.

    PubMed

    Xu, Huilei; Baroukh, Caroline; Dannenfelser, Ruth; Chen, Edward Y; Tan, Christopher M; Kou, Yan; Kim, Yujin E; Lemischka, Ihor R; Ma'ayan, Avi

    2013-01-01

    High content studies that profile mouse and human embryonic stem cells (m/hESCs) using various genome-wide technologies such as transcriptomics and proteomics are constantly being published. However, efforts to integrate such data to obtain a global view of the molecular circuitry in m/hESCs are lagging behind. Here, we present an m/hESC-centered database called Embryonic Stem Cell Atlas from Pluripotency Evidence integrating data from many recent diverse high-throughput studies including chromatin immunoprecipitation followed by deep sequencing, genome-wide inhibitory RNA screens, gene expression microarrays or RNA-seq after knockdown (KD) or overexpression of critical factors, immunoprecipitation followed by mass spectrometry proteomics and phosphoproteomics. The database provides web-based interactive search and visualization tools that can be used to build subnetworks and to identify known and novel regulatory interactions across various regulatory layers. The web-interface also includes tools to predict the effects of combinatorial KDs by additive effects controlled by sliders, or through simulation software implemented in MATLAB. Overall, the Embryonic Stem Cell Atlas from Pluripotency Evidence database is a comprehensive resource for the stem cell systems biology community. Database URL: http://www.maayanlab.net/ESCAPE

  4. High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

    PubMed

    Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

    2016-10-01

    Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

  5. Electric-arc synthesis of soot with high content of higher fullerenes in parallel arc

    NASA Astrophysics Data System (ADS)

    Dutlov, A. E.; Nekrasov, V. M.; Sergeev, A. G.; Bubnov, V. P.; Kareev, I. E.

    2016-12-01

    Soot with a relatively high content of higher fullerenes (C76, C78, C80, C82, C84, C86, etc.) is synthesized in a parallel arc upon evaporation of pure carbon electrodes. The content of higher fullerenes in soot extract amounts to 13.8 wt % when two electrodes are simultaneously burnt in electric-arc reactor. Such a content is comparable with the content obtained upon evaporation of composite graphite electrodes with potassium carbonate impurity.

  6. Newborn Screening

    MedlinePlus

    ... Activities Importance of Newborn Screening Newborn Screening and Molecular Biology Branch Pulse Oximetry Screening for CCHDs Sickle Cell Disease Laboratory SCID Quality Assurance Training and Resources ...

  7. Rapid automated screening, identification and quantification of organic micro-contaminants and their main transformation products in wastewater and river waters using liquid chromatography-quadrupole-time-of-flight mass spectrometry with an accurate-mass database.

    PubMed

    Gómez, M J; Gómez-Ramos, M M; Malato, O; Mezcua, M; Férnandez-Alba, A R

    2010-11-05

    In this study we have developed and evaluated an analytical method for a rapid automated screening and confirmation of a large number of organic micro-contaminants (almost 400) and also the quantification of the positive findings in water samples of different types (surface and wastewaters) using liquid chromatography-electrospray quadrupole-time-of-flight mass spectrometry (LC-QTOFMS) based on the use of an accurate-mass database. The created database includes data not only on the accurate masses of the target ions but also on the characteristic in-source fragment ions, isotopic pattern and retention time data. This customized database was linked to commercially available software which extracted all the potential compounds of interest from the LC-QTOFMS raw data of each sample and matched them against the database to search for targeted compounds in the sample. The detailed fragmentation information has also been used as a powerful tool for the automatic identification of unknown compounds and/or transformation products with similar structures to those of known organic contaminants included in the database. The database can be continually enlarged. To confirm identification of compounds which have no fragment ions (or fragments with low intensity/relative abundance) from in-source CID fragmentation or isomers which are not distinguished within full single mass spectra, a "Targeted MS/MS" method is developed. Thereafter, these compounds can be further analyzed using the collision energy (CE) in QTOF-MS/MS mode. Linearity and limits of detection were studied. Method detection limits (MDLs) in effluent wastewater and river waters were, in most cases, lowers or equal to 5 and 2 ng/L, respectively. Only 15 compounds had MDLs between 5 and 50 ng/L in effluent wastewater matrix. We obtained a linearity of the calibration curves over two orders of magnitude. The method has been applied to real samples and the results obtained reveal that most of the pharmaceutically

  8. [From automation to robotics].

    PubMed

    1985-01-01

    The introduction of automation into the laboratory of biology seems to be unavoidable. But at which cost, if it is necessary to purchase a new machine for every new application? Fortunately the same image processing techniques, belonging to a theoretic framework called Mathematical Morphology, may be used in visual inspection tasks, both in car industry and in the biology lab. Since the market for industrial robotics applications is much higher than the market of biomedical applications, the price of image processing devices drops, and becomes sometimes less than the price of a complete microscope equipment. The power of the image processing methods of Mathematical Morphology will be illustrated by various examples, as automatic silver grain counting in autoradiography, determination of HLA genotype, electrophoretic gels analysis, automatic screening of cervical smears... Thus several heterogeneous applications may share the same image processing device, provided there is a separate and devoted work station for each of them.

  9. High-Content Imaging Reveals Expansion of the Endosomal Compartment during Coxiella burnetii Parasitophorous Vacuole Maturation

    PubMed Central

    Larson, Charles L.; Heinzen, Robert A.

    2017-01-01

    Coxiella burnetii is an obligate intracellular pathogen and the causative agent of human Q fever. Replication of the bacterium within a large parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. PV biogenesis is a pathogen driven process that requires engagement of several host cell vesicular trafficking pathways to acquire vacuole components. The goal of this study was to determine if infection by C. burnetii modulates endolysosomal flux to potentially benefit PV formation. HeLa cells, infected with C. burnetii or left uninfected, were incubated with fluorescent transferrin (Tf) for 0–30 min, and the amount of Tf internalized by cells quantitated by high-content imaging. At 3 and 5 days, but not 1 day post-infection, the maximal amounts of fluorescent Tf internalized by infected cells were significantly greater than uninfected cells. The rates of Tf uptake and recycling were the same for infected and uninfected cells; however, residual Tf persisted in EEA.1 positive compartments adjacent to large PV after 30 min of recycling in the absence of labeled Tf. On average, C. burnetii-infected cells contained significantly more CD63-positive endosomes than uninfected cells. In contrast, cells containing large vacuoles generated by Chlamydia trachomatis exhibited increased rates of Tf internalization without increased CD63 expression. Our results suggest that C. burnetii infection expands the endosomal system to increase capacity for endocytic material. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to infection by intracellular pathogens. PMID:28293541

  10. Human pluripotent stem cells on artificial microenvironments: a high content perspective

    PubMed Central

    Viswanathan, Priyalakshmi; Gaskell, Terri; Moens, Nathalie; Culley, Oliver J.; Hansen, Darrick; Gervasio, Mia K. R.; Yeap, Yee J.; Danovi, Davide

    2014-01-01

    Self-renewing stem cell populations are increasingly considered as resources for cell therapy and tools for drug discovery. Human pluripotent stem (hPS) cells in particular offer a virtually unlimited reservoir of homogeneous cells and can be differentiated toward diverse lineages. Many diseases show impairment in self-renewal or differentiation, abnormal lineage choice or other aberrant cell behavior in response to chemical or physical cues. To investigate these responses, there is a growing interest in the development of specific assays using hPS cells, artificial microenvironments and high content analysis. Several hurdles need to be overcome that can be grouped into three areas: (i) availability of robust, homogeneous, and consistent cell populations as a starting point; (ii) appropriate understanding and use of chemical and physical microenvironments; (iii) development of assays that dissect the complexity of cell populations in tissues while mirroring specific aspects of their behavior. Here we review recent progress in the culture of hPS cells and we detail the importance of the environment surrounding the cells with a focus on synthetic material and suitable high content analysis approaches. The technologies described, if properly combined, have the potential to create a paradigm shift in the way diseases are modeled and drug discovery is performed. PMID:25071572

  11. Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells

    PubMed Central

    Kim, Joseph J.; Bennett, Neal K.; Devita, Mitchel S.; Chahar, Sanjay; Viswanath, Satish; Lee, Eunjee A.; Jung, Giyoung; Shao, Paul P.; Childers, Erin P.; Liu, Shichong; Kulesa, Anthony; Garcia, Benjamin A.; Becker, Matthew L.; Hwang, Nathaniel S.; Madabhushi, Anant; Verzi, Michael P.; Moghe, Prabhas V.

    2017-01-01

    While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks. PMID:28051095

  12. High-content analysis of single cells directly assembled on CMOS sensor based on color imaging.

    PubMed

    Tanaka, Tsuyoshi; Saeki, Tatsuya; Sunaga, Yoshihiko; Matsunaga, Tadashi

    2010-12-15

    A complementary metal oxide semiconductor (CMOS) image sensor was applied to high-content analysis of single cells which were assembled closely or directly onto the CMOS sensor surface. The direct assembling of cell groups on CMOS sensor surface allows large-field (6.66 mm×5.32 mm in entire active area of CMOS sensor) imaging within a second. Trypan blue-stained and non-stained cells in the same field area on the CMOS sensor were successfully distinguished as white- and blue-colored images under white LED light irradiation. Furthermore, the chemiluminescent signals of each cell were successfully visualized as blue-colored images on CMOS sensor only when HeLa cells were placed directly on the micro-lens array of the CMOS sensor. Our proposed approach will be a promising technique for real-time and high-content analysis of single cells in a large-field area based on color imaging.

  13. 78 FR 18221 - Disclosures at Automated Teller Machines (Regulation E)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-26

    ... Part 1005 RIN 3170-AA36 Disclosures at Automated Teller Machines (Regulation E) AGENCY: Bureau of... automated teller machines, leaving in place the requirement for a specific fee disclosure to appear on the screen of that machine or on paper issued from the machine. This final rule amends Regulation E...

  14. Quantification of Dynamic Morphological Drug Responses in 3D Organotypic Cell Cultures by Automated Image Analysis

    PubMed Central

    Härmä, Ville; Schukov, Hannu-Pekka; Happonen, Antti; Ahonen, Ilmari; Virtanen, Johannes; Siitari, Harri; Åkerfelt, Malin; Lötjönen, Jyrki; Nees, Matthias

    2014-01-01

    Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports

  15. OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

    NASA Astrophysics Data System (ADS)

    Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

    2016-02-01

    In the last two decades, <30% of drugs withdrawals from the market were due to cardiac toxicity, where unintended interactions with ion channels disrupt the heart's normal electrical function. Consequently, all new drugs must undergo preclinical testing for cardiac liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

  16. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

    To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

  17. AUTOMATED PLANT SAVES $30,000.

    ERIC Educational Resources Information Center

    1966

    AN AUTOMATED CENTRAL BUILDING CONTROL SYSTEM IS BEING INSTALLED AT THE UNIVERSITY OF UTAH PRIMARILY TO CONTROL HEATING IN 21 BUILDINGS. AN OPERATOR CAN SELECT A PARTICULAR SYSTEM FOR SURVEILLANCE OR ADJUSTMENT, AND AUTOMATICALLY A LARGE SCALE SCHEMATIC OF THE SYSTEM IS DISPLAYED ON A SCREEN AND ALL CONTROL CIRCUITS ARE SWITCHED TO THE SYSTEM UNDER…

  18. Northern and Southern Permafrost Regions on Mars with High Content of Water Ice: Similarities and Differences

    NASA Technical Reports Server (NTRS)

    Mitrofanov, I. G.; Litvak, M. L.; Kozyrev, A. S.; Sanin, A. B.; Tretyakov, V. I.; Kuzmin, R. O.; Boynton, W. V.; Hamara, D. K.; Shinohara, C.; Saunders, R. S.

    2004-01-01

    The measurements by neutron detectors on Odyssey have revealed two large poleward regions with large depression of flux of epithermal and high energy neutrons. The flux of neutrons from Mars is known to be produced by the bombardment of the surface layer by galactic cosmic rays. The leakage flux of epithermal and fast neutrons has regional variation by a factor of 10 over the surface of Mars. These variations are mainly produced by variations of hydrogen content in the shallow subsurface. On Mars hydrogen is associated with water. Therefore, the Northern and Southern depressions of neutron emission could be identified as permafrost regions with very high content of water ice. These regions are much larger than the residual polar caps, and could contain the major fraction of subsurface water ice. Here we present the results of HEND neutron data deconvolution for these regions and describe the similarities and differences between them.

  19. Northern and Southern Permafrost Regions on Mars with High Content of Water Ice: Similarities and Differences

    NASA Technical Reports Server (NTRS)

    Mitrofanov, I. G.; Litvak, M. L.; Kozyrev, A. S.; Sanin, A. B.; Tretyakov, V. I.; Kuzmin, R. O.; Boynton, W. V.; Hamara, D. K.; Shinohara, C.; Saunders, R. S.

    2004-01-01

    The measurements by neutron detectors on Odyssey have revealed two large poleward regions with large depression of flux of epithermal and high energy neutrons [1-3]. The flux of neutrons from Mars is known to be produced by the bombardment of the surface layer by galactic cosmic rays. The leakage flux of epithermal and fast neutrons has regional variation by a factor of 10 over the surface of Mars (e.g. see [3- 5]). These variations are mainly produced by variations of hydrogen content in the shallow subsurface. On Mars hydrogen is associated with water. Therefore, the Northern and Southern depressions of neutron emission could be identified as permafrost regions with very high content of water ice [1-5]. These regions are much larger than the residual polar caps, and could contain the major fraction of subsurface water ice. Here we present the results of HEND neutron data deconvolution for these regions and describe the similarities and differences between them.

  20. Depth-resolved incoherent and coherent wide-field high-content imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    So, Peter T.

    2016-03-01

    Recent advances in depth-resolved wide-field imaging technique has enabled many high throughput applications in biology and medicine. Depth resolved imaging of incoherent signals can be readily accomplished with structured light illumination or nonlinear temporal focusing. The integration of these high throughput systems with novel spectroscopic resolving elements further enable high-content information extraction. We will introduce a novel near common-path interferometer and demonstrate its uses in toxicology and cancer biology applications. The extension of incoherent depth-resolved wide-field imaging to coherent modality is non-trivial. Here, we will cover recent advances in wide-field 3D resolved mapping of refractive index, absorbance, and vibronic components in biological specimens.

