WorldWideScience

Sample records for automated cell-based assay

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

    International Nuclear Information System (INIS)

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

  2. Automated phantom assay system

    International Nuclear Information System (INIS)

    This paper describes an automated phantom assay system developed for assaying phantoms spiked with minute quantities of radionuclides. The system includes a computer-controlled linear-translation table that positions the phantom at exact distances from a spectrometer. A multichannel analyzer (MCA) interfaces with a computer to collect gamma spectral data. Signals transmitted between the controller and MCA synchronize data collection and phantom positioning. Measured data are then stored on disk for subsequent analysis. The automated system allows continuous unattended operation and ensures reproducible results

  3. Cell-based Assays to Identify Inhibitors of Viral Disease

    Science.gov (United States)

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong

    2009-01-01

    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  4. Radioligand assay: current trends in automation

    International Nuclear Information System (INIS)

    Using automatic equipment to perform routine RIA procedures can save time and money, improve precision, and free technologists to work on more-complex, non-routine assays. This article examines some of the instruments available for semiautomated and fully automated RIA testing. The pros and cons of automated testing are explored, and a framework for judging the true value and limitations of systems is presented

  5. Automated optical sensing system for biochemical assays

    Science.gov (United States)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  6. High content cell-based assay for the inflammatory pathway

    Science.gov (United States)

    Mukherjee, Abhishek; Song, Joon Myong

    2015-07-01

    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne;

    2005-01-01

    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...... 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...

  9. Optimising automation of a manual enzyme-linked immunosorbent assay

    OpenAIRE

    Corena de Beer; Monika Esser; Wolfgang Preiser

    2011-01-01

    Objective: Enzyme-linked immunosorbent assays (ELISAs) are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib) kit (MK016) from The Binding Site Company was optimised to be used on an automated BioRad...

  10. Optimising automation of a manual enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Corena de Beer

    2011-12-01

    Full Text Available Objective: Enzyme-linked immunosorbent assays (ELISAs are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib kit (MK016 from The Binding Site Company was optimised to be used on an automated BioRad PhD™ system in the Immunology Laboratory (National Health Laboratory Service in Tygerberg, South Africa.Methods: An automated ELISA system that uses individual well incubation was compared to a manual method that uses whole-plate incubation.Results: Results were calculated from calibration curves constructed with each assay. Marked differences in calibration curves were observed for the two methods. The automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. A comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. All incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. Several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. Calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid.Conclusion: Proper validation is vital before converting manual ELISA assays to automated or semi-automated methods. 

  11. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    Science.gov (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M

    2008-01-01

    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  12. Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

    Institute of Scientific and Technical Information of China (English)

    Jue WANG; Xin XIE

    2007-01-01

    Aim: To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR). Methods: Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptorligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated,optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results: The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L,which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified.One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK). Conclusion: This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.

  13. Automated saccharification assay for determination of digestibility in plant materials

    Directory of Open Access Journals (Sweden)

    Halpin Claire

    2010-10-01

    Full Text Available Abstract Background Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. Results We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment ( Conclusions The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

  14. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Science.gov (United States)

    Langer, Gernot

    2016-01-01

    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  15. Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays.

    Science.gov (United States)

    Johannessen, Erik A; Weaver, John M R; Bourova, Lenka; Svoboda, Petr; Cobbold, Peter H; Cooper, Jonathan M

    2002-05-01

    Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies. PMID:12033326

  16. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    OpenAIRE

    TimoYlikomi; JukkaUotila

    2011-01-01

    The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory pre-validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis, e.g., pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using six reference chemicals, which are widely used phar...

  17. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.

    Science.gov (United States)

    Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H; Xia, Menghang

    2016-04-01

    In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling. PMID:26820057

  18. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    Science.gov (United States)

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  19. A simple, versatile and sensitive cell-based assay for prions from various species.

    Directory of Open Access Journals (Sweden)

    Zaira E Arellano-Anaya

    Full Text Available Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

  20. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Science.gov (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  1. A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

    Science.gov (United States)

    Mentrup, Torben; Häsler, Robert; Fluhrer, Regina; Saftig, Paul; Schröder, Bernd

    2015-08-01

    During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes. PMID:25824657

  2. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Science.gov (United States)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  3. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Directory of Open Access Journals (Sweden)

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  4. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU

    2004-01-01

    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  5. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Institute of Scientific and Technical Information of China (English)

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU

    2005-01-01

    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  6. The automated counting of spots for the ELISpot assay.

    Science.gov (United States)

    Hawkins, Natalie; Self, Steve; Wakefield, Jon

    2006-10-20

    An automated method for counting spot-forming units in the ELISpot assay is described that uses a statistical model fit to training data that is based on counts from one or more experts. The method adapts to variable background intensities and provides considerable flexibility with respect to what image features can be used to model expert counts. Point estimates of spot counts are produced together with intervals that reflect the degree of uncertainty in the count. Finally, the approach is completely transparent and "open source" in contrast to methods embedded in current commercial software. An illustrative application to data from a study of the reactivity of T-cells from healthy human subjects to a pool of immunodominant peptides from CMV, EBV and flu is presented. PMID:17010368

  7. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  8. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna

    2013-01-01

    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  9. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Hondroulis, Evangelia; Liu Chang; Li Chenzhong, E-mail: licz@fiu.edu [Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, Miami, FL 33174 (United States)

    2010-08-06

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  10. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

    Science.gov (United States)

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua

    2015-12-01

    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  11. Multilayer-based lab-on-a-chip systems for perfused cell-based assays

    Science.gov (United States)

    Klotzbach, Udo; Sonntag, Frank; Grünzner, Stefan; Busek, Mathias; Schmieder, Florian; Franke, Volker

    2014-12-01

    A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

  12. Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

    Directory of Open Access Journals (Sweden)

    Haifeng Geng

    Full Text Available Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs, inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA activity found in patients with metachromatic leukodystrophy (MLD, a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS, detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an

  13. Automated assay of uranium solution concentration and enrichment

    International Nuclear Information System (INIS)

    For the first time, the concentration and enrichment of uranium solutions can be measured in one step. We have developed a new instrument to automatically measure the concentration and enrichment of uranium solutions through the adaptation of a commercial robot. Two identical solution enrichment systems are being installed in the Portsmouth Gaseous Diffusion Plant. These automated systems will reduce radiation exposure to personnel and increase the reliability and repeatability of the measurements. Each robotic system can process up to 40 batch and 8 priority samples in an unattended mode. Both passive gamma-ray and x-ray fluorescence (XRF) analyses are performed to determine total uranium concentration and 235U enrichment. Coded samples are read by a bar-code reader to determine measurement requirements, then assayed by either or both of the gamma-ray and XRF instruments. The robot moves the sample containers and operates all shield doors and shutters, reducing hardware complexity. If the robots is out of service, an operator can manually perform all operations

  14. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  15. Development of an automated scoring system for plant comet assay

    Directory of Open Access Journals (Sweden)

    Bertrand Pourrut

    2015-05-01

    -\tnucleus density: increase the density of nuclei is of importance to increase scoring reliability (Sharma et al., 2012. In conclusion, increasing plant nucleus extraction yield and automated scoring of nuclei do represent big challenges. However, our promising preliminary results open up the perspective of an automated high-throughput scoring of plant nuclei.

  16. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

    Directory of Open Access Journals (Sweden)

    Chan Koon H

    2010-09-01

    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  17. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan

    2011-01-01

    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

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

    OpenAIRE

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

    2015-01-01

    Background Automated Mediace Treponema pallidum latex agglutination (TPLA) and Mediace rapid plasma reagin (RPR) assays are used by many laboratories for syphilis diagnosis. This study compared the results of the traditional syphilis screening algorithm and a reverse algorithm using automated Mediace RPR or Mediace TPLA as first-line screening assays in subjects undergoing a health checkup. Methods Samples from 24,681 persons were included in this study. We routinely performed Mediace RPR and...

  19. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    Science.gov (United States)

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins. PMID:26003675

  20. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Science.gov (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  1. Automated assay data processing and quality control: A review and recommendations

    International Nuclear Information System (INIS)

    Automated data processing and quality control of assays offers not only increased speed but also a more thorough and statistically rigorous analysis of results. This review outlines the motivations, statistical definitions, and mathematical methods pertinent to assay data processing. The presentation concentrates on basic concepts rather than specific mathematical formulae. The numerous automated calibration procedures are discussed and summarized in tabular form. A comprehensive view of data processing is offered which includes much more than simple calibration and interpolation. A small number of calculator and computer programs which provide an acceptably detailed statistical analysis of assays are recommended. Finally, possible future developments in hardware and software are discussed. (author)

  2. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat

    2010-12-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  3. A Simple, Versatile and Sensitive Cell-Based Assay for Prions from Various Species

    OpenAIRE

    Arellano-Anaya, Zaira E.; Jimmy Savistchenko; Jacinthe Mathey; Alvina Huor; Caroline Lacroux; Olivier Andréoletti; Didier Vilette

    2011-01-01

    Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show ...

  4. A cell-based pharmacokinetics assay for evaluating tubulin-binding drugs.

    Science.gov (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W; Ying, Wenbin

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.

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

    NARCIS (Netherlands)

    Hodzic, J.; Dingjan, I.; Maas, M.J.M.; Meulen-Muileman, I.H. van der; Menezes, R.X. de; Heukelom, S.; Verheij, M.; Gerritsen, W.R.; Geldof, A.A.; Triest, B. van; Beusechem, V.W. van

    2015-01-01

    BACKGROUND: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit.

  6. Comparison of automated von Willebrand factor activity assays

    DEFF Research Database (Denmark)

    Timm, Annette; Hillarp, Andreas; Philips, Malou;

    2015-01-01

    INTRODUCTION: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. Measurement of von Willebrand factor (VWF) activity in plasma is often based on platelet agglutination stimulated by the ristocetin cofactor activity. Novel assays, based on latex beads with recombinant...

  7. Molecular design, synthesis and cell based HCV replicon assay of novel benzoxazole derivatives.

    Science.gov (United States)

    Ismail, M A H; Adel, M; Ismail, N S M; Abouzid, K A M

    2013-03-01

    Hepatitis C virus inhibitors based on benzoxazole scaffold were designed based on molecular modeling simulation study including docking into the NS5B polymerase active site. Several compounds showed significant high simulation docking scores relative to the assigned benzimidazole lead compound. The designed compounds were synthesized, structurally elucidated and their antiviral activity was evaluated through cell-based replicon in cultured Huh 5-2 cells. A number of the synthesized compounds showed significant inhibitory activity ranging from (52.2% inhibition up to 98% at<50 µg/mL). N-Benzyl-2-phenylbenzo[1,3]oxazole-5-carboxamide (8b) and N-Phenethyl-2-phenylbenzo[1,3] oxazole-5-carboxamide (8c) demonstrated genuine HCV inhibitory activity with EC50 values of 41.6 and 24.5 µg/mL respectively.

  8. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method...

  9. Nondestructive assay technology and automated ''real-time'' materials control

    International Nuclear Information System (INIS)

    Significant advances in nondestructive assay techniques and instrumentation now enable rapid, accurate and direct in-plant measurement of nuclear material on a continuous or ''real-time'' basis as it progresses through a nuclear facility. A variety of passive and active assay instruments are required for the broad range of materials measurement problems encountered by safeguards inspectors and facility operators in various types of nuclear plants. Representative NDA techniques and instruments are presented and reviewed with special attention to their assay capabilities and areas of applicability in the nuclear fuel cycle. An advanced system of materials control - called ''DYMAC'', for Dynamic Materials Control - is presently under development by the U.S. Energy Research and Development Administration; the DYMAC program integrates new nondestructive assay instrumentation and modern data-processing methods, with the overall objective of demonstrating a workable, cost-effective system of stringent safeguards and materials control in various generic types of facilities found in the nuclear fuel cycle. Throughout the program, emphasis will be placed on devloping practical solutions to generic measurement problems so that resulting techniques and instrumentation will have widespread utility. Projected levels of safeguards assurance, together with other vital - and cost-sensitive - plant operational factors such as process and quality control, criticality safety and waste management are examined in an evaluation of the impact of future advanced materials control systems on overall plant operations, efficiency and productivity. The task of implementing effective and stringent safeguards includes the transfer of new safeguards technology to the nuclear industry. Clearly the training of inspectors (both IAEA and national), plant people, etc., in the effective use of new NDA equipment is of paramount importance; thus in the United States, the Energy Research and Development

  10. [Development of a functional cell-based HTS assay for the identification mGluR4 modulators].

    Science.gov (United States)

    Zhang, Yaling; Bai, Yanqiu

    2009-03-01

    To identify metabotropic glutamate receptor 4 (mGluR4) modulators by Ca2+ influx assay, we developed the functional cell-based high throughput-screening (HTS) assay. The human mGluR4 cDNA was transfected into HEK-293 stably expressing promiscuous G-protein (Ga alpha15) cells. Recombinant stable mGluR4 cell line was selected under Zeocin and validated by Ca2+ influx assay. The assay was optimized on loading time of Fluo Calcium Indicator, Dimethyl sulfoxide (DMSO) tolerance and sodium hydroxide (NaOH) tolerance using agonist (L-Glutamic acid (L-Glu)) of mGluR4. The rank order of the agonist potency for the stable human mGluR4 cell line was L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) > L-Serine-O-phosphate (L-SOP) > L-Glu, and of the antagonist potency was (RS)-alpha-Methylserine-O-phosphate (MSOP) > (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG). Z' factor value of the cell line in 96- and 384-well plate format was 0.80 and 0.65. Our data indicate a successful development of functional human mGluR4 recombinant stable cell line that was suitable for high throughput screening to identify mGluR4 agonist/antagonist. PMID:19621591

  11. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

    Science.gov (United States)

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J

    2016-01-01

    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  12. Automated conductimetric assay of human serum cholinesterase activity.

    Science.gov (United States)

    Duffy, P; Wallach, J M

    1989-01-01

    Serum cholinesterase activity was determined by conductimetry using samples in the microliter range. Butyrylcholine iodide was demonstrated to be a convenient substrate for the conductimetric assay. Validation of the microassay was made by using either purified enzyme or control serum. In the range of 0-60 U/l, a linear relationship was demonstrated. Correlation with a reference spectrophotometric method was obtained with a slope of 1.18. An explanation of this value is proposed, as different hydrolysis rates were obtained with human sera, depending on the substrate used (butyrylthio- or butyryl-choline ester).

  13. Universal electronics for miniature and automated chemical assays.

    Science.gov (United States)

    Urban, Pawel L

    2015-02-21

    This minireview discusses universal electronic modules (generic programmable units) and their use by analytical chemists to construct inexpensive, miniature or automated devices. Recently, open-source platforms have gained considerable popularity among tech-savvy chemists because their implementation often does not require expert knowledge and investment of funds. Thus, chemistry students and researchers can easily start implementing them after a few hours of reading tutorials and trial-and-error. Single-board microcontrollers and micro-computers such as Arduino, Teensy, Raspberry Pi or BeagleBone enable collecting experimental data with high precision as well as efficient control of electric potentials and actuation of mechanical systems. They are readily programmed using high-level languages, such as C, C++, JavaScript or Python. They can also be coupled with mobile consumer electronics, including smartphones as well as teleinformatic networks. More demanding analytical tasks require fast signal processing. Field-programmable gate arrays enable efficient and inexpensive prototyping of high-performance analytical platforms, thus becoming increasingly popular among analytical chemists. This minireview discusses the advantages and drawbacks of universal electronic modules, considering their application in prototyping and manufacture of intelligent analytical instrumentation. PMID:25535820

  14. Nanoparticle-based assays in automated flow systems: A review

    International Nuclear Information System (INIS)

    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section

  15. Universal electronics for miniature and automated chemical assays.

    Science.gov (United States)

    Urban, Pawel L

    2015-02-21

    This minireview discusses universal electronic modules (generic programmable units) and their use by analytical chemists to construct inexpensive, miniature or automated devices. Recently, open-source platforms have gained considerable popularity among tech-savvy chemists because their implementation often does not require expert knowledge and investment of funds. Thus, chemistry students and researchers can easily start implementing them after a few hours of reading tutorials and trial-and-error. Single-board microcontrollers and micro-computers such as Arduino, Teensy, Raspberry Pi or BeagleBone enable collecting experimental data with high precision as well as efficient control of electric potentials and actuation of mechanical systems. They are readily programmed using high-level languages, such as C, C++, JavaScript or Python. They can also be coupled with mobile consumer electronics, including smartphones as well as teleinformatic networks. More demanding analytical tasks require fast signal processing. Field-programmable gate arrays enable efficient and inexpensive prototyping of high-performance analytical platforms, thus becoming increasingly popular among analytical chemists. This minireview discusses the advantages and drawbacks of universal electronic modules, considering their application in prototyping and manufacture of intelligent analytical instrumentation.

  16. Development of a high-throughput cell-based reporter assay for screening JAK3 inhibitors

    OpenAIRE

    Yin, Chang-Hong; Bach, Erika A.; Baeg, Gyeong-Hun

    2011-01-01

    JAK3 has become an ideal target for the therapeutic treatment of immune-related diseases, as well as for the prevention of organ allograft rejection. A number of JAK3 inhibitors have been identified by in vitro biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is an urgent need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we showed that in 32D/IL-2Rβ cells, STAT5 becomes phospho...

  17. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    oxidoreductase along with minor whey constituents like osteopontin, EPV20 etc. The enterocyte migration rate is a key parameter in maintaining intestinal homeostasis and intestinal repair when recovering from infection or intestinal diseases like Crohns and ulcerative colitis. We developed a novel in vitro wound...... healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  18. A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery

    OpenAIRE

    Yi Huan; Linyi Li; Quan Liu; Shuainan Liu; Zhufang Shen

    2013-01-01

    The sodium/glucose cotransporter 2 (SGLT2) is responsible for the majority of glucose reabsorption in the kidney, and currently, SGLT2 inhibitors are considered as promising hypoglycemic agents for the treatment of type 2 diabetes mellitus. By constructing CHO cell lines that stably express the human SGLT2 transmembrane protein, along with a fluorescent glucose transporter assay that uses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]2-deoxyglucose (2-NBDG) as a glucose analog, we have develo...