  1. tranSMART: An Open Source Knowledge Management and High Content Data Analytics Platform

    PubMed Central

    Scheufele, Elisabeth; Aronzon, Dina; Coopersmith, Robert; McDuffie, Michael T.; Kapoor, Manish; Uhrich, Christopher A.; Avitabile, Jean E.; Liu, Jinlei; Housman, Dan; Palchuk, Matvey B.

    2014-01-01

    The tranSMART knowledge management and high-content analysis platform is a flexible software framework featuring novel research capabilities. It enables analysis of integrated data for the purposes of hypothesis generation, hypothesis validation, and cohort discovery in translational research. tranSMART bridges the prolific world of basic science and clinical practice data at the point of care by merging multiple types of data from disparate sources into a common environment. The application supports data harmonization and integration with analytical pipelines. The application code was released into the open source community in January 2012, with 32 instances in operation. tranSMART’s extensible data model and corresponding data integration processes, rapid data analysis features, and open source nature make it an indispensable tool in translational or clinical research. PMID:25717408

  2. tranSMART: An Open Source Knowledge Management and High Content Data Analytics Platform.

    PubMed

    Scheufele, Elisabeth; Aronzon, Dina; Coopersmith, Robert; McDuffie, Michael T; Kapoor, Manish; Uhrich, Christopher A; Avitabile, Jean E; Liu, Jinlei; Housman, Dan; Palchuk, Matvey B

    2014-01-01

    The tranSMART knowledge management and high-content analysis platform is a flexible software framework featuring novel research capabilities. It enables analysis of integrated data for the purposes of hypothesis generation, hypothesis validation, and cohort discovery in translational research. tranSMART bridges the prolific world of basic science and clinical practice data at the point of care by merging multiple types of data from disparate sources into a common environment. The application supports data harmonization and integration with analytical pipelines. The application code was released into the open source community in January 2012, with 32 instances in operation. tranSMART's extensible data model and corresponding data integration processes, rapid data analysis features, and open source nature make it an indispensable tool in translational or clinical research.

  3. Profiling stem cell states in three-dimensional biomaterial niches using high content image informatics.

    PubMed

    Dhaliwal, Anandika; Brenner, Matthew; Wolujewicz, Paul; Zhang, Zheng; Mao, Yong; Batish, Mona; Kohn, Joachim; Moghe, Prabhas V

    2016-11-01

    A predictive framework for the evolution of stem cell biology in 3-D is currently lacking. In this study we propose deep image informatics of the nuclear biology of stem cells to elucidate how 3-D biomaterials steer stem cell lineage phenotypes. The approach is based on high content imaging informatics to capture minute variations in the 3-D spatial organization of splicing factor SC-35 in the nucleoplasm as a marker to classify emergent cell phenotypes of human mesenchymal stem cells (hMSCs). The cells were cultured in varied 3-D culture systems including hydrogels, electrospun mats and salt leached scaffolds. The approach encompasses high resolution 3-D imaging of SC-35 domains and high content image analysis (HCIA) to compute quantitative 3-D nuclear metrics for SC-35 organization in single cells in concert with machine learning approaches to construct a predictive cell-state classification model. Our findings indicate that hMSCs cultured in collagen hydrogels and induced to differentiate into osteogenic or adipogenic lineages could be classified into the three lineages (stem, adipogenic, osteogenic) with ⩾80% precision and sensitivity, within 72h. Using this framework, the augmentation of osteogenesis by scaffold design exerted by porogen leached scaffolds was also profiled within 72h with ∼80% high sensitivity. Furthermore, by employing 3-D SC-35 organizational metrics, differential osteogenesis induced by novel electrospun fibrous polymer mats incorporating decellularized matrix could also be elucidated and predictably modeled at just 3days with high precision. We demonstrate that 3-D SC-35 organizational metrics can be applied to model the stem cell state in 3-D scaffolds. We propose that this methodology can robustly discern minute changes in stem cell states within complex 3-D architectures and map single cell biological readouts that are critical to assessing population level cell heterogeneity.

  4. Inventory management and reagent supply for automated chemistry.

    PubMed

    Kuzniar, E

    1999-08-01

    Developments in automated chemistry have kept pace with developments in HTS such that hundreds of thousands of new compounds can be rapidly synthesized in the belief that the greater the number and diversity of compounds that can be screened, the more successful HTS will be. The increasing use of automation for Multiple Parallel Synthesis (MPS) and the move to automated combinatorial library production is placing an overwhelming burden on the management of reagents. Although automation has improved the efficiency of the processes involved in compound synthesis, the bottleneck has shifted to ordering, collating and preparing reagents for automated chemistry resulting in loss of time, materials and momentum. Major efficiencies have already been made in the area of compound management for high throughput screening. Most of these efficiencies have been achieved with sophisticated library management systems using advanced engineering and data handling for the storage, tracking and retrieval of millions of compounds. The Automation Partnership has already provided many of the top pharmaceutical companies with modular automated storage, preparation and retrieval systems to manage compound libraries for high throughput screening. This article describes how these systems may be implemented to solve the specific problems of inventory management and reagent supply for automated chemistry.

  5. High throughput screening for anti-Trypanosoma cruzi drug discovery.

    PubMed

    Alonso-Padilla, Julio; Rodríguez, Ana

    2014-12-01

    The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.

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

    EPA Science Inventory

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

  7. Functional characterization of putative cilia genes by high-content analysis

    PubMed Central

    Lai, Cary K.; Gupta, Nidhi; Wen, Xiaohui; Rangell, Linda; Chih, Ben; Peterson, Andrew S.; Bazan, J. Fernando; Li, Li; Scales, Suzie J.

    2011-01-01

    Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates. PMID:21289087

  8. High-Content Analysis of Breast Cancer Using Single-Cell Deep Transfer Learning.

    PubMed

    Kandaswamy, Chetak; Silva, Luís M; Alexandre, Luís A; Santos, Jorge M

    2016-03-01

    High-content analysis has revolutionized cancer drug discovery by identifying substances that alter the phenotype of a cell, which prevents tumor growth and metastasis. The high-resolution biofluorescence images from assays allow precise quantitative measures enabling the distinction of small molecules of a host cell from a tumor. In this work, we are particularly interested in the application of deep neural networks (DNNs), a cutting-edge machine learning method, to the classification of compounds in chemical mechanisms of action (MOAs). Compound classification has been performed using image-based profiling methods sometimes combined with feature reduction methods such as principal component analysis or factor analysis. In this article, we map the input features of each cell to a particular MOA class without using any treatment-level profiles or feature reduction methods. To the best of our knowledge, this is the first application of DNN in this domain, leveraging single-cell information. Furthermore, we use deep transfer learning (DTL) to alleviate the intensive and computational demanding effort of searching the huge parameter's space of a DNN. Results show that using this approach, we obtain a 30% speedup and a 2% accuracy improvement.

  9. Predicting In Vivo Anti-Hepatofibrotic Drug Efficacy Based on In Vitro High-Content Analysis

    PubMed Central

    Zheng, Baixue; Tan, Looling; Mo, Xuejun; Yu, Weimiao; Wang, Yan; Tucker-Kellogg, Lisa; Welsch, Roy E.; So, Peter T. C.; Yu, Hanry

    2011-01-01

    Background/Aims Many anti-fibrotic drugs with high in vitro efficacies fail to produce significant effects in vivo. The aim of this work is to use a statistical approach to design a numerical predictor that correlates better with in vivo outcomes. Methods High-content analysis (HCA) was performed with 49 drugs on hepatic stellate cells (HSCs) LX-2 stained with 10 fibrotic markers. ∼0.3 billion feature values from all cells in >150,000 images were quantified to reflect the drug effects. A systematic literature search on the in vivo effects of all 49 drugs on hepatofibrotic rats yields 28 papers with histological scores. The in vivo and in vitro datasets were used to compute a single efficacy predictor (Epredict). Results We used in vivo data from one context (CCl4 rats with drug treatments) to optimize the computation of Epredict. This optimized relationship was independently validated using in vivo data from two different contexts (treatment of DMN rats and prevention of CCl4 induction). A linear in vitro-in vivo correlation was consistently observed in all the three contexts. We used Epredict values to cluster drugs according to efficacy; and found that high-efficacy drugs tended to target proliferation, apoptosis and contractility of HSCs. Conclusions The Epredict statistic, based on a prioritized combination of in vitro features, provides a better correlation between in vitro and in vivo drug response than any of the traditional in vitro markers considered. PMID:22073152

  10. Analysis of AQP4 Trafficking Vesicle Dynamics Using a High-Content Approach

    PubMed Central

    Mazzaferri, Javier; Costantino, Santiago; Lefrancois, Stephane

    2013-01-01

    Aquaporin-4 (AQP4) is found on the basolateral plasma membrane of a variety of epithelial cells, and it is widely accepted that microtubules play an important role in protein trafficking to the plasma membrane. In the particular case of polarized trafficking, however, most evidence on the involvement of microtubules has been obtained via biochemistry experiments and single-shot microscopy. These approaches have provided essential information, even though they neglect the dynamical details of microtubule transport. In this work, we present a high-content framework in which time-lapse imaging, and single-particle-tracking algorithms were used to study a large number (∼104) of GFP-AQP4-carrying vesicles on a large number of cells (∼170). By analyzing several descriptors in this large sample of trajectories, we were able to obtain highly statistically significant results. Our results support the hypothesis that AQP4 is transported along microtubules, but to our surprise, this transport is not directed straight to the basolateral plasma membrane. On the contrary, these vesicles move stochastically along microtubules, changing direction repeatedly. We propose that the role of microtubules in the basolateral trafficking of AQP4 is to increase the efficiency, rather than determine the specificity of the target. PMID:23870254

  11. Preparation of high concentration polyaluminum chloride with high content of Alb or Alc.

    PubMed

    Zhao, Changwei; Zhang, Jingbo; Luan, Zhaokun; Peng, Xianjia; Ren, Xiaojing

    2009-01-01

    A novel membrane distillation concentration method was used to prepare high concentration polyaluminum chloride (PACl) with high content of Alb or Alc. 2.52 mol/L PACl1 with 88% Alb and 2.38 mol/L PACl2 with 61% Alc were successfully prepared. Three coagulants, AlCl3, PACl1 and PACl2 were investigated on their hydrolysis behavior and speciation under different conditions. The effects of pH and dilution ratio on Al species distribution were investigated by ferron assay. Experimental result showed that pH had a significant effect on Al species distribution for the three coagulants. Dilution ratio had little effects on Alb and Alc distribution in whole dilution process except the beginning for PACl1 and PACl2. The results indicated that transformation of Al depends largely on their original composition. AlCl3 was the most unstable coagulant among these three coagulants during hydrolysis process. PACl1 and PACl2 with significant amounts of highly charged and stable polynuclear aluminum hydrolysis products were less affected by the hydrolysis conditions and could maintain high speciation stability under various conditions.

  12. A high-content platform to characterise human induced pluripotent stem cell lines

    PubMed Central

    Leha, Andreas; Moens, Nathalie; Meleckyte, Ruta; Culley, Oliver J.; Gervasio, Mia K.; Kerz, Maximilian; Reimer, Andreas; Cain, Stuart A.; Streeter, Ian; Folarin, Amos; Stegle, Oliver; Kielty, Cay M.; Durbin, Richard; Watt, Fiona M.; Danovi, Davide

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components. Here we report the establishment of a high-content platform for phenotypic analysis of human iPSC lines. In the described assay, cells are dissociated and seeded as single cells onto 96-well plates coated with fibronectin at three different concentrations. This method allows assessment of cell number, proliferation, morphology and intercellular adhesion. Altogether, our strategy delivers robust quantification of phenotypic diversity within complex cell populations facilitating future identification of the genetic, biological and technical determinants of variance. Approaches such as the one described can be used to benchmark iPSCs from multiple donors and create novel platforms that can readily be tailored for disease modelling and drug discovery. PMID:26608109

  13. A high-content platform to characterise human induced pluripotent stem cell lines.

    PubMed

    Leha, Andreas; Moens, Nathalie; Meleckyte, Ruta; Culley, Oliver J; Gervasio, Mia K; Kerz, Maximilian; Reimer, Andreas; Cain, Stuart A; Streeter, Ian; Folarin, Amos; Stegle, Oliver; Kielty, Cay M; Durbin, Richard; Watt, Fiona M; Danovi, Davide

    2016-03-01

    Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components. Here we report the establishment of a high-content platform for phenotypic analysis of human iPSC lines. In the described assay, cells are dissociated and seeded as single cells onto 96-well plates coated with fibronectin at three different concentrations. This method allows assessment of cell number, proliferation, morphology and intercellular adhesion. Altogether, our strategy delivers robust quantification of phenotypic diversity within complex cell populations facilitating future identification of the genetic, biological and technical determinants of variance. Approaches such as the one described can be used to benchmark iPSCs from multiple donors and create novel platforms that can readily be tailored for disease modelling and drug discovery.

  14. Phoenito experiments: combining the strengths of commercial crystallization automation.

    PubMed

    Newman, Janet; Pham, Tam M; Peat, Thomas S

    2008-11-01

    The use of crystallization robots for initial screening in macromolecular crystallization is well established. This paper describes how four general optimization techniques, growth-rate modulation, fine screening, seeding and additive screening, have been adapted for automation in a medium-throughput crystallization service facility. The use of automation for more challenging optimization experiments is discussed, as is a novel way of using both the Mosquito and the Phoenix nano-dispensing robots during the setup of a single crystallization plate. This dual-dispenser technique plays to the strengths of both machines.

  15. Autonomy and Automation

    NASA Technical Reports Server (NTRS)

    Shively, Jay

    2017-01-01

    A significant level of debate and confusion has surrounded the meaning of the terms autonomy and automation. Automation is a multi-dimensional concept, and we propose that Remotely Piloted Aircraft Systems (RPAS) automation should be described with reference to the specific system and task that has been automated, the context in which the automation functions, and other relevant dimensions. In this paper, we present definitions of automation, pilot in the loop, pilot on the loop and pilot out of the loop. We further propose that in future, the International Civil Aviation Organization (ICAO) RPAS Panel avoids the use of the terms autonomy and autonomous when referring to automated systems on board RPA. Work Group 7 proposes to develop, in consultation with other workgroups, a taxonomy of Levels of Automation for RPAS.

  16. Workflow automation architecture standard

    SciTech Connect

    Moshofsky, R.P.; Rohen, W.T.

    1994-11-14

    This document presents an architectural standard for application of workflow automation technology. The standard includes a functional architecture, process for developing an automated workflow system for a work group, functional and collateral specifications for workflow automation, and results of a proof of concept prototype.

  17. Identification of Inhibitors of Triacylglyceride Accumulation in Muscle Cells: Comparing HTS Results from 1536-well Plate-Based and High-Content Platforms

    PubMed Central

    Sugarman, Eliot; Koo, Ada; Suyama, Eigo; Ruidiaz, Manuel E.; Heynen-Genel, Susanne; Nguyen, Kevin H.; Vasile, Stefan; Soundarapandian, Mangala M.; Vega, Rick B.; Kelly, Daniel P.; Smith, Layton H.; Malany, Siobhan

    2014-01-01

    Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate-reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the NIH Library. The screening data from the plate-reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate-reader assay is a powerful HTS platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity. PMID:23989452

  18. Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

    PubMed

    Carty, Nikisha; Berson, Nadège; Tillack, Karsten; Thiede, Christina; Scholz, Diana; Kottig, Karsten; Sedaghat, Yalda; Gabrysiak, Christina; Yohrling, George; von der Kammer, Heinz; Ebneth, Andreas; Mack, Volker; Munoz-Sanjuan, Ignacio; Kwak, Seung

    2015-01-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

  19. A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies

    PubMed Central

    Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

    2016-01-01

    Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2′-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell–based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs. PMID:27909235

  20. Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging

    PubMed Central

    Santulli-Marotto, Sandra; Gervais, Alexis; Fisher, Jamie; Strake, Brandy; Ogden, Carol Anne; Riveley, Chelsea; Giles-Komar, Jill

    2015-01-01

    Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the “professional phagocyte” of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity in vivo. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways. PMID:26674639

  1. Assessment of Cr(VI)-Induced Cytotoxicity and Genotoxicity Using High Content Analysis

    PubMed Central

    Thompson, Chad M.; Fedorov, Yuriy; Brown, Daniel D.; Suh, Mina; Proctor, Deborah M.; Kuriakose, Liz; Haws, Laurie C.; Harris, Mark A.

    2012-01-01

    Oral exposure to high concentrations of hexavalent chromium [Cr(VI)] induces intestinal redox changes, villus cytotoxicity, crypt hyperplasia, and intestinal tumors in mice. To assess the effects of Cr(VI) in a cell model relevant to the intestine, undifferentiated (proliferating) and differentiated (confluent) Caco-2 cells were treated with Cr(VI), hydrogen peroxide or rotenone for 2–24 hours. DNA damage was then assessed by nuclear staining intensity of 8-hydroxydeoxyguanosine (8-OHdG) and phosphorylated histone variant H2AX (γ-H2AX) measured by high content analysis methods. In undifferentiated Caco-2, all three chemicals increased 8-OHdG and γ-H2AX staining at cytotoxic concentrations, whereas only 8-OHdG was elevated at non-cytotoxic concentrations at 24 hr. Differentiated Caco-2 were more resistant to cytotoxicity and DNA damage than undifferentiated cells, and there were no changes in apoptotic markers p53 or annexin-V. However, Cr(VI) induced a dose-dependent translocation of the unfolded protein response transcription factor ATF6 into the nucleus. Micronucleus (MN) formation was assessed in CHO-K1 and A549 cell lines. Cr(VI) increased MN frequency in CHO-K1 only at highly cytotoxic concentrations. Relative to the positive control Mitomycin-C, Cr(VI) only slightly increased MN frequency in A549 at mildly cytotoxic concentrations. The results demonstrate that Cr(VI) genotoxicity correlates with cytotoxic concentrations, and that H2AX phosphorylation occurs at higher concentrations than oxidative DNA damage in proliferating Caco-2 cells. The findings suggest that in vitro genotoxicity of Cr(VI) is primarily oxidative in nature at low concentrations. Implications for in vivo intestinal toxicity of Cr(VI) will be discussed. PMID:22905163

  2. A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

    PubMed

    Lessing, Derek; Dial, Thomas O; Wei, Chunyao; Payer, Bernhard; Carrette, Lieselot L G; Kesner, Barry; Szanto, Attila; Jadhav, Ajit; Maloney, David J; Simeonov, Anton; Theriault, Jimmy; Hasaka, Thomas; Bedalov, Antonio; Bartolomei, Marisa S; Lee, Jeannie T

    2016-12-13

    X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

  3. Automation in Clinical Microbiology

    PubMed Central

    Ledeboer, Nathan A.

    2013-01-01

    Historically, the trend toward automation in clinical pathology laboratories has largely bypassed the clinical microbiology laboratory. In this article, we review the historical impediments to automation in the microbiology laboratory and offer insight into the reasons why we believe that we are on the cusp of a dramatic change that will sweep a wave of automation into clinical microbiology laboratories. We review the currently available specimen-processing instruments as well as the total laboratory automation solutions. Lastly, we outline the types of studies that will need to be performed to fully assess the benefits of automation in microbiology laboratories. PMID:23515547

  4. Shoe-String Automation

    SciTech Connect

    Duncan, M.L.

    2001-07-30

    Faced with a downsizing organization, serious budget reductions and retirement of key metrology personnel, maintaining capabilities to provide necessary services to our customers was becoming increasingly difficult. It appeared that the only solution was to automate some of our more personnel-intensive processes; however, it was crucial that the most personnel-intensive candidate process be automated, at the lowest price possible and with the lowest risk of failure. This discussion relates factors in the selection of the Standard Leak Calibration System for automation, the methods of automation used to provide the lowest-cost solution and the benefits realized as a result of the automation.

  5. A high content assay for biosensor validation and for examining stimuli that affect biosensor activity

    PubMed Central

    Slattery, Scott D.; Hahn, Klaus M.

    2015-01-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor’s maximally activated and inactivated state, and examine response to specific proteins. This involves considerable labor and expense, as expression conditions must be optimized to saturate the biosensor with the regulator, and multiple replicates and controls are required. We describe here a protocol for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays allows visual inspection (eg for cell health and biosensor or regulator localization). Optimization of single chain and dual chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for variations in upstream molecules. PMID:25447074

  6. A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.

    PubMed

    Slattery, Scott D; Hahn, Klaus M

    2014-12-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules.

  7. Health Screening

    MedlinePlus

    Screenings are tests that look for diseases before you have symptoms. Screening tests can find diseases early, when they're easier ... Overweight and obesity Prostate cancer in men Which tests you need depends on your age, your sex, ...

  8. Depression Screening

    MedlinePlus

    ... Centers Diseases + Condition Centers Mental Health Medical Library Depression Screening (PHQ-9) - Instructions The following questions are ... this tool, there is also text-only version . Depression Screening - Manual Instructions The following questions are a ...

  9. Automated DNA Sequencing System

    SciTech Connect

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  10. Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

    EPA Science Inventory

    Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

  11. Automated static perimetry to evaluate diabetic retinopathy.

    PubMed Central

    Federman, J L; Lloyd, J

    1984-01-01

    The Octopus automated static perimeter was used to evaluate patients with early diabetic retinopathy. It showed islands of threshold sensitivity depression that were equal to areas of nonperfusion seen on fluorescein angiography. The geographic area of the fundus at risk of developing these field defects was found to be between 20 and 45 degrees, representing the central area of the midperiphery. This procedure has potential as an excellent screening test for early diabetic retinopathy. Images FIGURE 1 (Cont'd) C PMID:6549516

  12. Double screening

    SciTech Connect

    Gratia, Pierre; Hu, Wayne; Joyce, Austin; Ribeiro, Raquel H.

    2016-06-15

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  13. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

    EPA Science Inventory

    A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

  14. Management Planning for Workplace Automation.

    ERIC Educational Resources Information Center

    McDole, Thomas L.

    Several factors must be considered when implementing office automation. Included among these are whether or not to automate at all, the effects of automation on employees, requirements imposed by automation on the physical environment, effects of automation on the total organization, and effects on clientele. The reasons behind the success or…

  15. Laboratory Automation and Middleware.

    PubMed

    Riben, Michael

    2015-06-01

    The practice of surgical pathology is under constant pressure to deliver the highest quality of service, reduce errors, increase throughput, and decrease turnaround time while at the same time dealing with an aging workforce, increasing financial constraints, and economic uncertainty. Although not able to implement total laboratory automation, great progress continues to be made in workstation automation in all areas of the pathology laboratory. This report highlights the benefits and challenges of pathology automation, reviews middleware and its use to facilitate automation, and reviews the progress so far in the anatomic pathology laboratory.

  16. Colon cancer screening

    MedlinePlus

    Screening for colon cancer; Colonoscopy - screening; Sigmoidoscopy - screening; Virtual colonoscopy - screening; Fecal immunochemical test; Stool DNA test; sDNA test; Colorectal cancer - screening; Rectal ...

  17. Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

    PubMed

    Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

    2014-04-01

    Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

  18. High-Content Movement Analysis as a Diagnostic Tool in C. elegans

    NASA Astrophysics Data System (ADS)

    Winter, Peter; Lancichinetti, Andrea; Krevitt, Leah; Amaral, Luis; Morimoto, Rick

    2013-03-01

    Many neurodegenerative diseases manifest themselves through a loss of motor control and give us information about the underlying disease. This loss of coordination is observed in humans and in the model organisms used to study neurodegeneration. In Caenorhabditis elegans, there is an extensive genetic library of strains that lack functional neuronal signaling pathways and expressing proteins associated with neurodegenerative diseases. While most of these strains have decrease motility or cause paralysis, relatively few have been screened to look for more subtle changes in motor control such as stiffness, twitching, or other changes in behavior. we use high-resolution position and posture data to automatically analyze the movement of worms from different genetic backgrounds and characterize 14 movement characteristics. By creating a quantitative mapping between the movement characterization and an online database of gene annotation, gene expression, and anatomy, we aim to predict a likely set of cellular and molecular disruptions. This work provides a proof of concept for the use of detailed movement analysis to uncover novel disruptions in certain motor control processes. Knowledge of the molecular origin of these disruptions provided by our understanding of C. elegans genetics and physiology could lead to new diagnostic and therapeutic targets for neurodegenerative disease.

  19. Automated System for Early Breast Cancer Detection in Mammograms

    NASA Technical Reports Server (NTRS)

    Bankman, Isaac N.; Kim, Dong W.; Christens-Barry, William A.; Weinberg, Irving N.; Gatewood, Olga B.; Brody, William R.

    1993-01-01

    The increasing demand on mammographic screening for early breast cancer detection, and the subtlety of early breast cancer signs on mammograms, suggest an automated image processing system that can serve as a diagnostic aid in radiology clinics. We present a fully automated algorithm for detecting clusters of microcalcifications that are the most common signs of early, potentially curable breast cancer. By using the contour map of the mammogram, the algorithm circumvents some of the difficulties encountered with standard image processing methods. The clinical implementation of an automated instrument based on this algorithm is also discussed.

  20. Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

    PubMed Central

    Zidar, Nace; Kikelj, Danijel; Kirby, Robert W.

    2016-01-01

    Abstract The rodent neuroblastoma cell line, ND7-23, is used to express voltage-dependent sodium (Nav) and other neuronal ion channels resistant to heterologous expression in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. Their advantage is that they provide endogenous factors and signaling pathways to promote ion channel peptide folding, expression, and function at the cell surface and are also amenable to automated patch clamping. However, ND7-23 cells exhibit endogenous tetrodotoxin (TTX)-sensitive Nav currents, and molecular profiling has revealed the presence of Nav1.2, Nav1.3, Nav1.6, and Nav1.7 transcripts, but no study has determined which subtypes contribute to functional channels at the cell surface. We profiled the repertoire of functional Nav channels endogenously expressed in ND7-23 cells using the QPatch automated patch clamp platform and selective toxins and small molecules. The potency and subtype selectivity of the ligands (Icagen compound 68 from patent US-20060025415-A1-20060202, 4,9 anhydro TTX, and Protoxin-II) were established in human Nav1.3, Nav1.6, and Nav1.7 channel cell lines before application of selective concentrations to ND7-23 cells. Our data confirm previous studies that >97% of macroscopic Nav current in ND7-23 cells is carried by TTX-sensitive channels (300 nM TTX) and that Nav1.7 is the predominant channel contributing to this response (65% of peak inward current), followed by Nav1.6 (∼20%) and negligible Nav1.3 currents (∼2%). In addition, our data are the first to assess the Nav1.6 potency (50% inhibitory concentration [IC50] of 33 nM) and selectivity (50-fold over Nav1.7) of 4,9 anhydro TTX in human Nav channels expressed in mammalian cells, confirming previous studies of rodent Nav channels expressed in oocytes and HEK cells. PMID:26991361

  1. Simultaneous multi-parametric analysis of Leishmania and of its hosting mammal cells: A high content imaging-based method enabling sound drug discovery process.

    PubMed

    Forestier, Claire-Lise; Späth, Gerald Frank; Prina, Eric; Dasari, Sreekanth

    2015-11-01

    Leishmaniasis is a vector-borne disease for which only limited therapeutic options are available. The disease is ranked among the six most important tropical infectious diseases and represents the second-largest parasitic killer in the world. The development of new therapies has been hampered by the lack of technologies and methodologies that can be integrated into the complex physiological environment of a cell or organism and adapted to suitable in vitro and in vivo Leishmania models. Recent advances in microscopy imaging offer the possibility to assess the efficacy of potential drug candidates against Leishmania within host cells. This technology allows the simultaneous visualization of relevant phenotypes in parasite and host cells and the quantification of a variety of cellular events. In this review, we present the powerful cellular imaging methodologies that have been developed for drug screening in a biologically relevant context, addressing both high-content and high-throughput needs. Furthermore, we discuss the potential of intra-vital microscopy imaging in the context of the anti-leishmanial drug discovery process.

  2. Automating checks of plan check automation.

    PubMed

    Halabi, Tarek; Lu, Hsiao-Ming

    2014-07-08

    While a few physicists have designed new plan check automation solutions for their clinics, fewer, if any, managed to adapt existing solutions. As complex and varied as the systems they check, these programs must gain the full confidence of those who would run them on countless patient plans. The present automation effort, planCheck, therefore focuses on versatility and ease of implementation and verification. To demonstrate this, we apply planCheck to proton gantry, stereotactic proton gantry, stereotactic proton fixed beam (STAR), and IMRT treatments.

  3. WANTED: Fully Automated Indexing.

    ERIC Educational Resources Information Center

    Purcell, Royal

    1991-01-01

    Discussion of indexing focuses on the possibilities of fully automated indexing. Topics discussed include controlled indexing languages such as subject heading lists and thesauri, free indexing languages, natural indexing languages, computer-aided indexing, expert systems, and the need for greater creativity to further advance automated indexing.…

  4. The Automated Office.

    ERIC Educational Resources Information Center

    Naclerio, Nick

    1979-01-01

    Clerical personnel may be able to climb career ladders as a result of office automation and expanded job opportunities in the word processing area. Suggests opportunities in an automated office system and lists books and periodicals on word processing for counselors and teachers. (MF)

  5. Planning for Office Automation.

    ERIC Educational Resources Information Center

    Sherron, Gene T.