  19. Cell-based assays and animal models for GPCR drug screening.

    Science.gov (United States)

    Takakura, Hideo; Hattori, Mitsuru; Tanaka, Miho; Ozawa, Takeaki

    2015-01-01

    The family of G protein-coupled receptors (GPCRs) remains a central focus of basic pharmacology and drug discovery efforts. Convenient methods to assess the efficacy of potentially therapeutic reagents for GPCRs are strongly required for high-throughput screening (HTS) assay. We recently developed a rapid, sensitive, and quantitative method for detecting potential chemicals that act on GPCRs using split luciferase complementation. In principle, this is based on the detection of interactions of GPCR with β-arrestin, which translocates to the activated GPCRs. This method can facilitate the construction of HTS systems in a multi-well plate format. Particularly, the method is compatible with single-cell imaging and animal models and even deeper tissues such as organs, because of its high sensitivity, suggesting that promising candidates from HTS assay can be moved easily to the next phase for additional analysis. This system can contribute to the effective evaluation of potentially therapeutic reagents and expedite the development of new drugs for GPCRs. PMID:25563190

  20. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Directory of Open Access Journals (Sweden)

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  1. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

    Science.gov (United States)

    Fu, Ying; Lu, Danqin; Lin, Bin; Sun, Qianqian; Liu, Kai; Xu, Lili; Zhang, Shengping; Hu, Chen; Wang, Chuangui; Xu, Zhiai; Zhang, Wen

    2013-11-21

    Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

  2. Development of PDMS-based Microfluidic Device for Cell-based Assays

    Institute of Scientific and Technical Information of China (English)

    LI Chenuk-Wing; YANG Jun; TZANG Chi-Hung; YANG Meng-Su

    2004-01-01

    In a single step photolithography, muhi-level microfluidic device is fabricated by printing novel architectures on a film photomasks. The whole fabrication process is executed by classical PCB technology without the need to access clean room facilities. Different levels of protruding features on PCB master are produced by exposing a photomask with specifically arranged "windows and rims" architectures, followed by chemical wet etching. Poly(dimethylsiloxane)(PDMS) is then molded against the positive relief master to generate microfluidic device featured with multi-level sandbag structure and peripheral microchannels. This sandbag structure is an analog to traditional dam or weir for particle entrapment. The microstructure does not collapse when subjected to applied pressure, which is suitable for operation on elastic PDMS substrate.Typical immunocytochemcial staining assays were performed in the microdevice to demonstrate the applicability of the sandbag structure for cellular analysis. This simplified microfabrication process employs low-cost materials and minimal specialized equipment and can reproducibly produce mask lines with about 20 μm in width, which is sufficient for most microfluidic applications.

  3. Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

    Directory of Open Access Journals (Sweden)

    Frick A

    2015-02-01

    were generated using GraphPad Prism 6. Results: Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion: Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies. Keywords: immunomodulation, cytotoxicity, chemotherapy, precision medicine

  4. Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

    Institute of Scientific and Technical Information of China (English)

    Hong-Xia Mao; Shui-Yun Lan; Yun-Wen Hu; Li Xiang; Zheng-Hong Yuan

    2003-01-01

    AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents.METHOOS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems.RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems,and was apparently decreased after Pefabloc SC treatment.In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a.CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.

  5. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    Directory of Open Access Journals (Sweden)

    Novielli Nicole M

    2011-10-01

    Full Text Available Abstract A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells, images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P

  6. Rapid detection of significant bacteriuria by use of an automated Limulus amoebocyte lysate assay.

    OpenAIRE

    Jorgensen, J H; Alexander, G A

    1982-01-01

    Previous studies have demonstrated that significant gram-negative bacteriuria can be detected by using the Limulus amoebocyte lysate test. A series of 580 urine specimens were tested in parallel with the automated MS-2 (Abbott Laboratories) assay and with quantitative urine bacterial cultures. The overall ability of the MS-2 Limulus amoebocyte lysate test to correctly classify urine specimens as containing either greater than or equal to 10(5) organisms or less than 10(5) organisms per ml dur...

  7. Cell-based optical assay for amyloid β-induced neuronal cell dysfunction using femtosecond-pulsed laser

    Science.gov (United States)

    Lee, Seunghee; Yoon, Jonghee; Choi, Chulhee

    2015-03-01

    Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer's disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson's disease in a preclinical study.

  8. Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

    Science.gov (United States)

    Anderson, Courtney M; Zhang, Bingqing; Miller, Melanie; Butko, Emerald; Wu, Xingyong; Laver, Thomas; Kernag, Casey; Kim, Jeffrey; Luo, Yuling; Lamparski, Henry; Park, Emily; Su, Nan; Ma, Xiao-Jun

    2016-10-01

    Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201-2208, 2016. © 2016 Wiley Periodicals, Inc. PMID:27191821

  9. OpenComet: An automated tool for comet assay image analysis

    Directory of Open Access Journals (Sweden)

    Benjamin M. Gyori

    2014-01-01

    Full Text Available Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

  10. Automated Ex Situ Assays of Amyloid Formation on a Microfluidic Platform.

    Science.gov (United States)

    Saar, Kadi-Liis; Yates, Emma V; Müller, Thomas; Saunier, Séverine; Dobson, Christopher M; Knowles, Tuomas P J

    2016-02-01

    Increasingly prevalent neurodegenerative diseases are associated with the formation of nanoscale amyloid aggregates from normally soluble peptides and proteins. A widely used strategy for following the aggregation process and defining its kinetics involves the use of extrinsic dyes that undergo a spectral shift when bound to β-sheet-rich aggregates. An attractive route to carry out such studies is to perform ex situ assays, where the dye molecules are not present in the reaction mixture, but instead are only introduced into aliquots taken from the reaction at regular time intervals to avoid the possibility that the dye molecules interfere with the aggregation process. However, such ex situ measurements are time-consuming to perform, require large sample volumes, and do not provide for real-time observation of aggregation phenomena. To overcome these limitations, here we have designed and fabricated microfluidic devices that offer continuous and automated real-time ex situ tracking of the protein aggregation process. This device allows us to improve the time resolution of ex situ aggregation assays relative to conventional assays by more than one order of magnitude. The availability of an automated system for tracking the progress of protein aggregation reactions without the presence of marker molecules in the reaction mixtures opens up the possibility of routine noninvasive study of protein aggregation phenomena. PMID:26840721

  11. Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

    Directory of Open Access Journals (Sweden)

    Schmid Michael

    2004-06-01

    Full Text Available Abstract Background Noroviruses (NoV have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements. Methods We have designed a new real-time RT-PCR assay on the LightCycler (LC with SYBR Green detection and melting curve analysis (Tm to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens. Results Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens. Conclusions The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

  12. A high sensitivity, high throughput, automated single-cell gel electrophoresis ('Comet') DNA damage assay

    International Nuclear Information System (INIS)

    A fully automated microscopy machine vision image capture and analysis system for the collection of data from slides of 'comets' has been developed. The novel image processing algorithms employed in delineating the 'comet head' from the 'comet tail' allow us to determine accurately very low levels of damage. In conjunction with calibrated and automated image capture methods, we are able to eliminate operator subjectivity and analyse large numbers of cells (>2500) in a short time (<1 hour). The image processing algorithm is designed to handle particularly difficult nuclei containing a high degree of structure, due to DNA clumping. We also present techniques used to extend the assay's dynamic range by removing interfering background fluorescence and to define a region of interest. If subtle biological variations are to be quantified (e.g. cell cycle dependant damage), then the use of large cell populations is dictated. Under those circumstances, the use of a fully automated system is particularly advantageous providing that the manner in which data is extracted does not introduce any inadvertent bias. In practice, it is essential that the image processing steps are geared towards the correct recognition of an acceptable cell nucleus, i.e. comet 'head'. We acknowledge the financial support of CRUK, Programme Grant C133/A1812 - SP 2195-01/02 and the US Department of Energy Low Dose Radiation Research Program grant DE-FG07-99ER62878

  13. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    Directory of Open Access Journals (Sweden)

    Renata Baronaite

    2014-01-01

    Full Text Available Anti-nuclear antibodies (ANA have traditionally been evaluated using indirect fluorescence assays (IFA with HEp-2 cells. Quantitative immunoassays (EIA have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA, from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique patients were compared. The majority of the results were the same between the two methods (n=325, 84%; however, 8% (n=30 yielded equivocal results (equivocal-negative and equivocal-positive and 8% (n=31 yielded divergent results (positive-negative. The results showed fairly good agreement, with Cohen’s kappa value of 0.30 (95% confidence interval (CI = 0.14–0.46, which decreased to 0.23 (95% CI = 0.06–0.40 when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test.

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

    Directory of Open Access Journals (Sweden)

    Asther Marcel

    2010-07-01

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

  15. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

  16. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection. PMID:27002718

  17. Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound

    International Nuclear Information System (INIS)

    Allergic contact dermatitis is the result of an adaptive immune response of the skin to direct exposure to an allergen. Since many chemicals are also allergens, European regulations require strict screening of all ingredients in consumer products. Until recently, identifying a potential allergen has completely relied on animal testing (e.g.: Local Lymph Node Assay). In addition to the ethical problems, both the 7th Amendment to the Cosmetics Directive and REACH have stimulated the development of alternative tests for the assessment of potential sensitizers. This review is aimed at summarising the progress on cell based assays, in particular dendritic cell based assays, being developed as animal alternatives. Primary cells (CD34+ derived dendritic cells, monocyte derived dendritic cells) as well as dendritic cell-like cell lines (THP-1, U-937, MUTZ-3, KG-1, HL-60, and K562) are extensively described along with biomarkers such as cell surface markers, cytokines, chemokines and kinases. From this review, it can be concluded that no single cell based assay nor single marker is yet able to distinguish all sensitizers from non-sensitizers in a test panel of chemicals, nor is it possible to rank the sensitizing potential of the test chemicals. This suggests that sensitivity and specificity may be increased by a tiered assay approach. Only a limited number of genomic and proteomic studies have been completed until now. Such studies have the potential to identify novel biomarkers for inclusion in future assay development. Although progress is promising, this review suggests that it may be difficult to meet the up and coming European regulatory deadlines.

  18. Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials

    Institute of Scientific and Technical Information of China (English)

    HUANG Chun-mei; YU Lian-hui; PU Cheng-wei; WANG Xin; WANG Geng; SHEN Li-song; WANG Jian-zhong

    2013-01-01

    Background White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review.However,this last step is time consuming and requires experienced personnel.We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.Methods A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials.WBC differentials of 622 samples were also determined using both a cell counter and FCM.These results were then confirmed using manual microscopic methods.Results The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%.The resulting WBC differentials were well correlated between FCM and the cell counter (r >0.88,P <0.001),except in the case of basophils.Neutrophils,lymphocytes,and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r >0.80,P <0.001).The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%,respectively) were higher than those of the cell counter method (44.92% and 11.11%,respectively).The specificities of FCM were all above 85%,substantially better than those of the cell counter method.Conclusion These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

  19. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Science.gov (United States)

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-01

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  20. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

    Directory of Open Access Journals (Sweden)

    van Remmerden Yvonne

    2012-10-01

    Full Text Available Abstract Background Virus neutralizing antibodies against respiratory syncytial virus (RSV are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. Conclusions The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale vaccine clinical trials.

  1. High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1

    Science.gov (United States)

    Wang, Mengqiao; Xu, Miao; Long, Yan; Fargue, Sonia; Southall, Noel; Hu, Xin; McKew, John C.; Danpure, Christopher J.; Zheng, Wei

    2016-01-01

    Glycolate oxidase (GO) and alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular oxalate and the primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the glyoxylate metabolic pathway, we developed an indirect glycolate cytotoxicity assay in a 1,536-well plate format for high throughput screening. This assay can be used to identify compounds that reduce indirect glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate salt and colistimethate. We also developed a GO enzyme assay using the hydrogen peroxide-Amplex red reporter system. The IC50 values of potassium dichromate, sodium dichromate, and colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO enzyme assay, respectively. Further enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based assay and GO enzyme assay developed in this study are useful for further screening of large compound libraries for drug development to treat PH1. PMID:27670739

  2. Evaluation of an Automated Liquid-Handling System (Tecan Genesis RSP 100) in the Abbott LCx Assay for Chlamydia trachomatis

    OpenAIRE

    Hanson, Kevan L.; Cartwright, Charles P.

    2001-01-01

    The present study investigated the feasibility of automating the specimen-pipetting component of sample preparation in the LCx Chlamydia assay (LCx-CT assay; Abbott Laboratories, Chicago, Ill.) by using a commercially available liquid-handling system (Tecan Genesis RSP100; Tecan Inc., Research Triangle Park, N.C.). The Tecan instrument proved to be comparable in both precision and accuracy to a manual multipipettor (Eppendorf model 4850; Eppendorf Scientific, Westbury, N.Y.). The Tecan instru...

  3. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

    OpenAIRE

    van Remmerden Yvonne; Xu Fang; van Eldik Mandy; Heldens Jacco GM; Huisman Willem; Widjojoatmodjo Myra N

    2012-01-01

    Abstract Background Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results The fluorescence plaque reduction a...

  4. Tay-Sachs disease heterozygote detection: use of a centrifugal analyser for automation of hexosaminidase assays with two different artificial substrates.

    OpenAIRE

    Landels, E C; Ellis, I H; Bobrow, M; Fensom, A H

    1991-01-01

    An assay for measuring hexosaminidase A in serum and leucocytes is described in which a centrifugal analyser is used for automation of the enzyme assays after manual heat inactivation. The assay was used in a screening programme to identify heterozygotes for Tay-Sachs disease in Ashkenazi Jewish subjects in the UK. The first results from this programme indicate a carrier frequency of 1 in 27. Automation of an assay for direct measurement of hexosaminidase A in serum using 4-methyl-umbellifery...

  5. An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

    Science.gov (United States)

    Shah, Pranav; Kerns, Edward; Nguyen, Dac-Trung; Obach, R. Scott; Wang, Amy Q.; Zakharov, Alexey; McKew, John; Simeonov, Anton; Hop, Cornelis E. C. A.

    2016-01-01

    Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes. PMID:27417180

  6. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    OpenAIRE

    Ruchy Jain; Orawan Monthakantirat; Parkpoom Tengamnuay; Wanchai De-Eknamkul

    2014-01-01

    Avicennia marina (AM) exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R) [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT) causing androgenic alopecia (AGA). An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs), the main regulator of ha...

  7. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    NARCIS (Netherlands)

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    2015-01-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  8. Searching for anti-prion compounds: cell-based high-throughput in vitro assays and animal testing strategies.

    Science.gov (United States)

    Kocisko, David A; Caughey, Byron

    2006-01-01

    The transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurodegenerative diseases of mammals. Protease-resistant prion protein (PrP-res) is only associated with TSEs and thus has been a target for therapeutic intervention. The most effective compounds known against scrapie in vivo are inhibitors of PrP-res in infected cells. Mouse neuroblastoma (N2a) cells have been chronically infected with several strains of mouse scrapie including RML and 22L. Also, rabbit epithelial cells that produce sheep prion protein in the presence of doxycycline (Rov9) have been infected with sheep scrapie. Here a high-throughput 96-well plate PrP-res inhibition assay is described for each of these scrapie-infected cell lines. With this dot-blot assay, thousands of compounds can easily be screened for inhibition of PrP-res formation. This assay is designed to find new PrP-res inhibitors, which may make good candidates for in vivo anti-scrapie testing. However, an in vitro assay can only suggest that a given compound might have in vivo anti-scrapie activity, which is typically measured as increased survival times. Methods for in vivo testing of compounds for anti-scrapie activity in transgenic mice, a much more lengthy and expensive process, are also discussed. PMID:17046661

  9. Evaluation of a CLEIA automated assay system for the detection of a panel of tumor markers.

    Science.gov (United States)

    Falzarano, Renato; Viggiani, Valentina; Michienzi, Simona; Longo, Flavia; Tudini, Silvestra; Frati, Luigi; Anastasi, Emanuela

    2013-10-01

    Tumor markers are commonly used to detect a relapse of disease in oncologic patients during follow-up. It is important to evaluate new assay systems for a better and more precise assessment, as a standardized method is currently lacking. The aim of this study was to assess the concordance between an automated chemiluminescent enzyme immunoassay system (LUMIPULSE® G1200) and our reference methods using seven tumor markers. Serum samples from 787 subjects representing a variety of diagnoses, including oncologic, were analyzed using LUMIPULSE® G1200 and our reference methods. Serum values were measured for the following analytes: prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and cytokeratin 19 fragment (CYFRA 21-1). For the determination of CEA, AFP, and PSA, an automatic analyzer based on chemiluminescence was applied as reference method. To assess CYFRA 21-1, CA125, CA19-9, and CA15-3, an immunoradiometric manual system was employed. Method comparison by Passing-Bablok analysis resulted in slopes ranging from 0.9728 to 1.9089 and correlation coefficients from 0.9977 to 0.9335. The precision of each assay was assessed by testing six serum samples. Each sample was analyzed for all tumor biomarkers in duplicate and in three different runs. The coefficients of variation were less than 6.3 and 6.2 % for within-run and between-run variation, respectively. Our data suggest an overall good interassay agreement for all markers. The comparison with our reference methods showed good precision and reliability, highlighting its usefulness in clinical laboratory's routine.

  10. Evaluation of a CLEIA automated assay system for the detection of a panel of tumor markers.

    Science.gov (United States)

    Falzarano, Renato; Viggiani, Valentina; Michienzi, Simona; Longo, Flavia; Tudini, Silvestra; Frati, Luigi; Anastasi, Emanuela

    2013-10-01

    Tumor markers are commonly used to detect a relapse of disease in oncologic patients during follow-up. It is important to evaluate new assay systems for a better and more precise assessment, as a standardized method is currently lacking. The aim of this study was to assess the concordance between an automated chemiluminescent enzyme immunoassay system (LUMIPULSE® G1200) and our reference methods using seven tumor markers. Serum samples from 787 subjects representing a variety of diagnoses, including oncologic, were analyzed using LUMIPULSE® G1200 and our reference methods. Serum values were measured for the following analytes: prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and cytokeratin 19 fragment (CYFRA 21-1). For the determination of CEA, AFP, and PSA, an automatic analyzer based on chemiluminescence was applied as reference method. To assess CYFRA 21-1, CA125, CA19-9, and CA15-3, an immunoradiometric manual system was employed. Method comparison by Passing-Bablok analysis resulted in slopes ranging from 0.9728 to 1.9089 and correlation coefficients from 0.9977 to 0.9335. The precision of each assay was assessed by testing six serum samples. Each sample was analyzed for all tumor biomarkers in duplicate and in three different runs. The coefficients of variation were less than 6.3 and 6.2 % for within-run and between-run variation, respectively. Our data suggest an overall good interassay agreement for all markers. The comparison with our reference methods showed good precision and reliability, highlighting its usefulness in clinical laboratory's routine. PMID:23775009

  11. Establishment and validation of whole-cell based fluorescence assays to identify anti-mycobacterial compounds using the Acanthamoeba castellanii-Mycobacterium marinum host-pathogen system.

    Directory of Open Access Journals (Sweden)

    Sébastien Kicka

    Full Text Available Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.

  12. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    Science.gov (United States)

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  13. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    Science.gov (United States)

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm.

  14. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol.

    Science.gov (United States)

    Schulpen, Sjors H W; de Jong, Esther; de la Fonteyne, Liset J J; de Klerk, Arja; Piersma, Aldert H

    2015-04-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as βIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of βIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.

  15. Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson's disease and in hepatic encephalopathy.

    Science.gov (United States)

    Siotto, Mariacristina; Pasqualetti, Patrizio; Marano, Massimo; Squitti, Rosanna

    2014-10-01

    Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.

  16. Impedance sensor technology for cell-based assays in the framework of a high-content screening system

    International Nuclear Information System (INIS)

    Living cultured cells react to external influences, such as pharmaceutical agents, in an intricate manner due to their complex internal signal processing. Impedance sensing of cells on microelectrodes is a favored label-free technology to indicate cellular events, usually ascribed to morphologic alteration or changes in cellular adhesion, which is usually found in stand-alone systems that do not incorporate life support or additional sensor systems. However, only in symbiosis with metabolic activity sensing and picture documentation may a complete insight into cellular vitality be provided. This complement was created within the framework of an automated high-content screening system previously developed by our group, monitoring 24 cell culture chambers in parallel. The objective of this paper is the development of miniaturized electronics for impedance measurements and its system integration as a modular unit. In addition, it is shown how sensor electrodes were optimized by impedance matching such that spectroscopy and raw data analysis become feasible for every culture well. Undesired mechanical stress on cultured cells may arise from the medium and agent support system of the autonomous screening apparatus. This paper demonstrates how this hazard is treated with the simulation of microfluidics and impedance measurements. Physiological data are subsequently derived from the exemplary tumor cell line MCF-7 both during treatment with the agent doxorubicin and through the impact of natural killer cells. This correlates the information content of complex impedance spectra with cellular respiration as well as data from microscopy

  17. Processing nanoparticles with A4F-SAXS for toxicological studies: Iron oxide in cell-based assays.

    Science.gov (United States)

    Knappe, Patrick; Boehmert, Linda; Bienert, Ralf; Kamutzki, Silvana; Karmutzki, Silvana; Niemann, Birgit; Lampen, Alfonso; Thünemann, Andreas F

    2011-07-01

    Nanoparticles are not typically ready-to-use for in vitro cell culture assays. Prior to their use in assays, powder samples containing nanoparticles must be dispersed, de-agglomerated, fractionated by size, and characterized with respect to size and size distribution. For this purpose we report exemplarily on polyphosphate-stabilized iron oxide nanoparticles in aqueous suspension. Fractionation and online particle size analysis was performed in a time-saving procedure lasting 50 min by combining asymmetrical flow field-flow fractionation (A4F) and small-angle X-ray scattering (SAXS). Narrowly distributed nanoparticle fractions with radii of gyration (R(g)) from 7 to 21 nm were obtained from polydisperse samples. The A4F-SAXS combination is introduced for the preparation of well-characterized sample fractions originating from a highly polydisperse system as typically found in engineered nanoparticles. A4F-SAXS processed particles are ready-to-use for toxicological studies. The results of preliminary tests of the effects of fractionated iron oxide nanoparticles with a R(g) of 15 nm on a human colon model cell line are reported.

  18. Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation.

    Science.gov (United States)

    Shinde, Vaibhav; Klima, Stefanie; Sureshkumar, Perumal Srinivasan; Meganathan, Kesavan; Jagtap, Smita; Rempel, Eugen; Rahnenführer, Jörg; Hengstler, Jan Georg; Waldmann, Tanja; Hescheler, Jürgen; Leist, Marcel; Sachinidis, Agapios

    2015-01-01

    Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically. PMID:26132533

  19. A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

    Science.gov (United States)

    Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

    2015-12-01

    Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. PMID:26584897

  20. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others

    2015-08-15

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  1. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    International Nuclear Information System (INIS)

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced

  2. High-throughput sample processing and sample management; the functional evolution of classical cytogenetic assay towards automation.

    Science.gov (United States)

    Ramakumar, Adarsh; Subramanian, Uma; Prasanna, Pataje G S

    2015-11-01

    High-throughput individual diagnostic dose assessment is essential for medical management of radiation-exposed subjects after a mass casualty. Cytogenetic assays such as the Dicentric Chromosome Assay (DCA) are recognized as the gold standard by international regulatory authorities. DCA is a multi-step and multi-day bioassay. DCA, as described in the IAEA manual, can be used to assess dose up to 4-6 weeks post-exposure quite accurately but throughput is still a major issue and automation is very essential. The throughput is limited, both in terms of sample preparation as well as analysis of chromosome aberrations. Thus, there is a need to design and develop novel solutions that could utilize extensive laboratory automation for sample preparation, and bioinformatics approaches for chromosome-aberration analysis to overcome throughput issues. We have transitioned the bench-based cytogenetic DCA to a coherent process performing high-throughput automated biodosimetry for individual dose assessment ensuring quality control (QC) and quality assurance (QA) aspects in accordance with international harmonized protocols. A Laboratory Information Management System (LIMS) is designed, implemented and adapted to manage increased sample processing capacity, develop and maintain standard operating procedures (SOP) for robotic instruments, avoid data transcription errors during processing, and automate analysis of chromosome-aberrations using an image analysis platform. Our efforts described in this paper intend to bridge the current technological gaps and enhance the potential application of DCA for a dose-based stratification of subjects following a mass casualty. This paper describes one such potential integrated automated laboratory system and functional evolution of the classical DCA towards increasing critically needed throughput.

  3. High-throughput sample processing and sample management; the functional evolution of classical cytogenetic assay towards automation.

    Science.gov (United States)

    Ramakumar, Adarsh; Subramanian, Uma; Prasanna, Pataje G S

    2015-11-01

    High-throughput individual diagnostic dose assessment is essential for medical management of radiation-exposed subjects after a mass casualty. Cytogenetic assays such as the Dicentric Chromosome Assay (DCA) are recognized as the gold standard by international regulatory authorities. DCA is a multi-step and multi-day bioassay. DCA, as described in the IAEA manual, can be used to assess dose up to 4-6 weeks post-exposure quite accurately but throughput is still a major issue and automation is very essential. The throughput is limited, both in terms of sample preparation as well as analysis of chromosome aberrations. Thus, there is a need to design and develop novel solutions that could utilize extensive laboratory automation for sample preparation, and bioinformatics approaches for chromosome-aberration analysis to overcome throughput issues. We have transitioned the bench-based cytogenetic DCA to a coherent process performing high-throughput automated biodosimetry for individual dose assessment ensuring quality control (QC) and quality assurance (QA) aspects in accordance with international harmonized protocols. A Laboratory Information Management System (LIMS) is designed, implemented and adapted to manage increased sample processing capacity, develop and maintain standard operating procedures (SOP) for robotic instruments, avoid data transcription errors during processing, and automate analysis of chromosome-aberrations using an image analysis platform. Our efforts described in this paper intend to bridge the current technological gaps and enhance the potential application of DCA for a dose-based stratification of subjects following a mass casualty. This paper describes one such potential integrated automated laboratory system and functional evolution of the classical DCA towards increasing critically needed throughput. PMID:26520383

  4. An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening

    Directory of Open Access Journals (Sweden)

    Ruan Ke-He

    2011-02-01

    Full Text Available Abstract Background Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC. In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E1 (PGE1 and E2 (PGE2 can be identified in the water-soluble non-purified fraction. PGE1 is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE2 is an inflammatory molecule. Methods We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney. PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements were screened. Results After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration using fluorescence microscopy. Conclusions Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using

  5. [Use of procalcitonine in intensive care units: comparison of semi quantitative PCT-Q Brahms assay with automated PCT-Kryptor assay].

    Science.gov (United States)

    Schuch, Géraldine; Duc-Marchand, Catherine; Venet, Cyrille; Mann, Hubert; Tixier, Anne; Bionda, Clara

    2011-01-01

    Procalcitonine (PCT) is recognized as a major and specific biomarker in diagnosis of bacterial infection. Used early in sepsis, it allows immediate administration of antibiotics and monitoring its effectiveness. Confronted on systemic inflammation response syndrom (SIRS), physicians must react quickly and effectively to evaluate bacterial infection and sepsis. The objective of this study was to compare analytical and clinical performances of semi-quantitative PCT-Q assay (Brahms) with quantitative and automated assay such on Kryptor (Brahms). Fifty blood samples of intensive care patients were compared. The analytical performance observed with PCT-Q assay is accurate: linear ratio kappa of 0.912 (95% CI 0.61, 0.97) and a good correlation between these techniques (p < 0.0001) (MedCalc software) were observed. Three discordances were observed and confirm the difficulties of reading for values close to 0.5 ng/mL. For these patients, PCT result showed its interest to discriminate local infection of a sepsis, to stop antibiotherapy with broad spectrum and to consolidate a therapeutic effectiveness in multi-visceral failure context. The semi-quantitative assay seems adapted for a fast and reliable evaluation of PCT in a general-purpose laboratory, not requiring neither dedicated analyzer, nor complex technicality but a control of the visual evaluation of results. It could be used for diagnosis of sepsis without monitoring precisely therapeutic follow-up.

  6. [Use of procalcitonine in intensive care units: comparison of semi quantitative PCT-Q Brahms assay with automated PCT-Kryptor assay].

    Science.gov (United States)

    Schuch, Géraldine; Duc-Marchand, Catherine; Venet, Cyrille; Mann, Hubert; Tixier, Anne; Bionda, Clara

    2011-01-01

    Procalcitonine (PCT) is recognized as a major and specific biomarker in diagnosis of bacterial infection. Used early in sepsis, it allows immediate administration of antibiotics and monitoring its effectiveness. Confronted on systemic inflammation response syndrom (SIRS), physicians must react quickly and effectively to evaluate bacterial infection and sepsis. The objective of this study was to compare analytical and clinical performances of semi-quantitative PCT-Q assay (Brahms) with quantitative and automated assay such on Kryptor (Brahms). Fifty blood samples of intensive care patients were compared. The analytical performance observed with PCT-Q assay is accurate: linear ratio kappa of 0.912 (95% CI 0.61, 0.97) and a good correlation between these techniques (p < 0.0001) (MedCalc software) were observed. Three discordances were observed and confirm the difficulties of reading for values close to 0.5 ng/mL. For these patients, PCT result showed its interest to discriminate local infection of a sepsis, to stop antibiotherapy with broad spectrum and to consolidate a therapeutic effectiveness in multi-visceral failure context. The semi-quantitative assay seems adapted for a fast and reliable evaluation of PCT in a general-purpose laboratory, not requiring neither dedicated analyzer, nor complex technicality but a control of the visual evaluation of results. It could be used for diagnosis of sepsis without monitoring precisely therapeutic follow-up. PMID:22123565

  7. Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

    Science.gov (United States)

    Kutilek, Victoria D; Andrews, Christine L; Richards, Matthew P; Xu, Zangwei; Sun, Tianxiao; Chen, Yiping; Hashke, Andrew; Smotrov, Nadya; Fernandez, Rafael; Nickbarg, Elliott B; Chamberlin, Chad; Sauvagnat, Berengere; Curran, Patrick J; Boinay, Ryan; Saradjian, Peter; Allen, Samantha J; Byrne, Noel; Elsen, Nathaniel L; Ford, Rachael E; Hall, Dawn L; Kornienko, Maria; Rickert, Keith W; Sharma, Sujata; Shipman, Jennifer M; Lumb, Kevin J; Coleman, Kevin; Dandliker, Peter J; Kariv, Ilona; Beutel, Bruce

    2016-07-01

    The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway. PMID:26969322

  8. Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

    Science.gov (United States)

    Jang, Mi Seon; Sahastrabuddhe, Sushant; Yun, Cheol-Heui; Han, Seung Hyun; Yang, Jae Seung

    2016-08-01

    Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P typhoid vaccines.

  9. Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

    Science.gov (United States)

    Jang, Mi Seon; Sahastrabuddhe, Sushant; Yun, Cheol-Heui; Han, Seung Hyun; Yang, Jae Seung

    2016-08-01

    Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines. PMID:27216239

  10. Fluorescence-PCR Assays and Isolation of Luminescent Bacterial Clones Using an Automated Plate Reader

    Science.gov (United States)

    Crowley, Thomas E.

    2011-01-01

    The genes responsible for luminescence in various species of the marine microorganism "Photobacterium", have been used for many years as a tool by researchers and instructors. In particular, the "lux" operon of "Photobacterium fischeri" has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate…

  11. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    DEFF Research Database (Denmark)

    Baronaite, Renata; Engelhart, Merete; Mørk Hansen, Troels;

    2014-01-01

    using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique...

  12. Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay.

    Science.gov (United States)

    Chen, Catherine Z; Southall, Noel; Xiao, Jingbo; Marugan, Juan J; Ferrer, Marc; Hu, Xin; Jones, Raisa E; Feng, Shu; Agoulnik, Irina U; Zheng, Wei; Agoulnik, Alexander I

    2013-07-01

    The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  13. Avicequinone C isolated from Avicennia marina exhibits 5α-reductase-type 1 inhibitory activity using an androgenic alopecia relevant cell-based assay system.

    Science.gov (United States)

    Jain, Ruchy; Monthakantirat, Orawan; Tengamnuay, Parkpoom; De-Eknamkul, Wanchai

    2014-01-01

    Avicennia marina (AM) exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R) [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT) causing androgenic alopecia (AGA). An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs), the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC) detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1) inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C. PMID:24858268

  14. Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

    Science.gov (United States)

    Yamashita, Atsuya; Fujimoto, Yuusuke; Tamaki, Mayumi; Setiawan, Andi; Tanaka, Tomohisa; Okuyama-Dobashi, Kaori; Kasai, Hirotake; Watashi, Koichi; Wakita, Takaji; Toyama, Masaaki; Baba, Masanori; de Voogd, Nicole J; Maekawa, Shinya; Enomoto, Nobuyuki; Tanaka, Junichi; Moriishi, Kohji

    2015-11-01

    The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs. PMID:26561821

  15. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    Directory of Open Access Journals (Sweden)

    Ruchy Jain

    2014-05-01

    Full Text Available Avicennia marina (AM exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT causing androgenic alopecia (AGA. An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs, the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1 inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C.

  16. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    (STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains...

  17. Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

    Science.gov (United States)

    Börner, Tim; Grey, Carl; Adlercreutz, Patrick

    2016-08-01

    Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

  18. OpenComet: An automated tool for comet assay image analysis

    OpenAIRE

    Gyori, Benjamin M.; Gireedhar Venkatachalam; Thiagarajan, P. S.; David Hsu; Marie-Veronique Clement

    2014-01-01

    Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires ...

  19. Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay.

    Science.gov (United States)

    Hu, Zongyi; Hu, Xin; He, Shanshan; Yim, Hyung Joon; Xiao, Jingbo; Swaroop, Manju; Tanega, Cordelle; Zhang, Ya-qin; Yi, Guanghui; Kao, C Cheng; Marugan, Juan; Ferrer, Marc; Zheng, Wei; Southall, Noel; Liang, T Jake

    2015-12-01

    Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells. PMID:26515788

  20. Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay

    Directory of Open Access Journals (Sweden)

    Atsuya Yamashita

    2015-11-01

    Full Text Available The current treatments of chronic hepatitis B (CHB face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV. We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95% and low cytotoxicity (66% to 77%. Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy-phenol (compound 1 and 3,4,5-tribromo-2-(2,4-dibromophenoxy-phenol (compound 2, which are classified as polybrominated diphenyl ethers (PBDEs, were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

  1. Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis.

    Science.gov (United States)

    Nolte, Frederick S; Rogers, Beverly B; Tang, Yi-Wei; Oberste, M Steven; Robinson, Christine C; Kehl, K Sue; Rand, Kenneth A; Rotbart, Harley A; Romero, Jose R; Nyquist, Ann-Christine; Persing, David H

    2011-02-01

    Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.

  2. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available 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.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.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.

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

    Science.gov (United States)

    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

  4. Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine.

    Science.gov (United States)

    Said, R; Robinet, D; Barbier, C; Sartre, J; Huguet, C

    1990-08-24

    A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%.

  5. Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine.

    Science.gov (United States)

    Said, R; Robinet, D; Barbier, C; Sartre, J; Huguet, C

    1990-08-24

    A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%. PMID:2277100

  6. Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

    Directory of Open Access Journals (Sweden)

    Kramer Marianne F

    2007-10-01

    Full Text Available Abstract An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.

  7. Automated direct assay system for RU38486, an antiprogesterone-antiglucocorticoid agent, and its metabolites using high performance liquid chromatography.

    Directory of Open Access Journals (Sweden)

    Nagoshi,Kazusuke

    1991-04-01

    Full Text Available An automated direct assay system using high performance liquid chromatography was developed for the measurement of RU38486 and its three metabolites (RU42698, RU42848, RU42633 in human serum. Serum concentrations of these compounds were measured up to 144 h following single oral administration of 200 (200 mg group, n = 3 or 400 mg (400 mg group, n = 3 of RU38486 to healthy female volunteers. The serum half-lives (200 mg group-400 mg group of RU38486, RU42698, RU42848 and RU42633 were 31.8-33.1 h, 41.2-39.3 h, 33.9-36.6 h and 29.2-36.6 h, respectively. Our system could quantify them easily and simultaneously, and was considered to be valuable in studies on the relationship between the pharmacokinetics and the clinical effects of RU38486.

  8. Automated direct high-performance liquid chromatographic assay for estetrol, estriol, cortisone and cortisol in serum and amniotic fluid.

    Science.gov (United States)

    Noma, J; Hayashi, N; Sekiba, K

    1991-07-17

    An automated direct assay for the simultaneous determination of unconjugated estetrol, estriol, cortisone and cortisol in serum and amniotic fluid, using high-performance liquid chromatography with electrochemical detection and ultraviolet detection, has been developed. The analysis time is ca. 1 h. This system offers good reproducibility with low coefficients of variation (estetrol, 2.3%; estriol, 2.3%; cortisone, 2.6%; cortisol, 1.9%). Detection limits are low enough for routine determinations (estetrol and estriol, 150 pg; cortisone and cortisol, 5 ng). Comparison of the values measured by the present method and by radioimmunoassay revealed significant correlations for estetrol (r = 0.787, p less than 0.01), estriol (r = 0.957, p less than 0.01), cortisone (r = 0.956, p less than 0.01) and cortisol (r = 0.865, p less than 0.01). This system proved to be valuable in monitoring feto-placental function. PMID:1770108

  9. An automated electrophysiological assay for differentiating Ca(v)2.2 inhibitors based on state dependence and kinetics.

    Science.gov (United States)

    Swensen, Andrew M; Niforatos, Wende; Vortherms, Timothy A; Perner, Richard J; Li, Tao; Schrimpf, Michael R; Scott, Victoria E; Lee, Lance; Jarvis, Michael F; McGaraughty, Steve

    2012-12-01

    Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes. PMID:22428804

  10. Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

    OpenAIRE

    Pourmand, Nader; Caramuta, Stefano; Villablanca, Andrea; Mori, Silvia; Karhanek, Miloslav; Wang, Shan X.; Ronald W Davis

    2007-01-01

    The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics ...