    1982-01-01

    The steps taken toward office automation by the University of Maryland are described. Office automation is defined and some types of word processing systems are described. Policies developed in the writing of a campus plan are listed, followed by a section on procedures adopted to implement the plan. (Author/MLW)

  6. Work and Programmable Automation.

    ERIC Educational Resources Information Center

    DeVore, Paul W.

    A new industrial era based on electronics and the microprocessor has arrived, an era that is being called intelligent automation. Intelligent automation, in the form of robots, replaces workers, and the new products, using microelectronic devices, require significantly less labor to produce than the goods they replace. The microprocessor thus…

  7. Order Division Automated System.

    ERIC Educational Resources Information Center

    Kniemeyer, Justin M.; And Others

    This publication was prepared by the Order Division Automation Project staff to fulfill the Library of Congress' requirement to document all automation efforts. The report was originally intended for internal use only and not for distribution outside the Library. It is now felt that the library community at-large may have an interest in the…

  8. Automation and Cataloging.

    ERIC Educational Resources Information Center

    Furuta, Kenneth; And Others

    1990-01-01

    These three articles address issues in library cataloging that are affected by automation: (1) the impact of automation and bibliographic utilities on professional catalogers; (2) the effect of the LASS microcomputer software on the cost of authority work in cataloging at the University of Arizona; and (3) online subject heading and classification…

  9. INsPECT, an open-source and versatile software for automated quantification of (Leishmania) intracellular parasites.

    PubMed

    Yazdanparast, Ehsan; Dos Anjos, Antonio; Garcia, Deborah; Loeuillet, Corinne; Shahbazkia, Hamid Reza; Vergnes, Baptiste

    2014-05-01

    Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (4',6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.

  10. Automation in Immunohematology

    PubMed Central

    Bajpai, Meenu; Kaur, Ravneet; Gupta, Ekta

    2012-01-01

    There have been rapid technological advances in blood banking in South Asian region over the past decade with an increasing emphasis on quality and safety of blood products. The conventional test tube technique has given way to newer techniques such as column agglutination technique, solid phase red cell adherence assay, and erythrocyte-magnetized technique. These new technologies are adaptable to automation and major manufacturers in this field have come up with semi and fully automated equipments for immunohematology tests in the blood bank. Automation improves the objectivity and reproducibility of tests. It reduces human errors in patient identification and transcription errors. Documentation and traceability of tests, reagents and processes and archiving of results is another major advantage of automation. Shifting from manual methods to automation is a major undertaking for any transfusion service to provide quality patient care with lesser turnaround time for their ever increasing workload. This article discusses the various issues involved in the process. PMID:22988378

  11. Advances in inspection automation

    NASA Astrophysics Data System (ADS)

    Weber, Walter H.; Mair, H. Douglas; Jansen, Dion; Lombardi, Luciano

    2013-01-01

    This new session at QNDE reflects the growing interest in inspection automation. Our paper describes a newly developed platform that makes the complex NDE automation possible without the need for software programmers. Inspection tasks that are tedious, error-prone or impossible for humans to perform can now be automated using a form of drag and drop visual scripting. Our work attempts to rectify the problem that NDE is not keeping pace with the rest of factory automation. Outside of NDE, robots routinely and autonomously machine parts, assemble components, weld structures and report progress to corporate databases. By contrast, components arriving in the NDT department typically require manual part handling, calibrations and analysis. The automation examples in this paper cover the development of robotic thickness gauging and the use of adaptive contour following on the NRU reactor inspection at Chalk River.

  12. Robotic liquid handling and automation in epigenetics.

    PubMed

    Gaisford, Wendy

    2012-10-01

    Automated liquid-handling robots and high-throughput screening (HTS) are widely used in the pharmaceutical industry for the screening of large compound libraries, small molecules for activity against disease-relevant target pathways, or proteins. HTS robots capable of low-volume dispensing reduce assay setup times and provide highly accurate and reproducible dispensing, minimizing variation between sample replicates and eliminating the potential for manual error. Low-volume automated nanoliter dispensers ensure accuracy of pipetting within volume ranges that are difficult to achieve manually. In addition, they have the ability to potentially expand the range of screening conditions from often limited amounts of valuable sample, as well as reduce the usage of expensive reagents. The ability to accurately dispense lower volumes provides the potential to achieve a greater amount of information than could be otherwise achieved using manual dispensing technology. With the emergence of the field of epigenetics, an increasing number of drug discovery companies are beginning to screen compound libraries against a range of epigenetic targets. This review discusses the potential for the use of low-volume liquid handling robots, for molecular biological applications such as quantitative PCR and epigenetics.

  13. Internet-Based Cervical Cytology Screening Program

    DTIC Science & Technology

    2006-04-01

    Appendices……………………………………………………………………………14 Introduction Cervical cancer is theoretically completely preventable by effective...approaches to cervical cancer screening possible. In addition, advances in information technology have facilitated the Internet transmission and archival...to allow secure, automated reporting of cervical cancer screening results - completed f) Adapt commercial software (Wellogic) to integrate

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

    PubMed Central

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

    2016-01-01

    Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ∼4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ∼100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ∼1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays. PMID:27725672

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  16. Screening of over 100 drugs in horse urine using automated on-line solid-phase extraction coupled to liquid chromatography-high resolution mass spectrometry for doping control.

    PubMed

    Kwok, W H; Choi, Timmy L S; Tsoi, Yeuki Y K; Leung, Gary N W; Wan, Terence S M

    2017-02-14

    A fast method for the direct analysis of enzyme-hydrolysed horse urine using an automated on-line solid-phase extraction (SPE) coupled to a liquid-chromatography/high resolution mass spectrometer was developed. Over 100 drugs of diverse drug classes could be simultaneously detected in horse urine at sub to low parts per billion levels. Urine sample was first hydrolysed by β-glucuronidase to release conjugated drugs, followed by centrifugal filtration. The filtrate (1mL) was directly injected into an on-line SPE system consisting of a pre-column filter and a SPE cartridge column for the separation of analytes from matrix components. Through valves-switching, the interfering matrix components were flushed to waste, and the analytes were eluted to a C18 analytical column for refocusing and chromatographic separation. Detections were achieved by full-scan HRMS in alternating positive and negative electrospray ionisation modes within a turn-around time of 16min, inclusive of on-line sample clean-up and post-run mobile phase equilibration. No significant matrix interference was observed at the expected retention times of the targeted masses. Over 90% of the drugs studied gave estimated limits of detection (LoDs) at or below 5ng/mL, with some LoDs reaching down to 0.05ng/mL. Data-dependent acquisition (DDA) was included to provide additional product-ion scan data to substantiate the presence of detected analytes. The resulting product-ion spectra can be searched against an in-house MS/MS library for identity verification. The applicability of the method has been demonstrated by the detection of drugs in doping control samples.

  17. Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

    PubMed Central

    Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

    2016-01-01

    Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

  18. Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis

    SciTech Connect

    Tajbakhsh, Jian; Stefanovski, Darko; Tang, George; Wawrowsky, Kolja; Liu, Naiyou; Fair, Jeffrey H.

    2015-03-15

    Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU/day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17: 0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, global DNA methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC{sup +}/5mC{sup −}, 5hmC{sup +}/5mC{sup +}, and 5hmC{sup −}/5mC{sup +} cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC{sup +}/5mC{sup +} cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably

  19. Genetic screening

    PubMed Central

    Andermann, Anne; Blancquaert, Ingeborg

    2010-01-01

    Abstract OBJECTIVE To provide a primer for primary care professionals who are increasingly called upon to discuss the growing number of genetic screening services available and to help patients make informed decisions about whether to participate in genetic screening, how to interpret results, and which interventions are most appropriate. QUALITY OF EVIDENCE As part of a larger research program, a wide literature relating to genetic screening was reviewed. PubMed and Internet searches were conducted using broad search terms. Effort was also made to identify the gray literature. MAIN MESSAGE Genetic screening is a type of public health program that is systematically offered to a specified population of asymptomatic individuals with the aim of providing those identified as high risk with prevention, early treatment, or reproductive options. Ensuring an added benefit from screening, as compared with standard clinical care, and preventing unintended harms, such as undue anxiety or stigmatization, depends on the design and implementation of screening programs, including the recruitment methods, education and counseling provided, timing of screening, predictive value of tests, interventions available, and presence of oversight mechanisms and safeguards. There is therefore growing apprehension that economic interests might lead to a market-driven approach to introducing and expanding screening before program effectiveness, acceptability, and feasibility have been demonstrated. As with any medical intervention, there is a moral imperative for genetic screening to do more good than harm, not only from the perspective of individuals and families, but also for the target population and society as a whole. CONCLUSION Primary care professionals have an important role to play in helping their patients navigate the rapidly changing terrain of genetic screening services by informing them about the benefits and risks of new genetic and genomic technologies and empowering them to

  20. Systematic review automation technologies.

    PubMed

    Tsafnat, Guy; Glasziou, Paul; Choong, Miew Keen; Dunn, Adam; Galgani, Filippo; Coiera, Enrico

    2014-07-09

    Systematic reviews, a cornerstone of evidence-based medicine, are not produced quickly enough to support clinical practice. The cost of production, availability of the requisite expertise and timeliness are often quoted as major contributors for the delay. This detailed survey of the state of the art of information systems designed to support or automate individual tasks in the systematic review, and in particular systematic reviews of randomized controlled clinical trials, reveals trends that see the convergence of several parallel research projects.We surveyed literature describing informatics systems that support or automate the processes of systematic review or each of the tasks of the systematic review. Several projects focus on automating, simplifying and/or streamlining specific tasks of the systematic review. Some tasks are already fully automated while others are still largely manual. In this review, we describe each task and the effect that its automation would have on the entire systematic review process, summarize the existing information system support for each task, and highlight where further research is needed for realizing automation for the task. Integration of the systems that automate systematic review tasks may lead to a revised systematic review workflow. We envisage the optimized workflow will lead to system in which each systematic review is described as a computer program that automatically retrieves relevant trials, appraises them, extracts and synthesizes data, evaluates the risk of bias, performs meta-analysis calculations, and produces a report in real time.

  1. Systematic review automation technologies

    PubMed Central

    2014-01-01

    Systematic reviews, a cornerstone of evidence-based medicine, are not produced quickly enough to support clinical practice. The cost of production, availability of the requisite expertise and timeliness are often quoted as major contributors for the delay. This detailed survey of the state of the art of information systems designed to support or automate individual tasks in the systematic review, and in particular systematic reviews of randomized controlled clinical trials, reveals trends that see the convergence of several parallel research projects. We surveyed literature describing informatics systems that support or automate the processes of systematic review or each of the tasks of the systematic review. Several projects focus on automating, simplifying and/or streamlining specific tasks of the systematic review. Some tasks are already fully automated while others are still largely manual. In this review, we describe each task and the effect that its automation would have on the entire systematic review process, summarize the existing information system support for each task, and highlight where further research is needed for realizing automation for the task. Integration of the systems that automate systematic review tasks may lead to a revised systematic review workflow. We envisage the optimized workflow will lead to system in which each systematic review is described as a computer program that automatically retrieves relevant trials, appraises them, extracts and synthesizes data, evaluates the risk of bias, performs meta-analysis calculations, and produces a report in real time. PMID:25005128

  2. Managing laboratory automation

    PubMed Central

    Saboe, Thomas J.

    1995-01-01

    This paper discusses the process of managing automated systems through their life cycles within the quality-control (QC) laboratory environment. The focus is on the process of directing and managing the evolving automation of a laboratory; system examples are given. The author shows how both task and data systems have evolved, and how they interrelate. A BIG picture, or continuum view, is presented and some of the reasons for success or failure of the various examples cited are explored. Finally, some comments on future automation need are discussed. PMID:18925018

  3. A somaclonal variant of rose-scented geranium (Pelargonium spp.) with moderately high content of isomenthone in its essential oil.

    PubMed

    Kulkarni, Swaroop S; Ravindra, Nagawara S; Srinivas, Kalavagunta V N S; Kulkarni, Raghavendra N

    2012-09-01

    Rose-scented geranium (Pelargonium spp.), which is highly valued for its essential oil, is exclusively propagated vegetatively. Hence no genetic improvement work is possible through conventional breeding. Somaclonal variation was generated with and without in vitro mutagenesis using N-nitroso-N-methyl urea (NMU) in an Indian cultivar 'Bourbon', and a clone 'Narmada'. A somaclonal variant (N75) with a moderately high content of isomenthone in its essential oil was isolated from somaclones generated after treatment of internodal explants of clone, 'Narmada' with 0.25 mM NMU for 1 h. The contents of isomenthone in its essential oil were 26% and 35%, respectively, in SC2/VM2 and SC3/VM3 generations (second and third vegetative generations, respectively, after in vitro mutagen treatment) as compared with 0.7% and 0.3%, respectively, in the parental clone, 'Narmada'. The contents of alcohols and their esters (linalool, citronellol, geraniol, citronellyl formate and geranyl formate) in the essential oil of N75 in SC2/VM2 and SC3/VM3 generations were 49% and 35%, respectively, as compared with 69% and 63%, respectively, in the parental clone, 'Narmada'. This is the first report on a chemovariant of rose-scented geranium with a moderately high content of isomenthone. All earlier reported isomenthone-rich variants of rose-scented geranium had quite high contents of isomenthone (64-71%) in their essential oils. The probable modes of origin of this somaclonal variant, its parental clone 'Narmada' (with very low content of isomenthone) and four earlier reported isomenthone-rich variants of Indian cultivars of geranium are discussed.

  4. Automating the Media Center.

    ERIC Educational Resources Information Center

    Holloway, Mary A.

    1988-01-01

    Discusses the need to develop more efficient information retrieval skills by the use of new technology. Lists four stages used in automating the media center. Describes North Carolina's pilot programs. Proposes benefits and looks at the media center's future. (MVL)

  5. Planning for Office Automation.

    ERIC Educational Resources Information Center

    Mick, Colin K.