  11. Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

    Science.gov (United States)

    Galanti, L M; Dell'Omo, J; Wanet, B; Guarin, J L; Jamart, J; Garrino, M G; Masson, P L; Cambiaso, C L

    1997-09-24

    An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

  12. Detection of rubella-specific immunoglobulin G: comparison of the enzyme-linked immunosorbent assay and an automated microparticle enzyme immunoassay (IMx).

    OpenAIRE

    Skurrie, I J; Head, J L; Garland, S M

    1991-01-01

    An automated microparticle enzyme immunoassay (IMx Rubella IgG Antibody Assay; Abbott Laboratories, North Chicago, Ill.) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for detection of rubella-specific immunoglobulin G (IgG) in 400 consecutive antenatal patients. There was complete agreement between the two tests in this population, which had a positivity rate of 99% for rubella-specific IgG antibodies. The performance of the IMx was also evaluated at the cutoff zo...

  13. Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

    Science.gov (United States)

    Ramos, Inês I; Gregório, Bruno J R; Barreiros, Luísa; Magalhães, Luís M; Tóth, Ildikó V; Reis, Salette; Lima, José L F C; Segundo, Marcela A

    2016-04-01

    An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were high sampling frequency (29 h(-1)), low operating costs and low generation of waste. PMID:26838448

  14. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5 ' nuclease PCR assay

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    included assays for the detection of verocytotoxin genes (vtx1, vtx2), pO157-associated genes (ehxA, katP, espP, and etpD), a recently identified adhesin (saa), intimin (eae, all variants), seven subtypes of eae, four subtypes of tir, and three subtypes of espD. A number of reference strains (VTEC and......In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization...... of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5' nuclease PCR assay (real-time PCR) that targets vtx1 and vtx2. VTEC isolates were obtained by colony replication by use of...

  15. Performance of the new automated Abbott RealTime MTB assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens.

    Science.gov (United States)

    Chen, J H K; She, K K K; Kwong, T-C; Wong, O-Y; Siu, G K H; Leung, C-C; Chang, K-C; Tam, C-M; Ho, P-L; Cheng, V C C; Yuen, K-Y; Yam, W-C

    2015-09-01

    The automated high-throughput Abbott RealTime MTB real-time PCR assay has been recently launched for Mycobacterium tuberculosis complex (MTBC) clinical diagnosis. This study would like to evaluate its performance. We first compared its diagnostic performance with the Roche Cobas TaqMan MTB assay on 214 clinical respiratory specimens. Prospective analysis of a total 520 specimens was then performed to further evaluate the Abbott assay. The Abbott assay showed a lower limit of detection at 22.5 AFB/ml, which was more sensitive than the Cobas assay (167.5 AFB/ml). The two assays demonstrated a significant difference in diagnostic performance (McNemar's test; P = 0.0034), in which the Abbott assay presented significantly higher area under curve (AUC) than the Cobas assay (1.000 vs 0.880; P = 0.0002). The Abbott assay demonstrated extremely low PCR inhibition on clinical respiratory specimens. The automated Abbott assay required only very short manual handling time (0.5 h), which could help to improve the laboratory management. In the prospective analysis, the overall estimates for sensitivity and specificity of the Abbott assay were both 100 % among smear-positive specimens, whereas the smear-negative specimens were 96.7 and 96.1 %, respectively. No cross-reactivity with non-tuberculosis mycobacterial species was observed. The superiority in sensitivity of the Abbott assay for detecting MTBC in smear-negative specimens could further minimize the risk in MTBC false-negative detection. The new Abbott RealTime MTB assay has good diagnostic performance which can be a useful diagnostic tool for rapid MTBC detection in clinical laboratories.

  16. Automated low dose assay system for survival measurements of mammalian cells in vitro

    International Nuclear Information System (INIS)

    It has been suggested that at low doses of radiation both surviving cells (S) and inactivated cells (K) should be identified to obtain accurate data. One way to achieve this is to microscopically examine individual cells attached to a culture vessel, record their positions and observe and classify subsequent cell growth. In this way most systematic errors (counting, pipetting, diluting, etc.) are eliminated and since both S and K cells are scored, statistical accuracy (binomial) is also improved. For this purpose the authors have developed a semi automatic low dose assay system (ALDAS) whereby a microscope state was modified and equipped with two stepping motors under computer control. The computer automatically scans tissue culture flasks in which cells were plated after irradiation. When a cell is observed, the operator assumes command of the stage monitor, centres the cell in the field of view using a ''joystick'' control and signals the computer to record the cell's X-Y coordinates. After one week of incubation each cell location is revisited automatically and the operator scores the cells as S or K

  17. Implementation and development of an automated, ultra-high-capacity, acoustic, flexible dispensing platform for assay-ready plate delivery.

    Science.gov (United States)

    Griffith, Dylan; Northwood, Roger; Owen, Paul; Simkiss, Ellen; Brierley, Andrew; Cross, Kevin; Slaney, Andrew; Davis, Miranda; Bath, Colin

    2012-10-01

    Compound management faces the daily challenge of providing high-quality samples to drug discovery. The advent of new screening technologies has seen demand for liquid samples move toward nanoliter ranges, dispensed by contactless acoustic droplet ejection. Within AstraZeneca, a totally integrated assay-ready plate production platform has been created to fully exploit the advantages of this technology. This enables compound management to efficiently deliver large throughputs demanded by high-throughput screening while maintaining regular delivery of smaller numbers of compounds in varying plate formats for cellular or biochemical concentration-response curves in support of hit and lead optimization (structure-activity relationship screening). The automation solution, CODA, has the capability to deliver compounds on demand for single- and multiple-concentration ranges, in batch sizes ranging from 1 sample to 2 million samples, integrating seamlessly into local compound and test management systems. The software handles compound orders intelligently, grouping test requests together dependent on output plate type and serial dilution ranges so that source compound vessels are shared among numerous tests, ensuring conservation of sample, reduced labware and costs, and efficiency of work cell logistics. We describe the development of CODA to address the customer demand, challenges experienced, learning made, and subsequent enhancements. PMID:22922543

  18. An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum.

    Science.gov (United States)

    Chhatre, Sunil; Francis, Richard; Bracewell, Daniel G; Titchener-Hooker, Nigel J

    2010-11-15

    The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.

  19. In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

    Science.gov (United States)

    Hayashi, M; MacGregor, J T; Gatehouse, D G; Adler, I D; Blakey, D H; Dertinger, S D; Krishna, G; Morita, T; Russo, A; Sutou, S

    2000-01-01

    An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature

  20. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    Science.gov (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P HBcrAg assays.

  1. Additive mixture effects of estrogenic chemicals in human cell-based assays can be influenced by inclusion of chemicals with differing effect profiles.

    Directory of Open Access Journals (Sweden)

    Richard Mark Evans

    Full Text Available A growing body of experimental evidence indicates that the in vitro effects of mixtures of estrogenic chemicals can be well predicted from the estrogenicity of their components by the concentration addition (CA concept. However, some studies have observed small deviations from CA. Factors affecting the presence or observation of deviations could include: the type of chemical tested; number of mixture components; mixture design; and assay choice. We designed mixture experiments that address these factors, using mixtures with high numbers of components, chemicals from diverse chemical groups, assays with different in vitro endpoints and different mixture designs and ratios. Firstly, the effects of mixtures composed of up to 17 estrogenic chemicals were examined using estrogenicity assays with reporter-gene (ERLUX and cell proliferation (ESCREEN endpoints. Two mixture designs were used: 1 a 'balanced' design with components present in proportion to a common effect concentration (e.g. an EC(10 and 2 a 'non-balanced' design with components in proportion to potential human tissue concentrations. Secondly, the individual and simultaneous ability of 16 potential modulator chemicals (each with minimal estrogenicity to influence the assay outcome produced by a reference mixture of estrogenic chemicals was examined. Test chemicals included plasticizers, phthalates, metals, PCBs, phytoestrogens, PAHs, heterocyclic amines, antioxidants, UV filters, musks, PBDEs and parabens. In all the scenarios tested, the CA concept provided a good prediction of mixture effects. Modulation studies revealed that chemicals possessing minimal estrogenicity themselves could reduce (negatively modulate the effect of a mixture of estrogenic chemicals. Whether the type of modulation we observed occurs in practice most likely depends on the chemical concentrations involved, and better information is required on likely human tissue concentrations of estrogens and of potential

  2. Automated immunohistochemical assay for estrogen receptor status in breast cancer using monoclonal antibody CC4-5 on the Ventana ES.

    Science.gov (United States)

    Nichols, G E; Frierson, H F; Boyd, J C; Hanigan, M H

    1996-09-01

    Determination of breast cancer estrogen receptor (ER) status as a predictor of tumor response to adjuvant endocrine therapy remains a mainstay of breast cancer management. Recent second generation anti-ER antibodies and new epitope retrieval methods have produced paraffin-based immunohistochemical results that correlate closely with the dextran-coated charcoal (DCC) assay and appear to represent a superior method of ER assay. The authors determined the ER status of 103 invasive breast cancers by paraffin-based, automated immunohistochemistry on the Ventana ES using a new monoclonal antibody, CC4-5, and compared the results to those of parallel DCC biochemical analysis and manual immunohistochemical analysis using anti-ER monoclonal antibody ER1D5. The specificity of the CC4-5 antibody for ER protein was confirmed by Western blot analysis. Sixty of 103 cases were positive for ER by CC4-5 automated immunohistochemistry. With a ligand binding assay threshold value of 20 fmol/mg protein, there were 50 positive cases by biochemical assay. The biochemical results corresponded to an 88% rate of agreement with automated CC4-5 staining. Analysis of discordant cases revealed that the majority of CC4-5 immunopositive only cases (8 of 11) were strongly positive, stroma rich tumors, suggesting that corresponding biochemical measurements were diluted by non representative stromal tissue. There was only one immunonegative, biochemically positive case (27 fmol/mg protein). Semiquantitation of CC4-5 staining using percent positive tumor cells or weighted average staining intensity (HSCORE) showed moderate to good correlation with quantitative DCC results (r = 0.64 and 0.62, P Ventana ES, most likely due to temperature constraints of the instrument. By manual ER1D5 staining, 40 of 79 examined cases were positive corresponding to a 99% rate of agreement with automated CC4-5 staining. Semiquantitation of ER1D5 staining by percent positive tumor cells and weighted average staining

  3. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    Science.gov (United States)

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  4. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  5. Automation of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay for High-Throughput Screening of Nasal Swab Specimens▿

    OpenAIRE

    Wang, Xue-Ping; Ginocchio, Christine C.

    2009-01-01

    This study demonstrated that an automated version of the BD-GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay (BD-MRSA), using achromopeptidase sample lysis and PCR setup performed on the Hamilton MICROLAB STARlet (Auto-MRSA), gave results comparable to those obtained with BD-MRSA. The positive- and negative-result concordance rates and overall concordance of BD-MRSA and Auto-MRSA were 98.2, 97.7, and 97.6%, respectively. Auto-MRSA required 60% less technical time than BD-MRSA,...

  6. Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

    Science.gov (United States)

    Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

    2016-06-01

    Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    Directory of Open Access Journals (Sweden)

    Christiane Radzimski

    2013-01-01

    Full Text Available Autoantibodies against soluble liver antigen (SLA are specific markers for autoimmune hepatitis (AIH type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA, detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors. Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  8. Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

    Science.gov (United States)

    Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

    2016-06-01

    Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990224

  9. A new automated method for the determination of the Total Antioxidant Capacity (TAC of human plasma, based on the crocin bleaching assay

    Directory of Open Access Journals (Sweden)

    Notas George

    2002-08-01

    Full Text Available Abstract Background Antioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (polyaromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. Methods In the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay, based on the crocin bleaching (oxidation method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L, and performed using an end point measurement. Results The TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM, while a diet rich in antioxidants more than doubled this value. Conclusions The proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and

  10. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

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    Masayoshi Tojo

    Full Text Available We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA, an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods, Citrobacter spp. (7/7, Escherichia coli (87/87, Klebsiella oxytoca (13/13, and Proteus spp. (11/11; Enterobacter spp. (29/30; Klebsiella pneumoniae (62/72; Pseudomonas aeruginosa (124/125; and Serratia marcescens (18/21; respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes, including 54 bla(CTX-M, 119 bla(IMP, 8 bla(KPC, 16 bla(NDM, 24 bla(OXA-23, 1 bla(OXA-24/40, 1 bla(OXA-48, 4 bla(OXA-58, and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  11. Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora.

    Science.gov (United States)

    Cheng, Zhongzhe; Huang, Meilin; Chen, Guiying; Yang, Guangjie; Zhou, Xin; Chen, Chang; Zhang, Yang; Xu, Yong; Feng, Yulin; Zhang, Lin; Jiang, Hongliang

    2016-02-01

    Many plant-derived glycosides are used as medications. It is common that these glycosides show poor intestinal absorption but their metabolites generated by intestinal microflora demonstrate strong bioactivity. Hence, it is crucial to develop a method for the identification and characterization of the metabolites, and consequently reveal the pathway in which the glycosides are processed in gut. In this study, cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) were developed for rapid discovery and evaluation of the metabolites of a glycoside compound, capilliposide C (LC-C). 92.7% of LC-C was biotransformed by rat intestinal microflora after 36-h incubation at 37°C. Human cancer cell lines HepG2, PC-3 and A549 was treated with metabolites pool, respectively, which was followed by cell viability assays and characterization of metabolites using UHPLC-QTOF-MS/MS. As a result, significant cytotoxicity was observed for the metabolites pool, from which six metabolites were identified. Based on the metabolites identified, deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-C in rat intestinal microflora. In addition, M4, an esterolysis product of LC-C, was obtained and evaluated for its bioactivity in vitro. As a result, M4 exhibited a reduction in cell viability in HepG2 with an IC50 value of 17.46±1.55μg/mL.

  12. Development and validation of fluorescence-based and automated patch clamp-based functional assays for the inward rectifier potassium channel Kir4.1.

    Science.gov (United States)

    Raphemot, Rene; Kadakia, Rishin J; Olsen, Michelle L; Banerjee, Sreedatta; Days, Emily; Smith, Stephen S; Weaver, C David; Denton, Jerod S

    2013-01-01

    The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac(©)), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl(+)) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z'=0.75±0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC50 of ∼6 μM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble "loose patch" recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC50=10 μM), VU717 (IC50=6 μM), and structurally related calcium channel blocker prenylamine (IC50=6 μM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl(+) flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels. PMID:24266659

  13. Improving the measurement of longitudinal change in renal function: automated detection of changes in laboratory creatinine assay

    Directory of Open Access Journals (Sweden)

    Norman Poh

    2015-04-01

    Full Text Available IntroductionRenal function is reported using the estimates of glomerular filtration rate (eGFR. However, eGFR values are recorded without reference to the particular serum creatinine (SCr assays used to derive them, and newer assays were introduced at different time points across the laboratories in the United Kingdom. These changes may cause systematic bias in eGFR reported in routinely collected data, even though laboratory-reported eGFR values have a correction factor applied.DesignAn algorithm to detect changes in SCr that in turn affect eGFR calculation method was developed. It compares the mapping of SCr values on to eGFR values across a time series of paired eGFR and SCr measurements.SettingRoutinely collected primary care data from 20,000 people with the richest renal function data from the quality improvement in chronic kidney disease trial.ResultsThe algorithm identified a change in eGFR calculation method in 114 (90% of the 127 included practices. This change was identified in 4736 (23.7% patient time series analysed. This change in calibration method was found to cause a significant step change in the reported eGFR values, producing a systematic bias. The eGFR values could not be recalibrated by applying the Modification of Diet in Renal Disease equation to the laboratory reported SCr values.ConclusionsThis algorithm can identify laboratory changes in eGFR calculation methods and changes in SCr assay. Failure to account for these changes may misconstrue renal function changes over time. Researchers using routine eGFR data should account for these effects.  

  14. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

  15. Performance of an automated solid-phase red cell adherence system compared with that of a manual gel microcolumn assay for the identification of antibodies eluted from red blood cells.

    Science.gov (United States)

    Finck, R H; Davis, R J; Teng, S; Goldfinger, D; Ziman, A F; Lu, Q; Yuan, S

    2011-01-01

    IgG antibodies coating red blood cells (RBCs) can be removed by elution procedures and their specificity determined by antibody identification studies. Although such testing is traditionally performed using the tube agglutination assay, prior studies have shown that the gel microcolumn (GMC) assay may also be used with comparable results. The purpose of this study was to compare an automated solid-phase red cell adherence (SPRCA) system with a GMC assay for the detection of antibodies eluted from RBCs. Acid eluates from 51 peripheral blood (PB) and 7 cord blood (CB) samples were evaluated by both an automated SPRCA instrument and a manual GMC assay. The concordance rate between the two systems for peripheral RBC samples was 88.2 percent (45 of 51), including cases with alloantibodies (n = 8), warm autoantibodies (n = 12), antibodies with no identifiable specificity (n = 2), and negative results (n = 23). There were six discordant cases, of which four had alloantibodies (including anti-Jka, -E, and -e) demonstrable by the SPRCA system only. In the remaining 2 cases, anti-Fya and antibodies with no identifiable specificity were demonstrable by the GMC assay only. All seven CB specimens produced concordant results, showing anti-A (n = 3), -B (n = 1), maternal anti-Jka (n = 2), or a negative result (n = 1). Automated SPRCA technology has a performance that is comparable with that of a manual GMC assay for identifying antibodies eluted from PB and CB RBCs.

  16. In-syringe magnetic stirring-assisted dispersive liquid-liquid microextraction for automation and downscaling of methylene blue active substances assay.

    Science.gov (United States)

    Suárez, Ruth; Horstkotte, Burkhard; Cerdà, Victor

    2014-12-01

    A simple and rapid method for the determination of the methylene blue active substances assay based on in-syringe automation of magnetic stirring-assisted dispersive liquid-liquid microextraction was developed. The proposed method proved to be valid for the determination of anionic surfactant in waste, pond, well, tap, and drinking water samples. Sample mixing with reagents, extraction and phase separation were performed within the syringe of an automated syringe pump containing a magnetic stirring bar for homogenization and solvent dispersion. The syringe module was used upside-down to enable the use of chloroform as an extraction solvent of higher density than water. The calibration was found to be linear up to 0.3mg/L using only 200 µL of solvent and 4 mL of sample. The limits of detection (3σ) and quantification (10σ) were 7.0 µg/L and 22 µg/L, respectively. The relative standard deviation for 10 replicate determinations of 0.1mg/L SBDS was below 3%. Concentrations of anionic surfactants in natural water samples were in the range of 0.032-0.213 mg/L and no significant differences towards the standard method were found. Standard additions gave analyte recoveries between 95% and 106% proving the general applicability and adequateness of the system to MBSA index determination. Compared to the tedious standard method requiring up to 50 mL of chloroform, the entire procedure took only 345 s using 250-times less solvent.