    1983-01-01

    Outlines a practical approach to planning for office automation termed the "Focused Process Approach" (the "what" phase, "how" phase, "doing" phase) which is a synthesis of the problem-solving and participatory planning approaches. Thirteen references are provided. (EJS)

  6. Xenon International Automated Control

    SciTech Connect

    2016-08-05

    The Xenon International Automated Control software monitors, displays status, and allows for manual operator control as well as fully automatic control of multiple commercial and PNNL designed hardware components to generate and transmit atmospheric radioxenon concentration measurements every six hours.

  7. Quadruple screen test

    MedlinePlus

    Quad screen; Multiple marker screening; AFP plus; Triple screen test; AFP maternal; MSAFP; 4-marker screen; Down syndrome - quadruple; Trisomy 21 - quadruple; Turner syndrome - quadruple; Spina bifida - ...

  8. Automated Cyber Red Teaming

    DTIC Science & Technology

    2015-04-01

    possible attack paths for CRT. This report surveys the current state-of-the- art planning techniques, tools and frameworks, their performance at...6 3.3 State of the art Automated Planning ..................................................................... 7 3.3.1...automated planning to CRT problems. Finally, we recommend several state-of-the- art planning tools for trial and, more generally, when it is suitable to use

  9. Automating Index Preparation

    DTIC Science & Technology

    1987-03-23

    Automating Index Preparation* Pehong Chent Michael A. Harrison Computer Science Division University of CaliforniaI Berkeley, CA 94720 March 23, 1987...Abstract Index preparation is a tedious and time-consuming task. In this paper we indicate * how the indexing process can be automated in a way which...identified and analyzed. Specifically, we describe a framework for placing index commands in the document and a general purpose index processor which

  10. Automated Pilot Advisory System

    NASA Technical Reports Server (NTRS)

    Parks, J. L., Jr.; Haidt, J. G.

    1981-01-01

    An Automated Pilot Advisory System (APAS) was developed and operationally tested to demonstrate the concept that low cost automated systems can provide air traffic and aviation weather advisory information at high density uncontrolled airports. The system was designed to enhance the see and be seen rule of flight, and pilots who used the system preferred it over the self announcement system presently used at uncontrolled airports.

  11. Automated Lattice Perturbation Theory

    SciTech Connect

    Monahan, Christopher

    2014-11-01

    I review recent developments in automated lattice perturbation theory. Starting with an overview of lattice perturbation theory, I focus on the three automation packages currently "on the market": HiPPy/HPsrc, Pastor and PhySyCAl. I highlight some recent applications of these methods, particularly in B physics. In the final section I briefly discuss the related, but distinct, approach of numerical stochastic perturbation theory.

  12. Automated Status Notification System

    NASA Technical Reports Server (NTRS)

    2005-01-01

    NASA Lewis Research Center's Automated Status Notification System (ASNS) was born out of need. To prevent "hacker attacks," Lewis' telephone system needed to monitor communications activities 24 hr a day, 7 days a week. With decreasing staff resources, this continuous monitoring had to be automated. By utilizing existing communications hardware, a UNIX workstation, and NAWK (a pattern scanning and processing language), we implemented a continuous monitoring system.

  13. Evaluating the Validity of the Automated Working Memory Assessment

    ERIC Educational Resources Information Center

    Alloway, Tracy; Gathercole, Susan E.; Kirkwood, Hannah; Elliott, Julian

    2008-01-01

    The aim of the present study was to investigate the construct stability and diagnostic validity of a standardised computerised tool for assessing working memory: the Automated Working Memory Assessment (AWMA). The purpose of the AWMA is to provide educators with a quick and effective tool to screen for and support those with memory impairments.…

  14. MRSA Screening

    MedlinePlus

    ... Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising ... aureus Screening Related tests: Wound Culture All content on Lab Tests Online has been ...

  15. Hypertension screening

    NASA Technical Reports Server (NTRS)

    Foulke, J. M.

    1975-01-01

    An attempt was made to measure the response to an announcement of hypertension screening at the Goddard Space Center, to compare the results to those of previous statistics. Education and patient awareness of the problem were stressed.

  16. Developmental Screening

    MedlinePlus

    Learn More about Your Child’s Development: Developmental Monitoring and Screening Taking a first step, waving “bye-bye,” and pointing to something interesting are all developmental milestones, ...

  17. Get Screened

    MedlinePlus

    ... health. Depending on your age, sex, and medical history, you may need to be screened for things like: Certain types of cancer High blood pressure or high cholesterol Diabetes Osteoporosis (weak bones) ...

  18. Metrology automation reliability

    NASA Astrophysics Data System (ADS)

    Chain, Elizabeth E.

    1996-09-01

    At Motorola's MOS-12 facility automated measurements on 200- mm diameter wafers proceed in a hands-off 'load-and-go' mode requiring only wafer loading, measurement recipe loading, and a 'run' command for processing. Upon completion of all sample measurements, the data is uploaded to the factory's data collection software system via a SECS II interface, eliminating the requirement of manual data entry. The scope of in-line measurement automation has been extended to the entire metrology scheme from job file generation to measurement and data collection. Data analysis and comparison to part specification limits is also carried out automatically. Successful integration of automated metrology into the factory measurement system requires that automated functions, such as autofocus and pattern recognition algorithms, display a high degree of reliability. In the 24- hour factory reliability data can be collected automatically on every part measured. This reliability data is then uploaded to the factory data collection software system at the same time as the measurement data. Analysis of the metrology reliability data permits improvements to be made as needed, and provides an accurate accounting of automation reliability. This reliability data has so far been collected for the CD-SEM (critical dimension scanning electron microscope) metrology tool, and examples are presented. This analysis method can be applied to such automated in-line measurements as CD, overlay, particle and film thickness measurements.

  19. Elements of EAF automation processes

    NASA Astrophysics Data System (ADS)

    Ioana, A.; Constantin, N.; Dragna, E. C.

    2017-01-01

    Our article presents elements of Electric Arc Furnace (EAF) automation. So, we present and analyze detailed two automation schemes: the scheme of electrical EAF automation system; the scheme of thermic EAF automation system. The application results of these scheme of automation consists in: the sensitive reduction of specific consummation of electrical energy of Electric Arc Furnace, increasing the productivity of Electric Arc Furnace, increase the quality of the developed steel, increasing the durability of the building elements of Electric Arc Furnace.

  20. [HCC screening].

    PubMed

    Albrecht, T

    2008-01-01

    Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed tumour diseases throughout the world. In the vast majority of cases those affected are high-risk patients with chronic viral hepatitis and/or liver cirrhosis, which means there is a clearly identifiable target group for HCC screening. With resection, transplantation, and interventional procedures for local ablation, following early diagnosis curative treatment options are available with which 5-year survival rates of over 60% can be reached. Such early diagnosis is a reality only in a minority of patients, however, and in the majority of cases the disease is already in an advanced stage at diagnosis. One of the objects of HCC screening is diagnosis in an early stage when curative treatment is still possible. Precisely this is achieved by screening, so that the proportion of patients treated with curative intent is decisively higher. There is not yet any clear evidence as to whether this leads to a lowering of the mortality of HCC. As lower mortality is the decisive indicator of success for a screening programme the benefit of HCC screening has so far been neither documented nor refuted. Nonetheless, in large regions of the world it is the practice for high-risk patients to undergo HCC screening in the form of twice-yearly ultrasound examination and determination of AFP.

  1. An automated approach to configuration baseline documentation

    SciTech Connect

    Blake, G.D.; Khan, M.A.

    1990-01-01

    This paper presents Public Service Electric and Gas Company's (PSE and G's) automated approach to configuration base-line documentation (CBD) for Salem units 1 and 2 and Hope Creek. The CBD project is a proactive project, similar to what is commonly termed a design basis documentation program in the nuclear utility industry. The data information management system (DIMS) element of the CBD project is expected to automate the CBD development, review/approval, control, maintenance, and distribution of CBD and the subsequent integration of the CBD into the day-to-day design processes of PSE and G's nuclear engineering department. The DIMS project scope emphasizes streamlined, swift, and accurate design information retrieval system hardware and software; proper and controlled screening of stored design information; legible storage of design information; and more efficient and user-friendly information handling. This paper discusses the selection and implementation of an integrated optical imaging and textual search technology.

  2. Automated diagnostic kiosk for diagnosing diseases

    DOEpatents

    Regan, John Frederick; Birch, James Michael

    2014-02-11

    An automated and autonomous diagnostic apparatus that is capable of dispensing collection vials and collections kits to users interesting in collecting a biological sample and submitting their collected sample contained within a collection vial into the apparatus for automated diagnostic services. The user communicates with the apparatus through a touch-screen monitor. A user is able to enter personnel information into the apparatus including medical history, insurance information, co-payment, and answer a series of questions regarding their illness, which is used to determine the assay most likely to yield a positive result. Remotely-located physicians can communicate with users of the apparatus using video tele-medicine and request specific assays to be performed. The apparatus archives submitted samples for additional testing. Users may receive their assay results electronically. Users may allow the uploading of their diagnoses into a central databank for disease surveillance purposes.

  3. Multiparametric Cell Cycle Analysis Using the Operetta High-Content Imager and Harmony Software with PhenoLOGIC.

    PubMed

    Massey, Andrew J

    2015-01-01

    High-content imaging is a powerful tool for determining cell phenotypes at the single cell level. Characterising the effect of small molecules on cell cycle distribution is important for understanding their mechanism of action especially in oncology drug discovery but also for understanding potential toxicology liabilities. Here, a high-throughput phenotypic assay utilising the PerkinElmer Operetta high-content imager and Harmony software to determine cell cycle distribution is described. PhenoLOGIC, a machine learning algorithm within Harmony software was employed to robustly separate single cells from cell clumps. DNA content, EdU incorporation and pHH3 (S10) expression levels were subsequently utilised to separate cells into the various phases of the cell cycle. The assay is amenable to multiplexing with an additional pharmacodynamic marker to assess cell cycle changes within a specific cellular sub-population. Using this approach, the cell cycle distribution of γH2AX positive nuclei was determined following treatment with DNA damaging agents. Likewise, the assay can be multiplexed with Ki67 to determine the fraction of quiescent cells and with BrdU dual labelling to determine S-phase duration. This methodology therefore provides a relatively cheap, quick and high-throughput phenotypic method for determining accurate cell cycle distribution for small molecule mechanism of action and drug toxicity studies.

  4. Benzyl butyl phthalate promotes adipogenesis in 3T3-L1 preadipocytes: A High Content Cellomics and metabolomic analysis.

    PubMed

    Yin, Lei; Yu, Kevin Shengyang; Lu, Kun; Yu, Xiaozhong

    2016-04-01

    Benzyl butyl phthalate (BBP) has been known to induce developmental and reproductive toxicity. However, its association with dysregulation of adipogenesis has been poorly investigated. The present study aimed to examine the effect of BBP on the adipogenesis, and to elucidate the underlying mechanisms using the 3T3-L1 cells. The capacity of BBP to promote adipogenesis was evaluated by multiple staining approaches combined with a High Content Cellomics analysis. The dynamic changes of adipogenic regulatory genes and proteins were examined, and the metabolite profile was identified using GC/MC based metabolomic analysis. The High Content analysis showed BBP in contrast with Bisphenol A (BPA), a known environmental obesogen, increased lipid droplet accumulation in a similar dose-dependent manner. However, the size of the lipid droplets in BBP-treated cells was significantly larger than those in cells treated with BPA. BBP significantly induced mRNA expression of transcriptional factors C/EBPα and PPARγ, their downstream genes, and numerous adipogenic proteins in a dose and time-dependent manner. Furthermore, GC/MC metabolomic analysis revealed that BBP exposure perturbed the metabolic profiles that are associated with glyceroneogenesis and fatty acid synthesis. Altogether, our current study clearly demonstrates that BBP promoted the differentiation of 3T3-L1 through the activation of the adipogenic pathway and metabolic disturbance.

  5. Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds.

    PubMed

    Sum, Chi Shing; Nickischer, Debra; Lei, Ming; Weston, Andrea; Zhang, Litao; Schweizer, Liang

    2014-01-01

    Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and β-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.

  6. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    PubMed

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-02-19

    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  7. Studying mechanosensitive ion channels with an automated patch clamp.

    PubMed

    Barthmes, Maria; Jose, Mac Donald F; Birkner, Jan Peter; Brüggemann, Andrea; Wahl-Schott, Christian; Koçer, Armağan

    2014-03-01

    Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.

  8. ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS

    EPA Science Inventory

    There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures ...

  9. Automated macromolecular crystal detection system and method

    DOEpatents

    Christian, Allen T.; Segelke, Brent; Rupp, Bernard; Toppani, Dominique

    2007-06-05

    An automated macromolecular method and system for detecting crystals in two-dimensional images, such as light microscopy images obtained from an array of crystallization screens. Edges are detected from the images by identifying local maxima of a phase congruency-based function associated with each image. The detected edges are segmented into discrete line segments, which are subsequently geometrically evaluated with respect to each other to identify any crystal-like qualities such as, for example, parallel lines, facing each other, similarity in length, and relative proximity. And from the evaluation a determination is made as to whether crystals are present in each image.

  10. Power subsystem automation study

    NASA Technical Reports Server (NTRS)

    Imamura, M. S.; Moser, R. L.; Veatch, M.

    1983-01-01

    Generic power-system elements and their potential faults are identified. Automation functions and their resulting benefits are defined and automation functions between power subsystem, central spacecraft computer, and ground flight-support personnel are partitioned. All automation activities were categorized as data handling, monitoring, routine control, fault handling, planning and operations, or anomaly handling. Incorporation of all these classes of tasks, except for anomaly handling, in power subsystem hardware and software was concluded to be mandatory to meet the design and operational requirements of the space station. The key drivers are long mission lifetime, modular growth, high-performance flexibility, a need to accommodate different electrical user-load equipment, onorbit assembly/maintenance/servicing, and potentially large number of power subsystem components. A significant effort in algorithm development and validation is essential in meeting the 1987 technology readiness date for the space station.

  11. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  12. Automated telescope scheduling

    NASA Technical Reports Server (NTRS)

    Johnston, Mark D.

    1988-01-01

    With the ever increasing level of automation of astronomical telescopes the benefits and feasibility of automated planning and scheduling are becoming more apparent. Improved efficiency and increased overall telescope utilization are the most obvious goals. Automated scheduling at some level has been done for several satellite observatories, but the requirements on these systems were much less stringent than on modern ground or satellite observatories. The scheduling problem is particularly acute for Hubble Space Telescope: virtually all observations must be planned in excruciating detail weeks to months in advance. Space Telescope Science Institute has recently made significant progress on the scheduling problem by exploiting state-of-the-art artificial intelligence software technology. What is especially interesting is that this effort has already yielded software that is well suited to scheduling groundbased telescopes, including the problem of optimizing the coordinated scheduling of more than one telescope.

  13. Development of a beetroot-based nutritional gel containing high content of bioaccessible dietary nitrate and antioxidants.

    PubMed

    Morgado, Marina; de Oliveira, Gustavo Vieira; Vasconcellos, Julia; Monteiro, Maria Lucia; Conte-Junior, Carlos; Pierucci, Anna Paola Trindade Rocha; Alvares, Thiago Silveira

    2016-01-01

    Beetroot, a food rich in nitrate and antioxidants has gained attention because of its potential effect on improving cardiovascular health and exercise performance. This work had the purpose of developing a beetroot-based nutritional gel (BG) and estimating the in vitro bioaccessibility of the nitrate, total antioxidant capacity (TAC), total phenolic (TP) and potassium content, as compared to beetroot juice (BJ). Nitrate was assessed by a high-performance liquid chromatography system, TAC was assessed using the Trolox equivalent antioxidant capacity (TEAC) assay and TP was measured using the Folin-Ciocalteu method before and after an in vitro digestion. Significantly higher values of nitrate, TEAC, TP and potassium before and after digestion were observed in BG as compared to BJ. The results suggest a new nutritional strategy to give high contents of bioaccessible nutrients (nitrate, antioxidants and potassium) that are potentially relevant to improve cardiovascular health and exercise performance.

  14. Modification of chemical oxygen demand monitoring in the Yellow River, China, with a high content of sediments.

    PubMed

    Ni, Jinren; Sun, Liyiing; Sun, Weiling

    2007-10-01

    This paper presents a modification of chemical oxygen demand (COD) monitoring giving a better indication of the pollution level compared with the conventional COD method for rivers with a high content of sediments. The correlation between the sediment organic carbon and COD was investigated using sediments sampled in the middle Yellow River, China. Partitioning of the sediment organic carbon between the water and sediment phases was also investigated using batch experiments, with the sediment concentration varying from 20 to 400 g/L. As a result, the COD modification equations are proposed for both turbid water (mixture of water and sediment) and supematant water (filtrate using a 0.45-microm membrane). The modified COD in turbid water and supernatant water could be 40 and 10% less than the monitored COD values, respectively. These results may have a significant influence on the assessment of water quality class in the Yellow River.

  15. Isolation and culture of adult human microglia within mixed glial cultures for functional experimentation and high-content analysis.

    PubMed

    Smith, Amy M; Gibbons, Hannah M; Lill, Claire; Faull, Richard L M; Dragunow, Mike

    2013-01-01

    Microglia are thought to be involved in diseases of the adult human brain as well as normal aging processes. While neonatal and rodent microglia are often used in studies investigating microglial function, there are important differences between rodent microglia and their adult human counterparts. Human brain tissue provides a unique and valuable tool for microglial cell and molecular biology. Routine protocols can now enable use of this culture method in many laboratories. Detailed protocols and advice for culture of human brain microglia are provided here. We demonstrate the protocol for culturing human adult microglia within a mixed glial culture and use a phagocytosis assay as an example of the functional studies possible with these cells as well as a high-content analysis method of quantification.

  16. Abnormally High Content of Free Glucosamine Residues Identified in a Preparation of Commercially Available Porcine Intestinal Heparan Sulfate

    PubMed Central

    2016-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after heparin lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies. PMID:27295282

  17. Improvement of the agitation granulation method to prepare granules containing a high content of a very hygroscopic drug.

    PubMed

    Hirai, Nobuaki; Ishikawa, Kazuyuki; Takahashi, Koichi

    2006-11-01

    This study describes a new approach to the preparation of a granulate with a high content of a very hygroscopic powder or drug, using the agitation granulation method, and the development of a tablet formulation using these granulates. A Chinese medicine extract, Hatimi-zio-gan, was used as the model of a very hygroscopic drug. Among the several excipients tested, only porous calcium silicate could be used to prepare granules, with a mixing ratio (extract to porous calcium silicate) from 2:1 to 20:1. With other excipients, very large lumps were formed during the granulation process. The best mixing ratio of extract to porous calcium silicate was 6:1. For preparation of the granules, water could be added to the mixed powder within a range of 1- to 4-times the amount of porous calcium silicate. From these results, it was concluded that the ability of porous calcium silicate to hold large amounts of water in its numerous pores may allow for the preparation of granulates with a high content of very hygroscopic drugs. Starch with partial alpha-links, carboxymethyl starch sodium salt and crospovidone were used for selection of the disintegration agent. When crospovidone was used as a disintegration agent, tablets containing about 70% of the Chinese medicine extract disintegrated in less than 7 min, with good dissolution rates. The same process was applied to extracts of Hotyu-ekki-to, Syo-seiryu-to, Boi-ogi-to and Bohu-tusyo-san. The absorption of paeoniflorin, a characteristic monoterpene glucoside contained in Hatimi-zio-gan extract, was evaluated in beagle dogs after oral administration of the Hatimi-zio-gan tablets prepared in this study. The values of C(max) and AUC obtained after administration of the tablets prepared in this study were significantly greater than those obtained for commercial tablets.

  18. High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture.

    PubMed

    Stojiljković, A; Kuehni-Boghenbor, K; Gaschen, V; Schüpbach, G; Mevissen, M; Kinnear, C; Möller, A-M; Stoffel, M H

    2016-09-22

    Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.

  19. Automating the CMS DAQ

    SciTech Connect

    Bauer, G.; et al.

    2014-01-01

    We present the automation mechanisms that have been added to the Data Acquisition and Run Control systems of the Compact Muon Solenoid (CMS) experiment during Run 1 of the LHC, ranging from the automation of routine tasks to automatic error recovery and context-sensitive guidance to the operator. These mechanisms helped CMS to maintain a data taking efficiency above 90% and to even improve it to 95% towards the end of Run 1, despite an increase in the occurrence of single-event upsets in sub-detector electronics at high LHC luminosity.

  20. Automated Library System Specifications.

    DTIC Science & Technology

    1986-06-01

    AD-A78 95 AUTOMATED LIBRARY SYSTEM SPECIFICATIONS(U) ARMY LIBRARY /i MANAGEMENT OFFICE ALEXANDRIA VA ASSISTANT CHIEF OF STAFF FOR INFORMATION... MANAGEMENT M B BONNETT JUN 86 UNCLASSIFIED F/G 9/2 NLEElIIhllEEEEE IllEEEEEllllEI .1lm lliml * ~I fI.L25 MI, [OCM RL,;OCLUTO fl. ’N k~ AUTOMATED LIBRARY...SYSTEM SPECIFICATIONS .,I Prepared by Mary B. Bonnett ARMY LIBRARY MANAGEMENT OFFICE OFFICE OF THE ASSISTANT CHIEF OF STAFF FOR INFORMATION MANAGEMENT Lij

  1. Automated gas chromatography

    DOEpatents

    Mowry, Curtis D.; Blair, Dianna S.; Rodacy, Philip J.; Reber, Stephen D.

    1999-01-01

    An apparatus and process for the continuous, near real-time monitoring of low-level concentrations of organic compounds in a liquid, and, more particularly, a water stream. A small liquid volume of flow from a liquid process stream containing organic compounds is diverted by an automated process to a heated vaporization capillary where the liquid volume is vaporized to a gas that flows to an automated gas chromatograph separation column to chromatographically separate the organic compounds. Organic compounds are detected and the information transmitted to a control system for use in process control. Concentrations of organic compounds less than one part per million are detected in less than one minute.

  2. Automated knowledge generation

    NASA Technical Reports Server (NTRS)

    Myler, Harley R.; Gonzalez, Avelino J.

    1988-01-01

    The general objectives of the NASA/UCF Automated Knowledge Generation Project were the development of an intelligent software system that could access CAD design data bases, interpret them, and generate a diagnostic knowledge base in the form of a system model. The initial area of concentration is in the diagnosis of the process control system using the Knowledge-based Autonomous Test Engineer (KATE) diagnostic system. A secondary objective was the study of general problems of automated knowledge generation. A prototype was developed, based on object-oriented language (Flavors).

  3. Hearing Screening

    ERIC Educational Resources Information Center

    Johnson-Curiskis, Nanette

    2012-01-01

    Hearing levels are threatened by modern life--headsets for music, rock concerts, traffic noises, etc. It is crucial we know our hearing levels so that we can draw attention to potential problems. This exercise requires that students receive a hearing screening for their benefit as well as for making the connection of hearing to listening.

  4. Altering user' acceptance of automation through prior automation exposure.

    PubMed

    Bekier, Marek; Molesworth, Brett R C

    2016-08-22

    Air navigation service providers worldwide see increased use of automation as one solution to overcome the capacity constraints imbedded in the present air traffic management (ATM) system. However, increased use of automation within any system is dependent on user acceptance. The present research sought to determine if the point at which an individual is no longer willing to accept or cooperate with automation can be manipulated. Forty participants underwent training on a computer-based air traffic control programme, followed by two ATM exercises (order counterbalanced), one with and one without the aid of automation. Results revealed after exposure to a task with automation assistance, user acceptance of high(er) levels of automation ('tipping point') decreased; suggesting it is indeed possible to alter automation acceptance. Practitioner Summary: This paper investigates whether the point at which a user of automation rejects automation (i.e. 'tipping point') is constant or can be manipulated. The results revealed after exposure to a task with automation assistance, user acceptance of high(er) levels of automation decreased; suggesting it is possible to alter automation acceptance.

  5. The ToxCast Pathway Database for Identifying Toxicity Signatures and Potential Modes of Action from Chemical Screening Data

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA), through its ToxCast program, is developing predictive toxicity approaches that will use in vitro high-throughput screening (HTS), high-content screening (HCS) and toxicogenomic data to predict in vivo toxicity phenotypes. There are ...

  6. Automated conflict resolution issues

    NASA Technical Reports Server (NTRS)

    Wike, Jeffrey S.

    1991-01-01

    A discussion is presented of how conflicts for Space Network resources should be resolved in the ATDRSS era. The following topics are presented: a description of how resource conflicts are currently resolved; a description of issues associated with automated conflict resolution; present conflict resolution strategies; and topics for further discussion.

  7. Automated Accounting. Instructor Guide.

    ERIC Educational Resources Information Center

    Moses, Duane R.

    This curriculum guide was developed to assist business instructors using Dac Easy Accounting College Edition Version 2.0 software in their accounting programs. The module consists of four units containing assignment sheets and job sheets designed to enable students to master competencies identified in the area of automated accounting. The first…

  8. Automated Student Model Improvement

    ERIC Educational Resources Information Center

    Koedinger, Kenneth R.; McLaughlin, Elizabeth A.; Stamper, John C.

    2012-01-01

    Student modeling plays a critical role in developing and improving instruction and instructional technologies. We present a technique for automated improvement of student models that leverages the DataShop repository, crowd sourcing, and a version of the Learning Factors Analysis algorithm. We demonstrate this method on eleven educational…

  9. Personnel Department Automation.

    ERIC Educational Resources Information Center

    Wilkinson, David

    In 1989, the Austin Independent School District's Office of Research and Evaluation was directed to monitor the automation of personnel information and processes in the district's Department of Personnel. Earlier, a study committee appointed by the Superintendent during the 1988-89 school year identified issues related to Personnel Department…

  10. Validating Automated Speaking Tests

    ERIC Educational Resources Information Center

    Bernstein, Jared; Van Moere, Alistair; Cheng, Jian

    2010-01-01

    This paper presents evidence that supports the valid use of scores from fully automatic tests of spoken language ability to indicate a person's effectiveness in spoken communication. The paper reviews the constructs, scoring, and the concurrent validity evidence of "facility-in-L2" tests, a family of automated spoken language tests in Spanish,…

  11. Automated Essay Scoring

    ERIC Educational Resources Information Center

    Dikli, Semire

    2006-01-01

    The impacts of computers on writing have been widely studied for three decades. Even basic computers functions, i.e. word processing, have been of great assistance to writers in modifying their essays. The research on Automated Essay Scoring (AES) has revealed that computers have the capacity to function as a more effective cognitive tool (Attali,…

  12. Automated Microbial Genome Annotation

    SciTech Connect

    Land, Miriam

    2009-05-29

    Miriam Land of the DOE Joint Genome Institute at Oak Ridge National Laboratory gives a talk on the current state and future challenges of moving toward automated microbial genome annotation at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM

  13. Automated Administrative Data Bases

    NASA Technical Reports Server (NTRS)

    Marrie, M. D.; Jarrett, J. R.; Reising, S. A.; Hodge, J. E.

    1984-01-01

    Improved productivity and more effective response to information requirements for internal management, NASA Centers, and Headquarters resulted from using automated techniques. Modules developed to provide information on manpower, RTOPS, full time equivalency, and physical space reduced duplication, increased communication, and saved time. There is potential for greater savings by sharing and integrating with those who have the same requirements.

  14. Automated Management Of Documents

    NASA Technical Reports Server (NTRS)

    Boy, Guy

    1995-01-01

    Report presents main technical issues involved in computer-integrated documentation. Problems associated with automation of management and maintenance of documents analyzed from perspectives of artificial intelligence and human factors. Technologies that may prove useful in computer-integrated documentation reviewed: these include conventional approaches to indexing and retrieval of information, use of hypertext, and knowledge-based artificial-intelligence systems.

  15. Building Automation Systems.

    ERIC Educational Resources Information Center

    Honeywell, Inc., Minneapolis, Minn.

    A number of different automation systems for use in monitoring and controlling building equipment are described in this brochure. The system functions include--(1) collection of information, (2) processing and display of data at a central panel, and (3) taking corrective action by sounding alarms, making adjustments, or automatically starting and…

  16. Guide to Library Automation.

    ERIC Educational Resources Information Center

    Toohill, Barbara G.

    Directed toward librarians and library administrators who wish to procure automated systems or services for their libraries, this guide offers practical suggestions, advice, and methods for determining requirements, estimating costs and benefits, writing specifications procuring systems, negotiating contracts, and installing systems. The advice…

  17. Microcontroller for automation application

    NASA Technical Reports Server (NTRS)

    Cooper, H. W.

    1975-01-01

    The description of a microcontroller currently being developed for automation application was given. It is basically an 8-bit microcomputer with a 40K byte random access memory/read only memory, and can control a maximum of 12 devices through standard 15-line interface ports.

  18. Automated EEG acquisition

    NASA Technical Reports Server (NTRS)

    Frost, J. D., Jr.; Hillman, C. E., Jr.