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

    International Nuclear Information System (INIS)

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

  18. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  19. Biotoxin Detection Using Cell-Based Sensors

    OpenAIRE

    Pratik Banerjee; Spyridon Kintzios; Balabhaskar Prabhakarpandian

    2013-01-01

    Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results ob...

  20. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

    Directory of Open Access Journals (Sweden)

    Alexandre Harlé

    Full Text Available Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies.Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM PCR, Real-time allele-specific amplification (RT-ASA PCR, Next generation sequencing (NGS, immunohistochemistry (IHC and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.BRAF mutations were found in 28(47.5%, 29(49.2%, 31(52.5%, 29(49.2% and 27(45.8% samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2% samples were found bearing a c.1799T>A (p.Val600Glu mutation, three (9.4% with a c.1798_1799delinsAA (p.Val600Lys mutation and one with c.1789_1790delinsTC (p.Leu597Ser mutation. Two samples were found bearing complex mutations.HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.

  1. Development of fully automated determination of marker-specific immunoglobulin G (IgG) avidity based on the avidity competition assay format: application for Abbott Architect cytomegalovirus and Toxo IgG Avidity assays.

    Science.gov (United States)

    Curdt, Ingo; Praast, Gerald; Sickinger, Eva; Schultess, Jan; Herold, Iris; Braun, Hans Bertram; Bernhardt, Stephanie; Maine, Gregory T; Smith, Darwin D; Hsu, Stephen; Christ, Heike M; Pucci, Dominick; Hausmann, Michael; Herzogenrath, Jörg

    2009-03-01

    Determination of the avidity of immunoglobulin G (IgG) directed against a specific marker has become an established diagnostic tool for identifying or excluding acute infections with pathogens. A novel assay format termed AVIcomp (avidity competition based on mass action) circumventing the conventional chaotropic format has been developed for determination of the avidity of marker-specific IgG in patient specimens. Its applications for cytomegalovirus (CMV) and Toxoplasma gondii are presented. Specific high-avidity IgG from the patient specimen is selectively blocked using a soluble antigen in a sample pretreatment reagent, and the amount of remaining specific low-avidity IgG is determined relative to that in an untreated control. The comparison of the conventional chaotropic format, represented by the Radim CMV IgG Avidity assay, and the newly developed AVIcomp method, as exemplified by the Architect CMV IgG Avidity assay, on blood drawn within 4 months after seroconversion revealed a sensitivity of 100% (97.3% by an alternative calculation) for the AVIcomp format versus 87.5% (75.7% by an alternative calculation) for the chaotropic avidity assay. The specificity on 312 CMV IgG reactive and CMV IgM nonreactive specimens from pregnant women was 100% for the AVIcomp assay and 99.7% for the conventional avidity assay. The Architect Toxo IgG Avidity assay showed an agreement of 97.2% with the bioMérieux Vidas Toxo IgG Avidity Assay employing chaotropic reagents. These performance data suggest that the AVIcomp format shows superior sensitivity and equivalent specificity for the determination of IgG avidity to assays based on the chaotropic method and that the AVIcomp format may also be applicable to other disease states.

  2. Automation in immunohematology.

    Science.gov (United States)

    Bajpai, Meenu; Kaur, Ravneet; Gupta, Ekta

    2012-07-01

    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.

  3. Automation in Immunohematology

    Directory of Open Access Journals (Sweden)

    Meenu Bajpai

    2012-01-01

    Full Text Available 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.

  4. Automation in immunohematology.

    Science.gov (United States)

    Bajpai, Meenu; Kaur, Ravneet; Gupta, Ekta

    2012-07-01

    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

  5. A semi-automated system for quantifying the oxidative potential of ambient particles in aqueous extracts using the dithiothreitol (DTT assay: results from the Southeastern Center for Air Pollution and Epidemiology (SCAPE

    Directory of Open Access Journals (Sweden)

    T. Fang

    2014-07-01

    Full Text Available A variety of methods are used to measure the capability of particulate matter (PM to catalytically generate reactive oxygen species (ROS in vivo, also defined as the aerosol oxidative potential. A widely used measure of aerosol oxidative potential is the dithiothreitol (DTT assay, which monitors the depletion of DTT (a surrogate for cellular antioxidants as catalyzed by the redox-active species in PM. However, a major constraint in the routine use of the DTT assay for integrating it with the large-scale health studies is its labor-intensive and time-consuming protocol. To specifically address this concern, we have developed a semi-automated system for quantifying the oxidative potential of aerosol liquid extracts using the DTT assay. The system, capable of unattended analysis at one sample per hour, has a high analytical precision (Coefficient of Variation of 12% for standards, 4% for ambient samples, and reasonably low limit of detection (0.31 nmol min−1. Comparison of the automated approach with the manual method conducted on ambient samples yielded good agreement (slope = 1.08 ± 0.12, r2 = 0.92, N = 9. The system was utilized for the Southeastern Center for Air Pollution and Epidemiology (SCAPE to generate an extensive data set on DTT activity of ambient particles collected from contrasting environments (urban, road-side, and rural in the southeastern US. We find that water-soluble PM2.5 DTT activity on a per air volume basis was spatially uniform and often well correlated with PM2.5 mass (r = 0.49 to 0.88, suggesting regional sources contributing to the PM oxidative potential in southeast US. However, the greater heterogeneity in the intrinsic DTT activity (per PM mass basis across seasons indicates variability in the DTT activity associated with aerosols from sources that vary with season. Although developed for the DTT assay, the instrument can also be used to determine oxidative potential with other acellular assays.

  6. A semi-automated system for quantifying the oxidative potential of ambient particles in aqueous extracts using the dithiothreitol (DTT) assay: results from the Southeastern Center for Air Pollution and Epidemiology (SCAPE)

    Science.gov (United States)

    Fang, T.; Verma, V.; Guo, H.; King, L. E.; Edgerton, E. S.; Weber, R. J.

    2015-01-01

    A variety of methods are used to measure the capability of particulate matter (PM) to catalytically generate reactive oxygen species (ROS) in vivo, also defined as the aerosol oxidative potential. A widely used measure of aerosol oxidative potential is the dithiothreitol (DTT) assay, which monitors the depletion of DTT (a surrogate for cellular antioxidants) as catalyzed by the redox-active species in PM. However, a major constraint in the routine use of the DTT assay for integrating it with large-scale health studies is its labor-intensive and time-consuming protocol. To specifically address this concern, we have developed a semi-automated system for quantifying the oxidative potential of aerosol liquid extracts using the DTT assay. The system, capable of unattended analysis at one sample per hour, has a high analytical precision (coefficient of variation of 15% for positive control, 4% for ambient samples) and reasonably low limit of detection (0.31 nmol min-1). Comparison of the automated approach with the manual method conducted on ambient samples yielded good agreement (slope = 1.08 ± 0.12, r2 = 0.92, N = 9). The system was utilized for the Southeastern Center for Air Pollution & Epidemiology (SCAPE) to generate an extensive data set on DTT activity of ambient particles collected from contrasting environments (urban, roadside, and rural) in the southeastern US. We find that water-soluble PM2.5 DTT activity on a per-air-volume basis was spatially uniform and often well correlated with PM2.5 mass (r = 0.49 to 0.88), suggesting regional sources contributing to the PM oxidative potential in the southeastern US. The correlation may also suggest a mechanistic explanation (oxidative stress) for observed PM2.5 mass-health associations. The heterogeneity in the intrinsic DTT activity (per-PM-mass basis) across seasons indicates variability in the DTT activity associated with aerosols from sources that vary with season. Although developed for the DTT assay, the

  7. Design of a facility for the automated receipt inspection and characterization of LILW using integrated non-destructive examination and assay techniques

    International Nuclear Information System (INIS)

    KHNP is constructing two repositories, located in Bonggil-ri, Yangbukmyun, Gyeongju-si, Gyeongbuk, Korea, for Low and Intermediate Level radioactive waste (LILW). The waste received is required to be inspected and characterized prior to being placed into storage at the repository. This paper describes the design of an integrated system of non destructive examination (X-ray), and non destructive assay (radiometric) instrument systems, waste package integrity checking systems, waste handling mechanisms and data management and archival provisions, which comprise the Waste Receipt and Inspection Facility (WRIF). The WRIF is designed to uncap transport containers, unload waste drums from containers, safely examine the drums, to ensure they meet the waste acceptance criteria established by the LILW facility. The drums are then loaded into waste disposal containers for transfer to the repository. The WRIF also receives larger waste containers, which are characterized in a separate section of the WRIF, before being transferred to the repository. Remote control and handling of the waste is a design requirement, as well as the management, processing archival and retrieval system for all data generated during waste characterization. (authors)

  8. Mammalian Cell-Based Sensor System

    Science.gov (United States)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  9. Dendritic cell-based in vitro assays for vaccine immunogenicity

    OpenAIRE

    Vandebriel, Rob J.; Hoefnagel, Marcel H. N.

    2012-01-01

    Dendritic cells (DC) are pivotal in the induction of adaptive immune responses because they can activate naive T-cells. Moreover, they steer these adaptive immune responses by integrating various stimuli, such as from different pathogen associated molecular patterns and the cytokine milieu. Immature DC are very well capable of ingesting protein antigens, whereas mature DC are efficient presenters of peptides to naive T cells. Human DC can be readily cultured from peripheral blood mononuclear ...

  10. Home Automation

    OpenAIRE

    Ahmed, Zeeshan

    2010-01-01

    In this paper I briefly discuss the importance of home automation system. Going in to the details I briefly present a real time designed and implemented software and hardware oriented house automation research project, capable of automating house's electricity and providing a security system to detect the presence of unexpected behavior.

  11. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert;

    2016-01-01

    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  12. Automation of antimicrobial activity screening.

    Science.gov (United States)

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

    2016-03-01

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

  13. Digital microfluidics for automated hanging drop cell spheroid culture.

    Science.gov (United States)

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. PMID:25510471

  14. An automated imaging system for radiation biodosimetry.

    Science.gov (United States)

    Garty, Guy; Bigelow, Alan W; Repin, Mikhail; Turner, Helen C; Bian, Dakai; Balajee, Adayabalam S; Lyulko, Oleksandra V; Taveras, Maria; Yao, Y Lawrence; Brenner, David J

    2015-07-01

    We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT-a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay. PMID:25939519

  15. Sensitive-cell-based fish chromatophore biosensor

    Science.gov (United States)

    Plant, Thomas K.; Chaplen, Frank W.; Jovanovic, Goran; Kolodziej, Wojtek; Trempy, Janine E.; Willard, Corwin; Liburdy, James A.; Pence, Deborah V.; Paul, Brian K.

    2004-07-01

    A sensitive biosensor (cytosensor) has been developed based on color changes in the toxin-sensitive colored living cells of fish. These chromatophores are highly sensitive to the presence of many known and unknown toxins produced by microbial pathogens and undergo visible color changes in a dose-dependent manner. The chromatophores are immobilized and maintained in a viable state while potential pathogens multiply and fish cell-microbe interactions are monitored. Low power LED lighting is used to illuminate the chromatophores which are magnified using standard optical lenses and imaged onto a CCD array. Reaction to toxins is detected by observing changes is the total area of color in the cells. These fish chromatophores are quite sensitive to cholera toxin, Staphococcus alpha toxin, and Bordatella pertussis toxin. Numerous other toxic chemical and biological agents besides bacterial toxins also cause readily detectable color effects in chromatophores. The ability of the chromatophore cell-based biosensor to distinguish between different bacterial pathogens was examined. Toxin producing strains of Salmonella enteritis, Vibrio parahaemolyticus, and Bacillus cereus induced movement of pigmented organelles in the chromatophore cells and this movement was measured by changes in the optical density over time. Each bacterial pathogen elicited this measurable response in a distinctive and signature fashion. These results suggest a chromatophore cell-based biosensor assay may be applicable for the detection and identification of virulence activities associated with certain air-, food-, and water-borne bacterial pathogens.

  16. Biotoxin Detection Using Cell-Based Sensors

    Directory of Open Access Journals (Sweden)

    Pratik Banerjee

    2013-11-01

    Full Text Available Cell-based biosensors (CBBs utilize the principles of cell-based assays (CBAs by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results obtained from CBBs are based on the toxin-cell interactions, and therefore, reveal functional information (such as mode of action, toxic potency, bioavailability, target tissue or organ, etc. about the toxin. CBBs incorporate both prokaryotic (bacteria and eukaryotic (yeast, invertebrate and vertebrate cells. To create CBB devices, living cells are directly integrated onto the biosensor platform. The sensors report the cellular responses upon exposures to toxins and the resulting cellular signals are transduced by secondary transducers generating optical or electrical signals outputs followed by appropriate read-outs. Examples of the layout and operation of cellular biosensors for detection of selected biotoxins are summarized.

  17. Biotoxin detection using cell-based sensors.

    Science.gov (United States)

    Banerjee, Pratik; Kintzios, Spyridon; Prabhakarpandian, Balabhaskar

    2013-12-01

    Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results obtained from CBBs are based on the toxin-cell interactions, and therefore, reveal functional information (such as mode of action, toxic potency, bioavailability, target tissue or organ, etc.) about the toxin. CBBs incorporate both prokaryotic (bacteria) and eukaryotic (yeast, invertebrate and vertebrate) cells. To create CBB devices, living cells are directly integrated onto the biosensor platform. The sensors report the cellular responses upon exposures to toxins and the resulting cellular signals are transduced by secondary transducers generating optical or electrical signals outputs followed by appropriate read-outs. Examples of the layout and operation of cellular biosensors for detection of selected biotoxins are summarized. PMID:24335754

  18. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  19. Live-cell luciferase assay of drug resistant cells

    OpenAIRE

    sprotocols

    2015-01-01

    To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes u...

  20. Process automation

    International Nuclear Information System (INIS)

    Process automation technology has been pursued in the chemical processing industries and to a very limited extent in nuclear fuel reprocessing. Its effective use has been restricted in the past by the lack of diverse and reliable process instrumentation and the unavailability of sophisticated software designed for process control. The Integrated Equipment Test (IET) facility was developed by the Consolidated Fuel Reprocessing Program (CFRP) in part to demonstrate new concepts for control of advanced nuclear fuel reprocessing plants. A demonstration of fuel reprocessing equipment automation using advanced instrumentation and a modern, microprocessor-based control system is nearing completion in the facility. This facility provides for the synergistic testing of all chemical process features of a prototypical fuel reprocessing plant that can be attained with unirradiated uranium-bearing feed materials. The unique equipment and mission of the IET facility make it an ideal test bed for automation studies. This effort will provide for the demonstration of the plant automation concept and for the development of techniques for similar applications in a full-scale plant. A set of preliminary recommendations for implementing process automation has been compiled. Some of these concepts are not generally recognized or accepted. The automation work now under way in the IET facility should be useful to others in helping avoid costly mistakes because of the underutilization or misapplication of process automation. 6 figs

  1. A Cell-based β2-adrenergic Receptor Agonist Functional Screening Assay for Chinese Traditional Medicines%基于细胞的β2肾上腺素能受体激动剂功能性筛选体系在中草药中的应用

    Institute of Scientific and Technical Information of China (English)

    杨洋; 王超; 潘鹏炜; 白钢

    2009-01-01

    为了建立基于细胞的β2肾上腺素能受体激动剂筛选体系,将大鼠β2受体基因克隆人pcDNA3.1载体.转染具有cAMP应答元件调控的绿色荧光蛋白报告基因的中国仓鼠卵巢细胞(CHO),从得到的阳性单克隆细胞中筛选得到一个稳定表达β2受体,并且在受到激动剂刺激后发出剂量依赖绿色荧光强度的细胞株.将此筛选体系用于从中药中筛选具有β2受体激动剂活性的化合物.从几种芸香科药材中得到了辛弗林(synephine),从细辛和附子中得到了去甲乌药碱(higenamine),首次从受体水平证明了它们的β2激动剂活性.此筛选体系适合从大量天然化合物中快速筛选具有β2激动剂活性的成分.%To construct a cell-based agonists screening system for β2-adrenergic receptor (β2-AR) from chi- nese traditional medicines, β2-AR gene was amplified by PCR from rat genomic DNA. The PCR products were cloned into pcDNA3.1 and transfected into chinese hamster ovary (CHO) cell, which contained a cyclic AMP response element-linked enhanced green fluorescent protein reporter gene. One stable cell line was generated from the single-cell-derived colonies. It expressed β2-AR and provided green fluorescence when stimulated by ligands. Expression of β2-AR was monitored by RT-PCR and western blotting anti-β2-AR. The function of re- porter gene system was studied by flow cytometry. This cell-based screening system was applied to the extracts of chinese traditional medicines (TCMs). Synephine which was obtained from Rutaceae generic plant, and hige- namine which was obtained from Herba Asari or Radix Aconiti Lateralis Preparata were verified to have β2-AR agonist activity at receptor level. This assay could be applied in the pharmaceutical industry for screening thou- sands or indeed millions of natural compounds of TCMs for β2-AR agonists very rapidly.