    1977-01-01

    Automated self-contained portable device can be used by technicians with minimal training. Data acquired from patient at remote site are transmitted to centralized interpretation center using conventional telephone equipment. There, diagnostic information is analyzed, and results are relayed back to remote site.

  19. Automated detection of dilated capillaries on optical coherence tomography angiography

    PubMed Central

    Dongye, Changlei; Zhang, Miao; Hwang, Thomas S.; Wang, Jie; Gao, Simon S.; Liu, Liang; Huang, David; Wilson, David J.; Jia, Yali

    2017-01-01

    Automated detection and grading of angiographic high-risk features in diabetic retinopathy can potentially enhance screening and clinical care. We have previously identified capillary dilation in angiograms of the deep plexus in optical coherence tomography angiography as a feature associated with severe diabetic retinopathy. In this study, we present an automated algorithm that uses hybrid contrast to distinguish angiograms with dilated capillaries from healthy controls and then applies saliency measurement to map the extent of the dilated capillary networks. The proposed algorithm agreed well with human grading. PMID:28271005

  20. Automated Inadvertent Intruder Application

    SciTech Connect

    Koffman, Larry D.; Lee, Patricia L.; Cook, James R.; Wilhite, Elmer L.

    2008-01-15

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) conducts performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. These performance assessments, which result in limits on the amounts of radiological substances that can be placed in the waste disposal facilities, consider numerous potential exposure pathways that could occur in the future. One set of exposure scenarios, known as inadvertent intruder analysis, considers the impact on hypothetical individuals who are assumed to inadvertently intrude onto the waste disposal site. Inadvertent intruder analysis considers three distinct scenarios for exposure referred to as the agriculture scenario, the resident scenario, and the post-drilling scenario. Each of these scenarios has specific exposure pathways that contribute to the overall dose for the scenario. For the inadvertent intruder analysis, the calculation of dose for the exposure pathways is a relatively straightforward algebraic calculation that utilizes dose conversion factors. Prior to 2004, these calculations were performed using an Excel spreadsheet. However, design checks of the spreadsheet calculations revealed that errors could be introduced inadvertently when copying spreadsheet formulas cell by cell and finding these errors was tedious and time consuming. This weakness led to the specification of functional requirements to create a software application that would automate the calculations for inadvertent intruder analysis using a controlled source of input parameters. This software application, named the Automated Inadvertent Intruder Application, has undergone rigorous testing of the internal calculations and meets software QA requirements. The Automated Inadvertent Intruder Application was intended to replace the previous spreadsheet analyses with an automated application that was verified to produce the same calculations and

  1. Automating spectral measurements

    NASA Astrophysics Data System (ADS)

    Goldstein, Fred T.

    2008-09-01

    This paper discusses the architecture of software utilized in spectroscopic measurements. As optical coatings become more sophisticated, there is mounting need to automate data acquisition (DAQ) from spectrophotometers. Such need is exacerbated when 100% inspection is required, ancillary devices are utilized, cost reduction is crucial, or security is vital. While instrument manufacturers normally provide point-and-click DAQ software, an application programming interface (API) may be missing. In such cases automation is impossible or expensive. An API is typically provided in libraries (*.dll, *.ocx) which may be embedded in user-developed applications. Users can thereby implement DAQ automation in several Windows languages. Another possibility, developed by FTG as an alternative to instrument manufacturers' software, is the ActiveX application (*.exe). ActiveX, a component of many Windows applications, provides means for programming and interoperability. This architecture permits a point-and-click program to act as automation client and server. Excel, for example, can control and be controlled by DAQ applications. Most importantly, ActiveX permits ancillary devices such as barcode readers and XY-stages to be easily and economically integrated into scanning procedures. Since an ActiveX application has its own user-interface, it can be independently tested. The ActiveX application then runs (visibly or invisibly) under DAQ software control. Automation capabilities are accessed via a built-in spectro-BASIC language with industry-standard (VBA-compatible) syntax. Supplementing ActiveX, spectro-BASIC also includes auxiliary serial port commands for interfacing programmable logic controllers (PLC). A typical application is automatic filter handling.

  2. Vision Screening

    NASA Technical Reports Server (NTRS)

    1993-01-01

    The Visi Screen OSS-C, marketed by Vision Research Corporation, incorporates image processing technology originally developed by Marshall Space Flight Center. Its advantage in eye screening is speed. Because it requires no response from a subject, it can be used to detect eye problems in very young children. An electronic flash from a 35 millimeter camera sends light into a child's eyes, which is reflected back to the camera lens. The photorefractor then analyzes the retinal reflexes generated and produces an image of the child's eyes, which enables a trained observer to identify any defects. The device is used by pediatricians, day care centers and civic organizations that concentrate on children with special needs.

  3. Assessment of 16 chemicals on proliferation and apoptosis in human neuroprogenitor cells using high-content image analysis (HCA).

    EPA Science Inventory

    The need for efficient methods of screening chemicals for the potential to cause developmental neurotoxicity is paramount. We previously described optimization of an HCA assay for proliferation and apoptosis in ReNcell CX cells (ReN), identifying appropriate controls. Utility of ...

  4. Transparent screens.

    PubMed

    Rosenthal, R J

    1988-01-01

    There is a kind of transitional phenomenon found among certain borderline patients which is quite distinct from Winnicott's transitional object. These are patients who are preoccupied with maintaining proper physical distance from their objects, in order to regulate anxieties about isolation on the one hand, and identity-annihilating closeness on the other. Since they believe the activity of looking to be intrusive and devouring, hence dangerous, transparent screens are interposed between self and other, and serve as protective barriers. These screens function intrapsychically as well, to split off or hide those aspects of the self felt to be unacceptable. The analyst may witness the failure of the screen in several ways: it may create too great a distance, isolating the individual and keeping him from life; it may become contaminated by projections and turn into a persecutor, or trap the individual, a state of intolerable claustrophobia; most dramatically, it may suddenly shatter. The latter is associated with psychosis and death, and its appearance may be a harbinger of suicide.

  5. Automated linking of suspicious findings between automated 3D breast ultrasound volumes

    NASA Astrophysics Data System (ADS)

    Gubern-Mérida, Albert; Tan, Tao; van Zelst, Jan; Mann, Ritse M.; Karssemeijer, Nico

    2016-03-01

    Automated breast ultrasound (ABUS) is a 3D imaging technique which is rapidly emerging as a safe and relatively inexpensive modality for screening of women with dense breasts. However, reading ABUS examinations is very time consuming task since radiologists need to manually identify suspicious findings in all the different ABUS volumes available for each patient. Image analysis techniques to automatically link findings across volumes are required to speed up clinical workflow and make ABUS screening more efficient. In this study, we propose an automated system to, given the location in the ABUS volume being inspected (source), find the corresponding location in a target volume. The target volume can be a different view of the same study or the same view from a prior examination. The algorithm was evaluated using 118 linkages between suspicious abnormalities annotated in a dataset of ABUS images of 27 patients participating in a high risk screening program. The distance between the predicted location and the center of the annotated lesion in the target volume was computed for evaluation. The mean ± stdev and median distance error achieved by the presented algorithm for linkages between volumes of the same study was 7.75±6.71 mm and 5.16 mm, respectively. The performance was 9.54±7.87 and 8.00 mm (mean ± stdev and median) for linkages between volumes from current and prior examinations. The proposed approach has the potential to minimize user interaction for finding correspondences among ABUS volumes.

  6. Microplate cell-retaining methodology for high-content analysis of individual non-adherent unanchored cells in a population.

    PubMed

    Deutsch, Assaf; Zurgil, Naomi; Hurevich, Ihar; Shafran, Yana; Afrimzon, Elena; Lebovich, Pnina; Deutsch, Mordechai

    2006-12-01

    A high throughput Microtiter plate Cell Retainer (MCR) has been developed to enable, for the first time, high-content, time-dependent analysis of the same single non-adherent and non-anchored cells in a large cell population, while bio-manipulating the cells. The identity of each cell in the investigated population is secured, even during bio-manipulation, by cell retention in a specially designed concave microlens, acting as a picoliter well (PW). The MCR technique combines micro-optical features and microtiter plate methodology. The array of PWs serves as the bottom of a microtiter plate, fitted with a unique flow damper element. The latter enables rapid fluid exchange without dislodging the cells from their original PWs, thus maintaining the cells' identity. Loading cell suspensions and reagents into the MCR is performed by simple pouring, followed by gravitational sedimentation and settling of cells into the PWs. Cell viability and cell division within the MCR were shown to be similar to those obtained under similar conditions in a standard microtiter plate. The efficiency of single cell occupancy in the MCR exceeded 90%. No cell dislodging was observed when comparing images before and after bio-manipulations (rinsing, staining, etc.). The MCR permits the performance of kinetic measurements on an individual cell basis. Data acquisition is governed by software, controlling microscope performance, stage position and image acquisition and analysis. The PW's unique micro-optical features enable rapid, simultaneous signal analysis of each individual cell, bypassing lengthy image analysis.

  7. Gel spinning of PVA composite fibers with high content of multi-walled carbon nanotubes and graphene oxide hybrids

    NASA Astrophysics Data System (ADS)

    Wei, Yizhe; Lai, Dengpan; Zou, Liming; Ling, Xinlong; Lu, Hongwei; Xu, Yongjing

    2015-07-01

    In this report, poly (vinyl alcohol) (PVA) composite fibers with high content of multi-walled carbon nanotubes and graphene oxide (MWCNTs-GO) hybrids were prepared by gel spinning, and were characterized by TGA, DSC, SEM, XL-2 yarn strength tester and electrical conductivity measurement. The total content of MWCNTs-GO hybrids in the PVA composite fibers, which is up to 25 wt%, was confirmed by TGA analysis. The DSC measurement shows that the melting and crystallization peaks decreased after the addition of nano-fillers. This is due to the reason that the motion of PVA chains is completely confined by strong hydrogen bonding interaction between PVA and nano-fillers. After the addtion of GO, the dispersibility of MWCNTs in composite fibers improved slightly. And the tensile strength and Young's modulus increased by 38% and 67%, respectively. This is caused by the increased hydrogen bonding interaction and synergistic effect through hybridization of MWCNTs and GO. More significantly, the electrical conductivity of PVA/MWCNTs/GO composite fibers enhanced by three orders of magnitude with the addition of GO.

  8. Breast cancer screening

    MedlinePlus

    Mammogram - breast cancer screening; Breast exam - breast cancer screening; MRI - breast cancer screening ... performed to screen women to detect early breast cancer when it is more likely to be cured. ...

  9. Automated campaign system

    NASA Astrophysics Data System (ADS)

    Vondran, Gary; Chao, Hui; Lin, Xiaofan; Beyer, Dirk; Joshi, Parag; Atkins, Brian; Obrador, Pere

    2006-02-01

    To run a targeted campaign involves coordination and management across numerous organizations and complex process flows. Everything from market analytics on customer databases, acquiring content and images, composing the materials, meeting the sponsoring enterprise brand standards, driving through production and fulfillment, and evaluating results; all processes are currently performed by experienced highly trained staff. Presented is a developed solution that not only brings together technologies that automate each process, but also automates the entire flow so that a novice user could easily run a successful campaign from their desktop. This paper presents the technologies, structure, and process flows used to bring this system together. Highlighted will be how the complexity of running a targeted campaign is hidden from the user through technologies, all while providing the benefits of a professionally managed campaign.

  10. Terminal automation system maintenance

    SciTech Connect

    Coffelt, D.; Hewitt, J.

    1997-01-01

    Nothing has improved petroleum product loading in recent years more than terminal automation systems. The presence of terminal automation systems (TAS) at loading racks has increased operational efficiency and safety and enhanced their accounting and management capabilities. However, like all finite systems, they occasionally malfunction or fail. Proper servicing and maintenance can minimize this. And in the unlikely event a TAS breakdown does occur, prompt and effective troubleshooting can reduce its impact on terminal productivity. To accommodate around-the-clock loading at racks, increasingly unattended by terminal personnel, TAS maintenance, servicing and troubleshooting has become increasingly demanding. It has also become increasingly important. After 15 years of trial and error at petroleum and petrochemical storage and transfer terminals, a number of successful troubleshooting programs have been developed. These include 24-hour {open_quotes}help hotlines,{close_quotes} internal (terminal company) and external (supplier) support staff, and {open_quotes}layered{close_quotes} support. These programs are described.

  11. Automated Chromosome Breakage Assessment

    NASA Technical Reports Server (NTRS)

    Castleman, Kenneth

    1985-01-01

    An automated karyotyping machine was built at JPL in 1972. It does computerized karyotyping, but it has some hardware limitations. The image processing hardware that was available at a reasonable price in 1972 was marginal, at best, for this job. In the meantime, NASA has developed an interest in longer term spaceflights and an interest in using chromosome breakage studies as a dosimeter for radiation or perhaps other damage that might occur to the tissues. This uses circulating lymphocytes as a physiological dosimeter looking for chromosome breakage on long-term spaceflights. For that reason, we have reactivated the automated karyotyping work at JPL. An update on that work, and a description of where it appears to be headed is presented.

  12. The automation of science.

    PubMed

    King, Ross D; Rowland, Jem; Oliver, Stephen G; Young, Michael; Aubrey, Wayne; Byrne, Emma; Liakata, Maria; Markham, Magdalena; Pir, Pinar; Soldatova, Larisa N; Sparkes, Andrew; Whelan, Kenneth E; Clare, Amanda

    2009-04-03

    The basis of science is the hypothetico-deductive method and the recording of experiments in sufficient detail to enable reproducibility. We report the development of Robot Scientist "Adam," which advances the automation of both. Adam has autonomously generated functional genomics hypotheses about the yeast Saccharomyces cerevisiae and experimentally tested these hypotheses by using laboratory automation. We have confirmed Adam's conclusions through manual experiments. To describe Adam's research, we have developed an ontology and logical language. The resulting formalization involves over 10,000 different research units in a nested treelike structure, 10 levels deep, that relates the 6.6 million biomass measurements to their logical description. This formalization describes how a machine contributed to scientific knowledge.

  13. Automated gas chromatography

    DOEpatents

    Mowry, C.D.; Blair, D.S.; Rodacy, P.J.; Reber, S.D.

    1999-07-13

    An apparatus and process for the continuous, near real-time monitoring of low-level concentrations of organic compounds in a liquid, and, more particularly, a water stream. A small liquid volume of flow from a liquid process stream containing organic compounds is diverted by an automated process to a heated vaporization capillary where the liquid volume is vaporized to a gas that flows to an automated gas chromatograph separation column to chromatographically separate the organic compounds. Organic compounds are detected and the information transmitted to a control system for use in process control. Concentrations of organic compounds less than one part per million are detected in less than one minute. 7 figs.