  2. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  3. Rover waste assay system

    International Nuclear Information System (INIS)

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  4. Development of an upconverting chelate assay

    Science.gov (United States)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  5. Automating Finance

    Science.gov (United States)

    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:…

  6. Cell-Based Genotoxicity Testing

    Science.gov (United States)

    Reifferscheid, Georg; Buchinger, Sebastian

    Genotoxicity test systems that are based on bacteria display an important role in the detection and assessment of DNA damaging chemicals. They belong to the basic line of test systems due to their easy realization, rapidness, broad applicability, high sensitivity and good reproducibility. Since the development of the Salmonella microsomal mutagenicity assay by Ames and coworkers in the early 1970s, significant development in bacterial genotoxicity assays was achieved and is still a subject matter of research. The basic principle of the mutagenicity assay is a reversion of a growth inhibited bacterial strain, e.g., due to auxotrophy, back to a fast growing phenotype (regain of prototrophy). Deeper knowledge of the ­mutation events allows a mechanistic understanding of the induced DNA-damage by the utilization of base specific tester strains. Collections of such specific tester strains were extended by genetic engineering. Beside the reversion assays, test systems utilizing the bacterial SOS-response were invented. These methods are based on the fusion of various SOS-responsive promoters with a broad variety of reporter genes facilitating numerous methods of signal detection. A very important aspect of genotoxicity testing is the bioactivation of ­xenobiotics to DNA-damaging compounds. Most widely used is the extracellular metabolic activation by making use of rodent liver homogenates. Again, genetic engineering allows the construction of highly sophisticated bacterial tester strains with significantly enhanced sensitivity due to overexpression of enzymes that are involved in the metabolism of xenobiotics. This provides mechanistic insights into the toxification and detoxification pathways of xenobiotics and helps explaining the chemical nature of hazardous substances in unknown mixtures. In summary, beginning with "natural" tester strains the rational design of bacteria led to highly specific and sensitive tools for a rapid, reliable and cost effective

  7. Microsystems for cell-based electrophysiology

    Science.gov (United States)

    Yobas, Levent

    2013-08-01

    Among the electrophysiology techniques, the voltage clamp and its subsequent scaling to smaller mammalian cells, the so-called patch clamp, led to fundamental discoveries in the last century, revealing the ionic mechanisms and the role of single-ion channels in the generation and propagation of action potentials through excitable membranes (e.g. nerves and muscles). Since then, these techniques have gained a reputation as the gold standard of studying cellular ion channels owing to their high accuracy and rich information content via direct measurements under a controlled membrane potential. However, their delicate and skill-laden procedure has put a serious constrain on the throughput and their immediate utilization in the discovery of new cures targeting ion channels until researchers discovered ‘lab-on-a-chip’ as a viable platform for the automation of these techniques into a reliable high-throughput screening functional assay on ion channels. This review examines the innovative ‘lab-on-a-chip’ microtechnologies demonstrated towards this target over a period of slightly more than a decade. The technologies are categorically reviewed according to their considerations for design, fabrication, as well as microfluidic integration from a performance perspective with reference to their ability to secure G Ω seals (gigaseals) on cells, the norm broadly accepted among electrophysiologists for quality recordings that reflect ion-channel activity with high fidelity.

  8. Microsystems for cell-based electrophysiology

    International Nuclear Information System (INIS)

    Among the electrophysiology techniques, the voltage clamp and its subsequent scaling to smaller mammalian cells, the so-called patch clamp, led to fundamental discoveries in the last century, revealing the ionic mechanisms and the role of single-ion channels in the generation and propagation of action potentials through excitable membranes (e.g. nerves and muscles). Since then, these techniques have gained a reputation as the gold standard of studying cellular ion channels owing to their high accuracy and rich information content via direct measurements under a controlled membrane potential. However, their delicate and skill-laden procedure has put a serious constrain on the throughput and their immediate utilization in the discovery of new cures targeting ion channels until researchers discovered ‘lab-on-a-chip’ as a viable platform for the automation of these techniques into a reliable high-throughput screening functional assay on ion channels. This review examines the innovative ‘lab-on-a-chip’ microtechnologies demonstrated towards this target over a period of slightly more than a decade. The technologies are categorically reviewed according to their considerations for design, fabrication, as well as microfluidic integration from a performance perspective with reference to their ability to secure G Ω seals (gigaseals) on cells, the norm broadly accepted among electrophysiologists for quality recordings that reflect ion-channel activity with high fidelity. (topical review)

  9. Automation Security

    OpenAIRE

    Mirzoev, Dr. Timur

    2014-01-01

    Web-based Automated Process Control systems are a new type of applications that use the Internet to control industrial processes with the access to the real-time data. Supervisory control and data acquisition (SCADA) networks contain computers and applications that perform key functions in providing essential services and commodities (e.g., electricity, natural gas, gasoline, water, waste treatment, transportation) to all Americans. As such, they are part of the nation s critical infrastructu...

  10. Cell-Based Biosensors Principles and Applications

    CERN Document Server

    Wang, Ping

    2009-01-01

    Written by recognized experts the field, this leading-edge resource is the first book to systematically introduce the concept, technology, and development of cell-based biosensors. You find details on the latest cell-based biosensor models and novel micro-structure biosensor techniques. Taking an interdisciplinary approach, this unique volume presents the latest innovative applications of cell-based biosensors in a variety of biomedical fields. The book also explores future trends of cell-based biosensors, including integrated chips, nanotechnology and microfluidics. Over 140 illustrations hel

  11. Automated Budget System

    Data.gov (United States)

    Department of Transportation — The Automated Budget System (ABS) automates management and planning of the Mike Monroney Aeronautical Center (MMAC) budget by providing enhanced capability to plan,...

  12. Auxiliary pattern for cell-based OPC

    Science.gov (United States)

    Kahng, Andrew B.; Park, Chul-Hong

    2006-10-01

    The runtime of model-based optical proximity correction (OPC) tools has grown unacceptably with each successive technology generation, and has emerged as one of the major bottlenecks for turnaround time (TAT) of IC data preparation and manufacturing. The cell-based OPC approach improves runtime by performing OPC once per cell definition as opposed to once per cell instantiation in the layout. However, cell-based OPC does not comprehend inter-cell optical interactions that affect feature printability in a layout context. In this work, we propose auxiliary pattern-enabled cell-based OPC which can minimize the CD differences between cell-based OPC and model-based OPC. To enable effective insertion of auxiliary pattern (AP) in the design, we also propose a post-placement optimization of a standard cell block with respect to detailed placement. By dynamic programming-based placement perturbation, we achieve 100% AP applicability in designs with placement utilizations of cell-based OPC with AP can match gate edge placement error (EPE) count of model-based OPC within 4%. This is an improvement of 90%, on average, over cell-based OPC without APs. The AP-based OPC approach can reduce OPC runtimes versus model-based OPC by up to 40X in our benchmark designs. We can also achieve reduction of GDSII file size and ORC runtimes due to hierarchy maintenance of cell-based OPC.

  13. A quantitative comet infection assay for influenza virus

    OpenAIRE

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold in...

  14. A Cell-Based Protein-Protein Interaction Method Using a Permuted Luciferase Reporter

    OpenAIRE

    Eishingdrelo, Haifeng; Cai, Jidong; Weissensee, Paul; Sharma, Praveen; Tocci, Michael J; Wright, Paul S

    2011-01-01

    We have developed a novel cell-based protein-protein interaction assay method. The method relies on conversion of an inactive permuted luciferase containing a Tobacco Etch Virus protease (TEV) cleavage sequence fused onto protein (A) to an active luciferase upon interaction and cleavage by another protein (B) fused with the TEV protease. We demonstrate assay applicability for ligand-induced protein-protein interactions including G-protein coupled receptors, receptor tyrosine kinases and nucle...

  15. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John;

    2008-01-01

    BACKGROUND: The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample...... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1......-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...

  16. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    DEFF Research Database (Denmark)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen;

    2015-01-01

    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  17. Automated, Miniaturized and Integrated Quality Control-on-Chip (QC-on-a-Chip for Advanced Cell Therapy Applications

    Directory of Open Access Journals (Sweden)

    David eWartmann

    2015-09-01

    Full Text Available The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of evaluating cell cultures under defined, reproducible and standardizable measurement conditions. In the present review we describe recent lab-on-a-chip developments for cell analysis and how these methodologies could improve standard quality control in the field of manufacturing cell-based vaccines for clinical purposes. We highlight in particular the regulatory requirements for advanced cell therapy applications using as an example dendritic cell-based cancer vaccines to describe the tangible advantages of microfluidic devices that overcome most of the challenges associated with automation, miniaturization and integration of cell-based assays. As its main advantage lab-on-a-chip technology allows for precise regulation of culturing conditions, while simultaneously monitoring cell relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of cell cultures and their potential future applications for cell therapies and cancer immunotherapy are discussed in the present review.

  18. Automated, Miniaturized and Integrated Quality Control-on-Chip (QC-on-a-Chip) for Advanced Cell Therapy Applications

    Science.gov (United States)

    Wartmann, David; Rothbauer, Mario; Kuten, Olga; Barresi, Caterina; Visus, Carmen; Felzmann, Thomas; Ertl, Peter

    2015-09-01

    The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of evaluating cell cultures under defined, reproducible and standardizable measurement conditions. In the present review we describe recent lab-on-a-chip developments for cell analysis and how these methodologies could improve standard quality control in the field of manufacturing cell-based vaccines for clinical purposes. We highlight in particular the regulatory requirements for advanced cell therapy applications using as an example dendritic cell-based cancer vaccines to describe the tangible advantages of microfluidic devices that overcome most of the challenges associated with automation, miniaturization and integration of cell-based assays. As its main advantage lab-on-a-chip technology allows for precise regulation of culturing conditions, while simultaneously monitoring cell relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of cell cultures and their potential future applications for cell therapies and cancer immunotherapy are discussed in the present review.

  19. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  20. Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring

    DEFF Research Database (Denmark)

    Rodrigues de Sousa Nunes, Pedro André; Kjaerulff, S.; Dufva, Martin;

    2015-01-01

    and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells......The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control......, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells...

  1. Manufacturing and automation

    Directory of Open Access Journals (Sweden)

    Ernesto Córdoba Nieto

    2010-04-01

    Full Text Available The article presents concepts and definitions from different sources concerning automation. The work approaches automation by virtue of the author’s experience in manufacturing production; why and how automation prolects are embarked upon is considered. Technological reflection regarding the progressive advances or stages of automation in the production area is stressed. Coriat and Freyssenet’s thoughts about and approaches to the problem of automation and its current state are taken and examined, especially that referring to the problem’s relationship with reconciling the level of automation with the flexibility and productivity demanded by competitive, worldwide manufacturing.

  2. Cell-Based Therapies for Diabetic Complications

    Directory of Open Access Journals (Sweden)

    Stella Bernardi

    2012-01-01

    Full Text Available In recent years, accumulating experimental evidence supports the notion that diabetic patients may greatly benefit from cell-based therapies, which include the use of adult stem and/or progenitor cells. In particular, mesenchymal stem cells and the circulating pool of endothelial progenitor cells have so far been the most studied populations of cells proposed for the treatment of vascular complications affecting diabetic patients. We review the evidence supporting their use in this setting, the therapeutic benefits that these cells have shown so far as well as the challenges that cell-based therapies in diabetic complications put out.

  3. Time-Resolved Fluorescence Assays.

    Science.gov (United States)

    Ma, Chen-Ting; Sergienko, Eduard A

    2016-01-01

    Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities. PMID:27316992

  4. Cell-based screens and phenomics with fission yeast.

    Science.gov (United States)

    Rallis, Charalampos; Bähler, Jürg

    2016-01-01

    Next-generation sequencing approaches have considerably advanced our understanding of genome function and regulation. However, the knowledge of gene function and complex cellular processes remains a challenge and bottleneck in biological research. Phenomics is a rapidly emerging area, which seeks to rigorously characterize all phenotypes associated with genes or gene variants. Such high-throughput phenotyping under different conditions can be a potent approach toward gene function. The fission yeast Schizosaccharomyces pombe (S. pombe) is a proven eukaryotic model organism that is increasingly used for genomewide screens and phenomic assays. In this review, we highlight current large-scale, cell-based approaches used with S. pombe, including computational colony-growth measurements, genetic interaction screens, parallel profiling using barcodes, microscopy-based cell profiling, metabolomic methods and transposon mutagenesis. These diverse methods are starting to offer rich insights into the relationship between genotypes and phenotypes. PMID:26523839

  5. Cell-Based Microarrays for In Vitro Toxicology

    Science.gov (United States)

    Wegener, Joachim

    2015-07-01

    DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

  6. Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

    Directory of Open Access Journals (Sweden)

    Tae-Hee Kim

    Full Text Available Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

  7. Automated diagnostic kiosk for diagnosing diseases

    Science.gov (United States)

    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.

  8. Configuration Management Automation (CMA)

    Data.gov (United States)

    Department of Transportation — Configuration Management Automation (CMA) will provide an automated, integrated enterprise solution to support CM of FAA NAS and Non-NAS assets and investments. CMA...

  9. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  10. Workflow automation architecture standard

    Energy Technology Data Exchange (ETDEWEB)

    Moshofsky, R.P.; Rohen, W.T. [Boeing Computer Services Co., Richland, WA (United States)

    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.

  11. Shoe-String Automation

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Cell-based bioassays in microfluidic systems

    Science.gov (United States)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.

    2004-12-01

    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  13. Stem cell-based bone repair

    OpenAIRE

    Fei, Yurong; Xu, Ren-He; Hurley, Marja M.

    2012-01-01

    To accelerate bone repair, one strategy is to deliver the cells that make bone. The current review focuses on stem cell-based bone repair. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-renew unlimitedly and differentiate into the bone forming cells – osteoblasts. Scientists have been actively investigating culture conditions to stably and efficiently induce differentiation of these stem cells into osteoblasts. However, ESCs have the issues of ethnics, immune ...

  14. Validação laboratorial de um método automatizado de dosagem da atividade de adenosina desaminase em líquido pleural e em líquido cefalorraquidiano Laboratorial validation of an automated assay for the determination of adenosine deaminase activity in pleural fluid and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Márcia Cristina Feres

    2008-12-01

    Full Text Available OBJETIVO: A incidência global de tuberculose reforça a necessidade de melhores ensaios para o diagnóstico desta doença, principalmente da tuberculose extrapulmonar. O objetivo do trabalho foi validar o desempenho de um método automatizado para a determinação da atividade de adenosina desaminase (ADA no líquido pleural (LP e no líquido cefalorraquidiano (LCR, comparando-o com um método convencional (Giusti modificado. MÉTODOS: Selecionaram-se 134 amostras da rotina laboratorial: 94 de LP e 40 de LCR. Foram realizadas as determinações da atividade de ADA através dos dois métodos. Calculou-se a precisão inter- e intra-ensaios, análise de regressão linear, testes de concordância simples e médias das diferenças. RESULTADOS: Os coeficientes de correlação para as amostras de LP e LCR foram, respectivamente, 0,96 e 0,95. A precisão interensaio foi determinada pela média de 21 amostras replicadas em ensaios diferentes para 3 níveis de atividade: baixa, média e alta. Os coeficientes de variação em porcentagem (%CV foram, respectivamente, 5,9, 8,1 e 5,8 para amostras de LP; e 21,9, 18,6 e 13,8 para amostras de LCR, respectivamente. A precisão intra-ensaio em %CV foi, respectivamente, 1,3 e 11,7% para amostras de LP e LCR. A concordância entre os dois métodos em amostras de LP e LCR foi, respectivamente, 96,8% e 100%, considerando-se como valores de referência para o diagnóstico de TB 40 U/L (convencional e 30 U/L (automatizado em amostras de LP, e 9 U/L em amostras de LCR para os dois métodos. CONCLUSÕES: Os resultados validaram o método automatizado de determinação da atividade de ADA para o uso em amostras de LP e LCR como alternativa ao método convencional.OBJECTIVE: The incidence of tuberculosis worldwide has emphasized the need for better assays designed to diagnose the disease, principally the extrapulmonary form. The objective of the present study was to validate the performance of an automated method for the

  15. Cell-Based Therapy for Silicosis.

    Science.gov (United States)

    Lopes-Pacheco, Miquéias; Bandeira, Elga; Morales, Marcelo M

    2016-01-01

    Silicosis is the most common pneumoconiosis globally, with higher prevalence and incidence in developing countries. To date, there is no effective treatment to halt or reverse the disease progression caused by silica-induced lung injury. Significant advances have to be made in order to reduce morbidity and mortality related to silicosis. In this review, we have highlighted the main mechanisms of action that cause lung damage by silica particles and summarized the data concerning the therapeutic promise of cell-based therapy for silicosis. PMID:27066079

  16. Cell-Based Therapy for Silicosis

    Directory of Open Access Journals (Sweden)

    Miquéias Lopes-Pacheco

    2016-01-01

    Full Text Available Silicosis is the most common pneumoconiosis globally, with higher prevalence and incidence in developing countries. To date, there is no effective treatment to halt or reverse the disease progression caused by silica-induced lung injury. Significant advances have to be made in order to reduce morbidity and mortality related to silicosis. In this review, we have highlighted the main mechanisms of action that cause lung damage by silica particles and summarized the data concerning the therapeutic promise of cell-based therapy for silicosis.

  17. Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery

    NARCIS (Netherlands)

    Zimmer, B.; Pallocca, G.; Dreser, N.; Foerster, S.; Waldmann, T.; Westerhout, J.; Julien, S.; Krause, K.H.; Van Thriel, C.; Hengstler, J.G.; Sachinidis, A.; Bosgra, S.; Leist, M.

    2014-01-01

    Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay

  18. A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.

    Directory of Open Access Journals (Sweden)

    Roman Anton

    Full Text Available The fundamental role of p38 mitogen-activated protein kinases (MAPKs in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.

  19. A p38 Substrate-Specific MK2-EGFP Translocation Assay for Identification and Validation of New p38 Inhibitors in Living Cells: A Comprising Alternative for Acquisition of Cellular p38 Inhibition Data

    Science.gov (United States)

    Anton, Roman; Bauer, Silke M.; Keck, Peter R. W. E. F.; Laufer, Stefan; Rothbauer, Ulrich

    2014-01-01

    The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood. PMID:24743242

  20. Cell-based strategies for vascular regeneration.

    Science.gov (United States)

    Zou, Tongqiang; Fan, Jiabing; Fartash, Armita; Liu, Haifeng; Fan, Yubo

    2016-05-01

    Vascular regeneration is known to play an essential role in the repair of injured tissues mainly through accelerating the repair of vascular injury caused by vascular diseases, as well as the recovery of ischemic tissues. However, the clinical vascular regeneration is still challenging. Cell-based therapy is thought to be a promising strategy for vascular regeneration, since various cells have been identified to exert important influences on the process of vascular regeneration such as the enhanced endothelium formation on the surface of vascular grafts, and the induction of vessel-like network formation in the ischemic tissues. Here are a vast number of diverse cell-based strategies that have been extensively studied in vascular regeneration. These strategies can be further classified into three main categories, including cell transplantation, construction of tissue-engineered grafts, and surface modification of scaffolds. Cells used in these strategies mainly refer to terminally differentiated vascular cells, pluripotent stem cells, multipotent stem cells, and unipotent stem cells. The aim of this review is to summarize the reported research advances on the application of various cells for vascular regeneration, yielding insights into future clinical treatment for injured tissue/organ. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1297-1314, 2016. PMID:26864677

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

    OpenAIRE

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

    2011-01-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumpti...

  2. Automate functional testing

    Directory of Open Access Journals (Sweden)

    Ramesh Kalindri

    2014-06-01

    Full Text Available Currently, software engineers are increasingly turning to the option of automating functional tests, but not always have successful in this endeavor. Reasons range from low planning until over cost in the process. Some principles that can guide teams in automating these tests are described in this article.

  3. Automation in Warehouse Development

    NARCIS (Netherlands)

    Hamberg, R.; Verriet, J.

    2012-01-01

    The warehouses of the future will come in a variety of forms, but with a few common ingredients. Firstly, human operational handling of items in warehouses is increasingly being replaced by automated item handling. Extended warehouse automation counteracts the scarcity of human operators and support

  4. Work and Programmable Automation.

    Science.gov (United States)

    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…

  5. Library Automation Style Guide.

    Science.gov (United States)

    Gaylord Bros., Liverpool, NY.

    This library automation style guide lists specific terms and names often used in the library automation industry. The terms and/or acronyms are listed alphabetically and each is followed by a brief definition. The guide refers to the "Chicago Manual of Style" for general rules, and a notes section is included for the convenience of individual…

  6. Cell Migration and Invasion Assays as Tools for Drug Discovery

    Directory of Open Access Journals (Sweden)

    Keren I. Hulkower

    2011-03-01

    Full Text Available Cell migration and invasion are processes that offer rich targets for intervention in key physiologic and pathologic phenomena such as wound healing and cancer metastasis. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screening process. This article will review the effective use of several principle formats for studying cell motility: scratch assays, transmembrane assays, microfluidic devices and cell exclusion zone assays.

  7. The micronucleus assay in radiation accidents

    International Nuclear Information System (INIS)

    The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is a standardised and validated technique for bio dosimetry. Automated scoring of micronuclei allows large scale applications as in population triage in case of radiation accidents or malevolent use of radioactive sources. The dose detection limit (95% confidence) of the micronucleus assay for individual dose assessment is restricted to 0.2 Gy but can be decreased to 0.1 Gy by scoring centromeres in micronuclei using fluorescence in situ hybridization (FISH). In the past the micronucleus assay was applied for a number of large scale bio monitoring studies of nuclear power plant workers and hospital workers. Baseline micronucleus frequencies depend strongly on age and gender. The assay was also already used for bio dosimetry of radiation accidents. In a multiple endpoint bio dosimetry study for dose assessment of a worker exposed accidentally in 2003 to X-rays, a good agreement was obtained between dose estimates resulting from the micronucleus assay, the scoring of dicentrics and translocations. Automated scoring of micronuclei in combination with centromere signals, allowing systematic bio dosimetry of exposed populations, remains a challenge for the future.

  8. Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces

    OpenAIRE

    Edgeworth, J. A.; Jackson, G. S.; Clarke, A R; Weissmann, C; Collinge, J.

    2009-01-01

    Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental a...

  9. Urolithins, Intestinal Microbial Metabolites of Pomegranate Ellagitannins, Exhibit Potent Antioxidant Activity in Cell-Based Assay

    Science.gov (United States)

    Many health benefits of pomegranate products have been attributed to the potent antioxidant action of their tannin components, mainly punicalagins and ellagic acid. While moving through the intestines, ellagitannins are metabolized by gut bacteria into urolithins that readily enter systemic circulat...

  10. A Sensitive Cell-Based Assay to Measure the Doxycycline Concentration in Biological Samples

    NARCIS (Netherlands)

    W. Kleibeuker; X. Zhou; M. Centlivre; N. Legrand; M. Page; N. Almond; B. Berkhout; A.T. Das

    2009-01-01

    Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measu

  11. Automation in Warehouse Development

    CERN Document Server

    Verriet, Jacques

    2012-01-01

    The warehouses of the future will come in a variety of forms, but with a few common ingredients. Firstly, human operational handling of items in warehouses is increasingly being replaced by automated item handling. Extended warehouse automation counteracts the scarcity of human operators and supports the quality of picking processes. Secondly, the development of models to simulate and analyse warehouse designs and their components facilitates the challenging task of developing warehouses that take into account each customer’s individual requirements and logistic processes. Automation in Warehouse Development addresses both types of automation from the innovative perspective of applied science. In particular, it describes the outcomes of the Falcon project, a joint endeavour by a consortium of industrial and academic partners. The results include a model-based approach to automate warehouse control design, analysis models for warehouse design, concepts for robotic item handling and computer vision, and auton...

  12. Advances in inspection automation

    Science.gov (United States)

    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.

  13. Automated model building

    CERN Document Server

    Caferra, Ricardo; Peltier, Nicholas

    2004-01-01

    This is the first book on automated model building, a discipline of automated deduction that is of growing importance Although models and their construction are important per se, automated model building has appeared as a natural enrichment of automated deduction, especially in the attempt to capture the human way of reasoning The book provides an historical overview of the field of automated deduction, and presents the foundations of different existing approaches to model construction, in particular those developed by the authors Finite and infinite model building techniques are presented The main emphasis is on calculi-based methods, and relevant practical results are provided The book is of interest to researchers and graduate students in computer science, computational logic and artificial intelligence It can also be used as a textbook in advanced undergraduate courses

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

    Science.gov (United States)

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

    2016-08-01

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

  15. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  16. Toxicity assays in nanodrops combining bioassay and morphometric endpoints.

    OpenAIRE

    Lemaire, Frédéric; Mandon, Céline A; Reboud, Julien; Papine, Alexandre; Angulo, Jesus; Pointu, Hervé; Diaz-Latoud, Chantal; Lajaunie, Christian; Chatelain, François; Arrigo, André-Patrick; Schaack, Béatrice

    2007-01-01

    BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested ...

  17. Chef infrastructure automation cookbook

    CERN Document Server

    Marschall, Matthias

    2013-01-01

    Chef Infrastructure Automation Cookbook contains practical recipes on everything you will need to automate your infrastructure using Chef. The book is packed with illustrated code examples to automate your server and cloud infrastructure.The book first shows you the simplest way to achieve a certain task. Then it explains every step in detail, so that you can build your knowledge about how things work. Eventually, the book shows you additional things to consider for each approach. That way, you can learn step-by-step and build profound knowledge on how to go about your configuration management

  18. I-94 Automation FAQs

    Data.gov (United States)

    Department of Homeland Security — In order to increase efficiency, reduce operating costs and streamline the admissions process, U.S. Customs and Border Protection has automated Form I-94 at air and...

  19. Hydrometeorological Automated Data System

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Office of Hydrologic Development of the National Weather Service operates HADS, the Hydrometeorological Automated Data System. This data set contains the last...

  20. Automated Vehicles Symposium 2015

    CERN Document Server

    Beiker, Sven

    2016-01-01

    This edited book comprises papers about the impacts, benefits and challenges of connected and automated cars. It is the third volume of the LNMOB series dealing with Road Vehicle Automation. The book comprises contributions from researchers, industry practitioners and policy makers, covering perspectives from the U.S., Europe and Japan. It is based on the Automated Vehicles Symposium 2015 which was jointly organized by the Association of Unmanned Vehicle Systems International (AUVSI) and the Transportation Research Board (TRB) in Ann Arbor, Michigan, in July 2015. The topical spectrum includes, but is not limited to, public sector activities, human factors, ethical and business aspects, energy and technological perspectives, vehicle systems and transportation infrastructure. This book is an indispensable source of information for academic researchers, industrial engineers and policy makers interested in the topic of road vehicle automation.

  1. Automated Vehicles Symposium 2014

    CERN Document Server

    Beiker, Sven; Road Vehicle Automation 2

    2015-01-01

    This paper collection is the second volume of the LNMOB series on Road Vehicle Automation. The book contains a comprehensive review of current technical, socio-economic, and legal perspectives written by experts coming from public authorities, companies and universities in the U.S., Europe and Japan. It originates from the Automated Vehicle Symposium 2014, which was jointly organized by the Association for Unmanned Vehicle Systems International (AUVSI) and the Transportation Research Board (TRB) in Burlingame, CA, in July 2014. The contributions discuss the challenges arising from the integration of highly automated and self-driving vehicles into the transportation system, with a focus on human factors and different deployment scenarios. This book is an indispensable source of information for academic researchers, industrial engineers, and policy makers interested in the topic of road vehicle automation.

  2. Disassembly automation automated systems with cognitive abilities

    CERN Document Server

    Vongbunyong, Supachai

    2015-01-01

    This book presents a number of aspects to be considered in the development of disassembly automation, including the mechanical system, vision system and intelligent planner. The implementation of cognitive robotics increases the flexibility and degree of autonomy of the disassembly system. Disassembly, as a step in the treatment of end-of-life products, can allow the recovery of embodied value left within disposed products, as well as the appropriate separation of potentially-hazardous components. In the end-of-life treatment industry, disassembly has largely been limited to manual labor, which is expensive in developed countries. Automation is one possible solution for economic feasibility. The target audience primarily comprises researchers and experts in the field, but the book may also be beneficial for graduate students.

  3. Instant Sikuli test automation

    CERN Document Server

    Lau, Ben

    2013-01-01

    Get to grips with a new technology, understand what it is and what it can do for you, and then get to work with the most important features and tasks. A concise guide written in an easy-to follow style using the Starter guide approach.This book is aimed at automation and testing professionals who want to use Sikuli to automate GUI. Some Python programming experience is assumed.

  4. Automated security management

    CERN Document Server

    Al-Shaer, Ehab; Xie, Geoffrey

    2013-01-01

    In this contributed volume, leading international researchers explore configuration modeling and checking, vulnerability and risk assessment, configuration analysis, and diagnostics and discovery. The authors equip readers to understand automated security management systems and techniques that increase overall network assurability and usability. These constantly changing networks defend against cyber attacks by integrating hundreds of security devices such as firewalls, IPSec gateways, IDS/IPS, authentication servers, authorization/RBAC servers, and crypto systems. Automated Security Managemen

  5. Automated Lattice Perturbation Theory

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Monitoring environmental exposures with semen assays

    International Nuclear Information System (INIS)

    Semen studies in humans and animals have yielded extensive and compelling evidence that sperm can be used to assess reproductive potential and diagnose pathology. More recent studies on mutagens and carcinogens both at this and other laboratories suggest that a combination of mouse and human assays can be an efficient, effective approach to monitoring for reproductive hazards in the environment. We are investigating the potential of using variability in sperm morphology and DNA content to quantify and monitor the effects of environmental agents on the human testes. Here we review the status of human and mouse assays for environmental surveillance, discuss the genetic and fertility implications of chemically induced semen changes, and describe the high-speed flow methods being developed to automate sperm assays

  7. Optimization of cell motility evaluation in scratch assay

    Directory of Open Access Journals (Sweden)

    Gotsulyak N. Ya.

    2014-05-01

    Full Text Available A scratch test is one of the most popular methods of classical cell migration assay in a monolayer culture. At the same time, the scratch assay has some disadvantages that can be easily corrected. Aim. Optimization the existent scratch assay on the base of detection of scratch wound surface area and the length of the field of observation which is more objective and less time consuming. Methods. Scratch assay. Results. The modification of scratch assay enables to perform measurement more accurately and rapidly. This approach is more simple and eliminates the main disadvantages of the classical method. Conclusions. The procedure of scratch wound width measurement calculated on the base of detection of cell free area and the length of the field of observation is more effective than the classical wound healing assay. It will be useful for the estimation of cell migration dynamics in monolayer culture for a wide range of live cell based experiments.

  8. Serum indices: managing assay interference.

    Science.gov (United States)

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  9. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The use of the CBMN assay in in vitro genetic toxicology testing is well established and in fact it has become an accepted standard method to assess the genotoxic hazard of chemicals which led to the development of a special guideline by the Organization for Economic Cooperation and Development (OECD, the OECD 487 guideline (Kirsch-Volders et al., 2014. The CBMN assay is an effective tool for the study of cellular and nuclear dysfunction caused by in vitro or in vivo aging, micronutrient deficiency or excess, genotoxins exposure and genetic defects in genome maintenance. It is also fruitful in the emerging fields of nutrigenomics and toxicogenomics and their combinations, as it becomes increasingly clear that nutrient status also impacts on sensitivity to exogenous genotoxins (Fenech, 2005, 2007. Many results obtained by this assay indicate the potential predictive value of the CBMN assay with respect to cancer risk and validate its use as a test for detecting nutritional, environmental and genetic factors that are potentially carcinogenic. Also it is used by pharmaceutical industry, human biomonitoring of genotoxic exposures and its increasing application in preventive medicine and nutrition and the increased investment in the automation of the CBMN assay are indicative of the increasing importance of this test (Fenech, 2007. The comet assay or single-cell gel electrophoresis (SCGE is a simple, sensitive method for detecting DNA-strand breaks. Cells embedded in agarose on a microscope slide are lysed with detergent and 2.5 M NaCl and fresh Triton X-100 to remove membranes and soluble cell constituents, including most histones, leaving the DNA, still supercoiled and attached to a nuclear matrix, as a nucleoid. A break in one strand of a DNA loop is enough to release the supercoiling, and during electrophoresis the relaxed loops are able to extend towards the anode (Fairbairn et al., 1995; Collins et al., 1997; Moller et al., 2000; Azqueta et al., 2009; Collins

  10. Materials Testing and Automation

    Science.gov (United States)

    Cooper, Wayne D.; Zweigoron, Ronald B.

    1980-07-01

    The advent of automation in materials testing has been in large part responsible for recent radical changes in the materials testing field: Tests virtually impossible to perform without a computer have become more straightforward to conduct. In addition, standardized tests may be performed with enhanced efficiency and repeatability. A typical automated system is described in terms of its primary subsystems — an analog station, a digital computer, and a processor interface. The processor interface links the analog functions with the digital computer; it includes data acquisition, command function generation, and test control functions. Features of automated testing are described with emphasis on calculated variable control, control of a variable that is computed by the processor and cannot be read directly from a transducer. Three calculated variable tests are described: a yield surface probe test, a thermomechanical fatigue test, and a constant-stress-intensity range crack-growth test. Future developments are discussed.

  11. Development of a Radioactive Waste Assay System

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-12-31

    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  12. Automating the CMS DAQ

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Automated gas chromatography

    Science.gov (United States)

    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.

  14. A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

    Directory of Open Access Journals (Sweden)

    Albrekt Ann-Sofie

    2011-08-01

    Full Text Available Abstract Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.

  15. Quantitation of residual trypsin in cell-based therapeutics using immobilized α-1-antitrypsin or SBTI in an ELISA format.

    Science.gov (United States)

    Braatz, James A; Elias, Christopher; Finny, Joseph G; Tran, Huan; McCaman, Michael

    2015-02-01

    An Enzyme-Linked Immunosorbent Assay (ELISA) has been developed for the quantitation of porcine trypsin as a process residual in cell therapy products based on its capture by either of two immobilized anti-trypsins, α-1-antitrypsin (α1AT) or soybean trypsin inhibitor (SBTI) followed by detection with a polyclonal goat anti-porcine trypsin-IgG conjugated with peroxidase. It was demonstrated that an extended range of antigen quantitation could be achieved that covered nearly three orders of magnitude of trypsin concentration. The utility of the assay was demonstrated by its application to samples generated in a cell-based therapeutic manufacturing setting.

  16. High-Throughput Cell Toxicity Assays.

    Science.gov (United States)

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  17. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  18. Automated solvent concentrator

    Science.gov (United States)

    Griffith, J. S.; Stuart, J. L.

    1976-01-01

    Designed for automated drug identification system (AUDRI), device increases concentration by 100. Sample is first filtered, removing particulate contaminants and reducing water content of sample. Sample is extracted from filtered residue by specific solvent. Concentrator provides input material to analysis subsystem.

  19. Protokoller til Home Automation

    DEFF Research Database (Denmark)

    Kjær, Kristian Ellebæk

    2008-01-01

    computer, der kan skifte mellem foruddefinerede indstillinger. Nogle gange kan computeren fjernstyres over internettet, så man kan se hjemmets status fra en computer eller måske endda fra en mobiltelefon. Mens nævnte anvendelser er klassiske indenfor home automation, er yderligere funktionalitet dukket op...

  20. ELECTROPNEUMATIC AUTOMATION EDUCATIONAL LABORATORY

    OpenAIRE

    Dolgorukov, S. O.; National Aviation University; Roman, B. V.; National Aviation University

    2013-01-01

    The article reflects current situation in education regarding mechatronics learning difficulties. Com-plex of laboratory test benches on electropneumatic automation are considered as a tool in advancing through technical science. Course of laboratory works developed to meet the requirement of efficient and reliable way of practical skills acquisition is regarded the simplest way for students to learn the ba-sics of mechatronics.

  1. Building Automation Systems.

    Science.gov (United States)

    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…

  2. Test Construction: Automated

    NARCIS (Netherlands)

    Veldkamp, Bernard P.

    2014-01-01

    Optimal test construction deals with automated assembly of tests for educational and psychological measurement. Items are selected from an item bank to meet a predefined set of test specifications. Several models for optimal test construction are presented, and two algorithms for optimal test assemb

  3. Test Construction: Automated

    NARCIS (Netherlands)

    Veldkamp, Bernard P.

    2016-01-01

    Optimal test construction deals with automated assembly of tests for educational and psychological measurement. Items are selected from an item bank to meet a predefined set of test specifications. Several models for optimal test construction are presented, and two algorithms for optimal test assemb

  4. Automated Web Applications Testing

    Directory of Open Access Journals (Sweden)

    Alexandru Dan CĂPRIŢĂ

    2009-01-01

    Full Text Available Unit tests are a vital part of several software development practicesand processes such as Test-First Programming, Extreme Programming andTest-Driven Development. This article shortly presents the software quality andtesting concepts as well as an introduction to an automated unit testingframework for PHP web based applications.

  5. Automated Student Model Improvement

    Science.gov (United States)

    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…

  6. Myths in test automation

    Directory of Open Access Journals (Sweden)

    Jazmine Francis

    2015-01-01

    Full Text Available Myths in automation of software testing is an issue of discussion that echoes about the areas of service in validation of software industry. Probably, the first though that appears in knowledgeable reader would be Why this old topic again? What's New to discuss the matter? But, for the first time everyone agrees that undoubtedly automation testing today is not today what it used to be ten or fifteen years ago, because it has evolved in scope and magnitude. What began as a simple linear scripts for web applications today has a complex architecture and a hybrid framework to facilitate the implementation of testing applications developed with various platforms and technologies. Undoubtedly automation has advanced, but so did the myths associated with it. The change in perspective and knowledge of people on automation has altered the terrain. This article reflects the points of views and experience of the author in what has to do with the transformation of the original myths in new versions, and how they are derived; also provides his thoughts on the new generation of myths.

  7. A cell-based computational modeling approach for developing site-directed molecular probes.

    Directory of Open Access Journals (Sweden)

    Jing-Yu Yu

    Full Text Available Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.

  8. Automating spectral measurements

    Science.gov (United States)

    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.

  9. Development of robotic plasma radiochemical assays for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J. [Brookhaven National Lab., Upton, NY (United States). Dept. of Chemistry

    1995-12-01

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [{sup 11}C]Benztropine, [{sup 11}C]cocaine, [{sup 11}C]clorgyline, [{sup 11}C]deprenyl, [{sup 11}C]methadone, [{sup 11}C]methylphenidate, [{sup 11}C]raclorpride, and [{sup 11}C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers.

  10. Development of robotic plasma radiochemical assays for positron emission tomography

    International Nuclear Information System (INIS)

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [11C]Benztropine, [11C]cocaine, [11C]clorgyline, [11C]deprenyl, [11C]methadone, [11C]methylphenidate, [11C]raclorpride, and [11C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers

  11. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  12. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  13. Transient expression assays in tobacco protoplasts.