  14. Automated assembly in space

    NASA Technical Reports Server (NTRS)

    Srivastava, Sandanand; Dwivedi, Suren N.; Soon, Toh Teck; Bandi, Reddy; Banerjee, Soumen; Hughes, Cecilia

    1989-01-01

    The installation of robots and their use of assembly in space will create an exciting and promising future for the U.S. Space Program. The concept of assembly in space is very complicated and error prone and it is not possible unless the various parts and modules are suitably designed for automation. Certain guidelines are developed for part designing and for an easy precision assembly. Major design problems associated with automated assembly are considered and solutions to resolve these problems are evaluated in the guidelines format. Methods for gripping and methods for part feeding are developed with regard to the absence of gravity in space. The guidelines for part orientation, adjustments, compliances and various assembly construction are discussed. Design modifications of various fasteners and fastening methods are also investigated.

  15. Automated Assembly Center (AAC)

    NASA Technical Reports Server (NTRS)

    Stauffer, Robert J.

    1993-01-01

    The objectives of this project are as follows: to integrate advanced assembly and assembly support technology under a comprehensive architecture; to implement automated assembly technologies in the production of high-visibility DOD weapon systems; and to document the improved cost, quality, and lead time. This will enhance the production of DOD weapon systems by utilizing the latest commercially available technologies combined into a flexible system that will be able to readily incorporate new technologies as they emerge. Automated assembly encompasses the following areas: product data, process planning, information management policies and framework, three schema architecture, open systems communications, intelligent robots, flexible multi-ability end effectors, knowledge-based/expert systems, intelligent workstations, intelligent sensor systems, and PDES/PDDI data standards.

  16. The automated command transmission

    NASA Astrophysics Data System (ADS)

    Inoue, Y.; Satoh, S.

    A technique for automated command transmission (ACT) to GEO-stationed satellites is presented. The system is intended for easing the command center workload. The ACT system determines the relation of the commands to on-board units, connects the telemetry with on-board units, defines the control path on the spacecraft, identifies the correspondence of back-up units to primary units, and ascertains sunlight or eclipse conditions. The system also has the address of satellite and command decoders, the ID and content for the mission command sequence, group and inhibit codes, a listing of all available commands, and restricts the data to a command sequence. Telemetry supplies data for automated problem correction. All other missions operations are terminated during system recovery data processing after a crash. The ACT system is intended for use with the GMS spacecraft.

  17. Automated RSO Stability Analysis

    NASA Astrophysics Data System (ADS)

    Johnson, T.

    2016-09-01

    A methodology for assessing the attitude stability of a Resident Space Object (RSO) using visual magnitude data is presented and then scaled to run in an automated fashion across the entire satellite catalog. Results obtained by applying the methodology to the Commercial Space Operations Center (COMSpOC) catalog are presented and summarized, identifying objects that have changed stability. We also examine the timeline for detecting the transition from stable to unstable attitude

  18. Automation in Photogrammetry,

    DTIC Science & Technology

    1980-07-25

    Allam , 1978), and the OM-Bendix AS-lIB-X (Scarano and Bruma, 1976). The UNAMACE and GPM-2 employ analog (electronic) correlation technology. However...Survey (USGS) and the Surveys and Mapping Branch (Canada) have formed integrated systems based on the Gestalt GPM 2 (Brunson and Olson, 1978; Allam , 1978...ten years off, and the full automation of planimetric extraction may be more than 20 years in the future. REFERENCES Allam , M. M., 1978. The Role of

  19. Automated Nitrocellulose Analysis

    DTIC Science & Technology

    1978-12-01

    is acceptable. (4) As would be expected from the theory of osmosis , a high saline content in the dialysis recipient stream (countersolution) is of...Block 39, II different from Report; IS. SUPPLEMENTARY NOTES IS. KEY WOROS (Continue on rereri Analysis Automated analysis Dialysis Glyceryl...Technicon AutoAnalyzer, involves aspiration of a stirred nitrocellulose suspension, dialysis against 9 percent saline, and hydrolysis with 5N sodium

  20. Automated Cooperative Trajectories

    NASA Technical Reports Server (NTRS)

    Hanson, Curt; Pahle, Joseph; Brown, Nelson

    2015-01-01

    This presentation is an overview of the Automated Cooperative Trajectories project. An introduction to the phenomena of wake vortices is given, along with a summary of past research into the possibility of extracting energy from the wake by flying close parallel trajectories. Challenges and barriers to adoption of civilian automatic wake surfing technology are identified. A hardware-in-the-loop simulation is described that will support future research. Finally, a roadmap for future research and technology transition is proposed.

  1. Power subsystem automation study

    NASA Technical Reports Server (NTRS)

    Tietz, J. C.; Sewy, D.; Pickering, C.; Sauers, R.

    1984-01-01

    The purpose of the phase 2 of the power subsystem automation study was to demonstrate the feasibility of using computer software to manage an aspect of the electrical power subsystem on a space station. The state of the art in expert systems software was investigated in this study. This effort resulted in the demonstration of prototype expert system software for managing one aspect of a simulated space station power subsystem.

  2. Automated Microfluidics for Genomics

    DTIC Science & Technology

    2007-11-02

    the automation of it, see [4]. In the Genomation Laboratory at the Univ. of Washington (http://rcs.ee.washington.edu/GNL/genomation.html) and with Orca ...reproducible biology without contamination . The high throughput capability is competitive with large scale robotic batch processing. III. INSTRUMENTATION...essentially arbitrary low volume, and without any contact that might cause contamination . A. ACAPELLA-5K Core Processor The ACAPELLA-5K was designed with

  3. Automated RTOP Management System

    NASA Technical Reports Server (NTRS)

    Hayes, P.

    1984-01-01

    The structure of NASA's Office of Aeronautics and Space Technology electronic information system network from 1983 to 1985 is illustrated. The RTOP automated system takes advantage of existing hardware, software, and expertise, and provides: (1) computerized cover sheet and resources forms; (2) electronic signature and transmission; (3) a data-based information system; (4) graphics; (5) intercenter communications; (6) management information; and (7) text editing. The system is coordinated with Headquarters efforts in codes R,E, and T.

  4. Automated Microbial Metabolism Laboratory

    NASA Technical Reports Server (NTRS)

    1973-01-01

    Development of the automated microbial metabolism laboratory (AMML) concept is reported. The focus of effort of AMML was on the advanced labeled release experiment. Labeled substrates, inhibitors, and temperatures were investigated to establish a comparative biochemical profile. Profiles at three time intervals on soil and pure cultures of bacteria isolated from soil were prepared to establish a complete library. The development of a strategy for the return of a soil sample from Mars is also reported.

  5. Cavendish Balance Automation

    NASA Technical Reports Server (NTRS)

    Thompson, Bryan

    2000-01-01

    This is the final report for a project carried out to modify a manual commercial Cavendish Balance for automated use in cryostat. The scope of this project was to modify an off-the-shelf manually operated Cavendish Balance to allow for automated operation for periods of hours or days in cryostat. The purpose of this modification was to allow the balance to be used in the study of effects of superconducting materials on the local gravitational field strength to determine if the strength of gravitational fields can be reduced. A Cavendish Balance was chosen because it is a fairly simple piece of equipment for measuring gravity, one the least accurately known and least understood physical constants. The principle activities that occurred under this purchase order were: (1) All the components necessary to hold and automate the Cavendish Balance in a cryostat were designed. Engineering drawings were made of custom parts to be fabricated, other off-the-shelf parts were procured; (2) Software was written in LabView to control the automation process via a stepper motor controller and stepper motor, and to collect data from the balance during testing; (3)Software was written to take the data collected from the Cavendish Balance and reduce it to give a value for the gravitational constant; (4) The components of the system were assembled and fitted to a cryostat. Also the LabView hardware including the control computer, stepper motor driver, data collection boards, and necessary cabling were assembled; and (5) The system was operated for a number of periods, data collected, and reduced to give an average value for the gravitational constant.

  6. Autonomy, Automation, and Systems

    NASA Astrophysics Data System (ADS)

    Turner, Philip R.

    1987-02-01

    Aerospace industry interest in autonomy and automation, given fresh impetus by the national goal of establishing a Space Station, is becoming a major item of research and technology development. The promise of new technology arising from research in Artificial Intelligence (AI) has focused much attention on its potential in autonomy and automation. These technologies can improve performance in autonomous control functions that involve planning, scheduling, and fault diagnosis of complex systems. There are, however, many aspects of system and subsystem design in an autonomous system that impact AI applications, but do not directly involve AI technology. Development of a system control architecture, establishment of an operating system within the design, providing command and sensory data collection features appropriate to automated operation, and the use of design analysis tools to support system engineering are specific examples of major design issues. Aspects such as these must also receive attention and technology development support if we are to implement complex autonomous systems within the realistic limitations of mass, power, cost, and available flight-qualified technology that are all-important to a flight project.

  7. Automating existing stations

    SciTech Connect

    Little, J.E.

    1986-09-01

    The task was to automate 20 major compressor stations along ANR Pipeline Co.'s Southeastern and Southwestern pipelines in as many months. Meeting this schedule required standardized hardware and software design. Working with Bristol Babcock Co., ANR came up with an off-the-shelf station automation package suitable for a variety of compressor stations. The project involved 148 engines with 488,880-hp in the 20 stations. ANR Pipeline developed software for these engines and compressors, including horsepower prediction and efficiency. The system places processors ''intelligence'' at each station and engine to monitor and control operations. The station processor receives commands from the company's gas dispatch center at Detroit and informs dispatchers of alarms, conditions, and decision it makes. The automation system is controlled by the Detroit center through a central communications network. Operating orders from the center are sent to the station processor, which obeys orders using the most efficient means of operation at the station's disposal. In a malfunction, a control and communications backup system takes over. Commands and information are directly transmitted between the center and the individual compressor stations. Stations receive their orders based on throughput, with suction and discharge pressure overrides. Additionally, a discharge temperature override protects pipeline coatings.

  8. Automation of optical tweezers

    NASA Astrophysics Data System (ADS)

    Hsieh, Tseng-Ming; Chang, Bo-Jui; Hsu, Long

    2000-07-01

    Optical tweezers is a newly developed instrument, which makes possible the manipulation of micro-optical particles under a microscope. In this paper, we present the automation of an optical tweezers which consists of a modified optical tweezers, equipped with two motorized actuators to deflect a 1 W argon laser beam, and a computer control system including a joystick. The trapping of a single bead and a group of lactoacidofilus was shown, separately. With the aid of the joystick and two auxiliary cursers superimposed on the real-time image of a trapped bead, we demonstrated the simple and convenient operation of the automated optical tweezers. By steering the joystick and then pressing a button on it, we assign a new location for the trapped bead to move to. The increment of the motion 0.04 (mu) m for a 20X objective, is negligible. With a fast computer for image processing, the manipulation of the trapped bead is smooth and accurate. The automation of the optical tweezers is also programmable. This technique may be applied to accelerate the DNA hybridization in a gene chip. The combination of the modified optical tweezers with the computer control system provides a tool for precise manipulation of micro particles in many scientific fields.

  9. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.

    PubMed

    Love, Melissa S; Beasley, Federico C; Jumani, Rajiv S; Wright, Timothy M; Chatterjee, Arnab K; Huston, Christopher D; Schultz, Peter G; McNamara, Case W

    2017-02-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine

  10. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis

    PubMed Central

    Jumani, Rajiv S.; Wright, Timothy M.; Chatterjee, Arnab K.; Huston, Christopher D.; Schultz, Peter G.; McNamara, Case W.

    2017-01-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis–the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years

  11. Origin and evolution of high throughput screening

    PubMed Central

    Pereira, D A; Williams, J A

    2007-01-01

    This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100μl. A nominal 30mM source compound concentration provided high μM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for 125I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced ‘hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle. PMID:17603542

  12. Dynamic Heterogeneity of DNA Methylation and Hydroxymethylation in Embryonic Stem Cell Populations Captured by Single-Cell 3D High-Content Analysis

    PubMed Central

    Tajbakhsh, Jian; Stefanovski, Darko; Tang, George; Wawrowsky, Kolja; Liu, Naiyou; Fair, Jeffrey H.

    2015-01-01

    Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a ten-day differentiation course in vitro: by means of confocal and super-resolution imaging together with high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU per day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17:0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, DNA global methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC+/5mC−, 5hmC+/5mC+, and 5hmC−/5mC+ cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC+/5mC+ cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably delineating chromatin domains in remodeling. We

  13. Evaluation of the cytotoxicity of organic dust components on THP1 monocytes-derived macrophages using high content analysis.

    PubMed

    Ramery, Eve; O'Brien, Peter J

    2014-03-01

    Organic dust contains pathogen-associated molecular patterns (PAMPs) which can induce significant airway diseases following chronic exposure. Mononuclear phagocytes are key protecting cells of the respiratory tract. Several studies have investigated the effects of PAMPs and mainly endotoxins, on cytokine production. However the sublethal cytotoxicity of organic dust components on macrophages has not been tested yet. The novel technology of high content analysis (HCA) is already used to assess subclinical drug-induced toxicity. It combines the capabilities of flow cytometry, intracellular fluorescence probes, and image analysis and enables rapid multiple analyses in large numbers of samples. In this study, HCA was used to investigate the cytotoxicity of the three major PAMPs contained in organic dust, i.e., endotoxin (LPS), peptidoglycan (PGN) and β-glucans (zymosan) on THP-1 monocyte-derived macrophages. LPS was used at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1, and 1 μg/mL; PGN and zymosan were used at concentrations of 1, 5, 10, 50, 100, and 500 μg/mL. Cells were exposed to PAMPs for 24 h. In addition, the oxidative burst and the phagocytic capabilities of the cells were tested. An overlap between PGN intrinsic fluorescence and red/far-red fluorescent dyes occurred, rendering the evaluation of some parameters impossible for PGN. LPS induced sublethal cytotoxicity at the lowest dose (from 50 ng/mL). However, the greatest cytotoxic changes occurred with zymosan. In addition, zymosan, but not LPS, induced phagosome maturation and oxidative burst. Given the fact that β-glucans can be up to 100-fold more concentrated in organic dust than LPS, these results suggest that β-glucans could play a major role in macrophage impairment following heavy dust exposure and will merit further investigation in the near future.

  14. Towards semantic-driven high-content image analysis: an operational instantiation for mitosis detection in digital histopathology.