    Science.gov (United States)

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain

    2013-01-01

    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  14. Rapid automated nuclear chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, R.A.

    1979-05-31

    Rapid Automated Nuclear Chemistry (RANC) can be thought of as the Z-separation of Neutron-rich Isotopes by Automated Methods. The range of RANC studies of fission and its products is large. In a sense, the studies can be categorized into various energy ranges from the highest where the fission process and particle emission are considered, to low energies where nuclear dynamics are being explored. This paper presents a table which gives examples of current research using RANC on fission and fission products. The remainder of this text is divided into three parts. The first contains a discussion of the chemical methods available for the fission product elements, the second describes the major techniques, and in the last section, examples of recent results are discussed as illustrations of the use of RANC.

  15. Automated theorem proving.

    Science.gov (United States)

    Plaisted, David A

    2014-03-01

    Automated theorem proving is the use of computers to prove or disprove mathematical or logical statements. Such statements can express properties of hardware or software systems, or facts about the world that are relevant for applications such as natural language processing and planning. A brief introduction to propositional and first-order logic is given, along with some of the main methods of automated theorem proving in these logics. These methods of theorem proving include resolution, Davis and Putnam-style approaches, and others. Methods for handling the equality axioms are also presented. Methods of theorem proving in propositional logic are presented first, and then methods for first-order logic. WIREs Cogn Sci 2014, 5:115-128. doi: 10.1002/wcs.1269 CONFLICT OF INTEREST: The authors has declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website. PMID:26304304

  16. ATLAS Distributed Computing Automation

    CERN Document Server

    Schovancova, J; The ATLAS collaboration; Borrego, C; Campana, S; Di Girolamo, A; Elmsheuser, J; Hejbal, J; Kouba, T; Legger, F; Magradze, E; Medrano Llamas, R; Negri, G; Rinaldi, L; Sciacca, G; Serfon, C; Van Der Ster, D C

    2012-01-01

    The ATLAS Experiment benefits from computing resources distributed worldwide at more than 100 WLCG sites. The ATLAS Grid sites provide over 100k CPU job slots, over 100 PB of storage space on disk or tape. Monitoring of status of such a complex infrastructure is essential. The ATLAS Grid infrastructure is monitored 24/7 by two teams of shifters distributed world-wide, by the ATLAS Distributed Computing experts, and by site administrators. In this paper we summarize automation efforts performed within the ATLAS Distributed Computing team in order to reduce manpower costs and improve the reliability of the system. Different aspects of the automation process are described: from the ATLAS Grid site topology provided by the ATLAS Grid Information System, via automatic site testing by the HammerCloud, to automatic exclusion from production or analysis activities.

  17. Rapid automated nuclear chemistry

    International Nuclear Information System (INIS)

    Rapid Automated Nuclear Chemistry (RANC) can be thought of as the Z-separation of Neutron-rich Isotopes by Automated Methods. The range of RANC studies of fission and its products is large. In a sense, the studies can be categorized into various energy ranges from the highest where the fission process and particle emission are considered, to low energies where nuclear dynamics are being explored. This paper presents a table which gives examples of current research using RANC on fission and fission products. The remainder of this text is divided into three parts. The first contains a discussion of the chemical methods available for the fission product elements, the second describes the major techniques, and in the last section, examples of recent results are discussed as illustrations of the use of RANC

  18. The Automated Medical Office

    OpenAIRE

    Petreman, Mel

    1990-01-01

    With shock and surprise many physicians learned in the 1980s that they must change the way they do business. Competition for patients, increasing government regulation, and the rapidly escalating risk of litigation forces physicians to seek modern remedies in office management. The author describes a medical clinic that strives to be paperless using electronic innovation to solve the problems of medical practice management. A computer software program to automate information management in a c...

  19. A droplet-to-digital (D2D) microfluidic device for single cell assays.

    Science.gov (United States)

    Shih, Steve C C; Gach, Philip C; Sustarich, Jess; Simmons, Blake A; Adams, Paul D; Singh, Seema; Singh, Anup K

    2015-01-01

    in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays. PMID:25354549

  20. Automation in biological crystallization.

    Science.gov (United States)

    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.

  1. Cell Migration and Invasion Assays as Tools for Drug Discovery

    OpenAIRE

    Hulkower, Keren I.; Herber, Renee L.

    2011-01-01

    Cell migration and invasion are processes that offer rich targets for intervention in key physiologic and pathologic phenomena such as wound healing and cancer metastasis. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screeni...

  2. Genotoxicity Assessment of Erythritol by Using Short-term Assay

    OpenAIRE

    Chung, Young-Shin; Lee, Michael

    2013-01-01

    Erythritol is a sugar alcohol that is widely used as a natural sugar substitute. Thus, the safety of its usage is very important. In the present study, short-term genotoxicity assays were conducted to evaluate the potential genotoxic effects of erythritol. According to the OECD test guidelines, the maximum test dose was 5,000 μg/plate in bacterial reverse mutation tests, 5,000 μg/ml in cell-based assays, and 5,000 mg/kg for in vivo testing. An Ames test did not reveal any positive results. No...

  3. CPTAC Assay Portal: a repository of targeted proteomic assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  4. Automation in organizations: Eternal conflict

    Science.gov (United States)

    Dieterly, D. L.

    1981-01-01

    Some ideas on and insights into the problems associated with automation in organizations are presented with emphasis on the concept of automation, its relationship to the individual, and its impact on system performance. An analogy is drawn, based on an American folk hero, to emphasize the extent of the problems encountered when dealing with automation within an organization. A model is proposed to focus attention on a set of appropriate dimensions. The function allocation process becomes a prominent aspect of the model. The current state of automation research is mentioned in relation to the ideas introduced. Proposed directions for an improved understanding of automation's effect on the individual's efficiency are discussed. The importance of understanding the individual's perception of the system in terms of the degree of automation is highlighted.

  5. Automated Assessment, Face to Face

    OpenAIRE

    Rizik M. H. Al-Sayyed; Amjad Hudaib; Muhannad AL-Shboul; Yousef Majdalawi; Mohammed Bataineh

    2010-01-01

    This research paper evaluates the usability of automated exams and compares them with the paper-and-pencil traditional ones. It presents the results of a detailed study conducted at The University of Jordan (UoJ) that comprised students from 15 faculties. A set of 613 students were asked about their opinions concerning automated exams; and their opinions were deeply analyzed. The results indicate that most students reported that they are satisfied with using automated exams but they have sugg...

  6. Automation System Products and Research

    OpenAIRE

    Rintala, Mikko; Sormunen, Jussi; Kuisma, Petri; Rahkala, Matti

    2014-01-01

    Automation systems are used in most buildings nowadays. In the past they were mainly used in industry to control and monitor critical systems. During the past few decades the automation systems have become more common and are used today from big industrial solutions to homes of private customers. With the growing need for ecologic and cost-efficient management systems, home and building automation systems are becoming a standard way of controlling lighting, ventilation, heating etc. Auto...

  7. Test Automation of Online Games

    OpenAIRE

    Schoenfeldt, Alexander

    2015-01-01

    State of the art browser games are increasingly complex pieces of software with extensive code basis. With increasing complexity, a software becomes harder to maintain. Automated regression testing can simplify these maintenance processes and thereby enable developers as well as testers to spend their workforce more efficiently. This thesis addresses the utilization of automated tests in web applications. As a use case test automation is applied to an online-based strategy game for the bro...

  8. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  9. Lateral flow assays.

    Science.gov (United States)

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  10. New Rapid Spore Assay

    Science.gov (United States)

    Kminek, Gerhard; Conley, Catharine

    2012-07-01

    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  11. RNA-protein binding kinetics in an automated microfluidic reactor.

    Science.gov (United States)

    Ridgeway, William K; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R

    2009-11-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

  12. Mechatronic Design Automation

    DEFF Research Database (Denmark)

    Fan, Zhun

    This book proposes a novel design method that combines both genetic programming (GP) to automatically explore the open-ended design space and bond graphs (BG) to unify design representations of multi-domain Mechatronic systems. Results show that the method, formally called GPBG method, can...... successfully design analogue filters, vibration absorbers, micro-electro-mechanical systems, and vehicle suspension systems, all in an automatic or semi-automatic way. It also investigates the very important issue of co-designing plant-structures and dynamic controllers in automated design of Mechatronic...

  13. The automated medical office.

    Science.gov (United States)

    Petreman, M

    1990-08-01

    With shock and surprise many physicians learned in the 1980s that they must change the way they do business. Competition for patients, increasing government regulation, and the rapidly escalating risk of litigation forces physicians to seek modern remedies in office management. The author describes a medical clinic that strives to be paperless using electronic innovation to solve the problems of medical practice management. A computer software program to automate information management in a clinic shows that practical thinking linked to advanced technology can greatly improve office efficiency.

  14. AUTOMATED API TESTING APPROACH

    Directory of Open Access Journals (Sweden)

    SUNIL L. BANGARE

    2012-02-01

    Full Text Available Software testing is an investigation conducted to provide stakeholders with information about the quality of the product or service under test. With the help of software testing we can verify or validate the software product. Normally testing will be done after development of software but we can perform the software testing at the time of development process also. This paper will give you a brief introduction about Automated API Testing Tool. This tool of testing will reduce lots of headache after the whole development of software. It saves time as well as money. Such type of testing is helpful in the Industries & Colleges also.

  15. World-wide distribution automation systems

    Energy Technology Data Exchange (ETDEWEB)

    Devaney, T.M.

    1994-12-31

    A worldwide power distribution automation system is outlined. Distribution automation is defined and the status of utility automation is discussed. Other topics discussed include a distribution management system, substation feeder, and customer functions, potential benefits, automation costs, planning and engineering considerations, automation trends, databases, system operation, computer modeling of system, and distribution management systems.

  16. Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry.

    Science.gov (United States)

    Otvos, Reka A; van Nierop, Pim; Niessen, Wilfried M A; Kini, R Manjunatha; Somsen, Govert W; Smit, August B; Kool, Jeroen

    2016-05-01

    This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC-FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist-agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist-agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup. PMID:27046509

  17. Cell-based biosensors: Towards the development of cellular monitoring

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Cell-based biosensors (CBBs), a research hotspot of biosensors, which treat living cells as sensing elements, can detect the functional information of biologically active analytes. They characterize with high sensitivity, excellent selectivity and rapid response, and have been applied in many fields, such as biomedicine, environmental monitoring and pharmaceutical screening. Recently cell-cultured technology, silicon microfabrication technology and genetic technology have promoted exploration of CBBs dramatically. To elucidate the novel research findings and applications of cell- based biosensors, this paper summarizes various research approaches, presents some challenges and proposes the research trends.

  18. Automation from pictures

    International Nuclear Information System (INIS)

    The state transition diagram (STD) model has been helpful in the design of real time software, especially with the emergence of graphical computer aided software engineering (CASE) tools. Nevertheless, the translation of the STD to real time code has in the past been primarily a manual task. At Los Alamos we have automated this process. The designer constructs the STD using a CASE tool (Cadre Teamwork) using a special notation for events and actions. A translator converts the STD into an intermediate state notation language (SNL), and this SNL is compiled directly into C code (a state program). Execution of the state program is driven by external events, allowing multiple state programs to effectively share the resources of the host processor. Since the design and the code are tightly integrated through the CASE tool, the design and code never diverge, and we avoid design obsolescence. Furthermore, the CASE tool automates the production of formal technical documents from the graphic description encapsulated by the CASE tool. (author)

  19. Automated Test Case Generation

    CERN Document Server

    CERN. Geneva

    2015-01-01

    I would like to present the concept of automated test case generation. I work on it as part of my PhD and I think it would be interesting also for other people. It is also the topic of a workshop paper that I am introducing in Paris. (abstract below) Please note that the talk itself would be more general and not about the specifics of my PhD, but about the broad field of Automated Test Case Generation. I would introduce the main approaches (combinatorial testing, symbolic execution, adaptive random testing) and their advantages and problems. (oracle problem, combinatorial explosion, ...) Abstract of the paper: Over the last decade code-based test case generation techniques such as combinatorial testing or dynamic symbolic execution have seen growing research popularity. Most algorithms and tool implementations are based on finding assignments for input parameter values in order to maximise the execution branch coverage. Only few of them consider dependencies from outside the Code Under Test’s scope such...

  20. Automated Postediting of Documents

    CERN Document Server

    Knight, K; Knight, Kevin; Chander, Ishwar

    1994-01-01

    Large amounts of low- to medium-quality English texts are now being produced by machine translation (MT) systems, optical character readers (OCR), and non-native speakers of English. Most of this text must be postedited by hand before it sees the light of day. Improving text quality is tedious work, but its automation has not received much research attention. Anyone who has postedited a technical report or thesis written by a non-native speaker of English knows the potential of an automated postediting system. For the case of MT-generated text, we argue for the construction of postediting modules that are portable across MT systems, as an alternative to hardcoding improvements inside any one system. As an example, we have built a complete self-contained postediting module for the task of article selection (a, an, the) for English noun phrases. This is a notoriously difficult problem for Japanese-English MT. Our system contains over 200,000 rules derived automatically from online text resources. We report on l...

  1. Maneuver Automation Software

    Science.gov (United States)

    Uffelman, Hal; Goodson, Troy; Pellegrin, Michael; Stavert, Lynn; Burk, Thomas; Beach, David; Signorelli, Joel; Jones, Jeremy; Hahn, Yungsun; Attiyah, Ahlam; Illsley, Jeannette

    2009-01-01

    The Maneuver Automation Software (MAS) automates the process of generating commands for maneuvers to keep the spacecraft of the Cassini-Huygens mission on a predetermined prime mission trajectory. Before MAS became available, a team of approximately 10 members had to work about two weeks to design, test, and implement each maneuver in a process that involved running many maneuver-related application programs and then serially handing off data products to other parts of the team. MAS enables a three-member team to design, test, and implement a maneuver in about one-half hour after Navigation has process-tracking data. MAS accepts more than 60 parameters and 22 files as input directly from users. MAS consists of Practical Extraction and Reporting Language (PERL) scripts that link, sequence, and execute the maneuver- related application programs: "Pushing a single button" on a graphical user interface causes MAS to run navigation programs that design a maneuver; programs that create sequences of commands to execute the maneuver on the spacecraft; and a program that generates predictions about maneuver performance and generates reports and other files that enable users to quickly review and verify the maneuver design. MAS can also generate presentation materials, initiate electronic command request forms, and archive all data products for future reference.

  2. A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins

    Science.gov (United States)

    Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.

    2005-11-01

    A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

  3. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    Science.gov (United States)

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  4. A Colloidal Stability Assay Suitable for High-Throughput Screening.

    Science.gov (United States)

    Abarca, Carla; Ali, M Monsur; Yang, Songtao; Dong, Xiaofei; Pelton, Robert H

    2016-03-01

    A library of 32 polystyrene copolymer latexes, with diameters ranging between 53 and 387 nm, was used to develop and demonstrate a high-throughput assay using a 96-well microplate platform to measure critical coagulation concentrations, a measure of colloidal stability. The most robust assay involved an automated centrifugation-decantation step to remove latex aggregates before absorbance measurements, eliminating aggregate interference with optical measurements made through the base of the multiwell plates. For smaller nanoparticles (diameter aggregation; however, the results were less sensitive than the absorbance measurements. PMID:26857643

  5. Assay method and compositions

    International Nuclear Information System (INIS)

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine(3H)-methyl. The O-methylated (3H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin-3H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin-3H and raising the pH of the aqueous periodate phase from which O-methylated (3H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  6. Non-separation assay for glycohemoglobin.

    Science.gov (United States)

    Blincko, S; Edwards, R

    1998-06-01

    The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD +/-0.03)%HbA1 +0.6(SD +/-0.3), S(y/x) = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD +/-0.04)%HbA1c + 1.8(SD +/-0.3), S(y/x) = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was <2% (intraassay, range 5-22% GHb) and <4.2% (interassay, range 4-16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 micromol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results. PMID:9625057

  7. Multisite Analytical Evaluation of the Abbott ARCHITECT Cyclosporine Assay.

    OpenAIRE

    Wallemacq, Pierre; Maine, Gregory,; Berg, Keith; Rosiere, Thomas; Marquet, Pierre; Aimo, Giuseppe; Mengozzi, Giulio; Young, Juliana; Wonigert, Kurt; Krestchmer, Robert; Wermuth, Bendicht; Schmid, Rainer,

    2010-01-01

    International audience The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Cyclosporine (CsA) immunoassay in 7 clinical laboratories in comparison to liquid chromatography/tandem mass spectrometry (LC/MS/MS), Abbott TDx, Cobas Integra 800, and the Dade Dimension Xpand immunoassay. The ARCHITECT assay uses a whole blood specimen, a pretreatment step with organic reagents to precipitate proteins and extract the drug, followed by a 2-step automated i...

  8. Get smart! automate your house!

    NARCIS (Netherlands)

    Van Amstel, P.; Gorter, N.; De Rouw, J.

    2016-01-01

    This "designers' manual" is made during the TIDO-course AR0531 Innovation and Sustainability This manual will help you in reducing both energy usage and costs by automating your home. It gives an introduction to a number of home automation systems that every homeowner can install.

  9. Automated Methods Of Corrosion Measurements

    DEFF Research Database (Denmark)

    Bech-Nielsen, Gregers; Andersen, Jens Enevold Thaulov; Reeve, John Ch;

    1997-01-01

    The chapter describes the following automated measurements: Corrosion Measurements by Titration, Imaging Corrosion by Scanning Probe Microscopy, Critical Pitting Temperature and Application of the Electrochemical Hydrogen Permeation Cell.......The chapter describes the following automated measurements: Corrosion Measurements by Titration, Imaging Corrosion by Scanning Probe Microscopy, Critical Pitting Temperature and Application of the Electrochemical Hydrogen Permeation Cell....

  10. Automated separation for heterogeneous immunoassays

    OpenAIRE

    Truchaud, A.; Barclay, J; Yvert, J. P.; Capolaghi, B.

    1991-01-01

    Beside general requirements for modern automated systems, immunoassay automation involves specific requirements as a separation step for heterogeneous immunoassays. Systems are designed according to the solid phase selected: dedicated or open robots for coated tubes and wells, systems nearly similar to chemistry analysers in the case of magnetic particles, and a completely original design for those using porous and film materials.

  11. Automated Test-Form Generation

    Science.gov (United States)

    van der Linden, Wim J.; Diao, Qi

    2011-01-01

    In automated test assembly (ATA), the methodology of mixed-integer programming is used to select test items from an item bank to meet the specifications for a desired test form and optimize its measurement accuracy. The same methodology can be used to automate the formatting of the set of selected items into the actual test form. Three different…

  12. Translation: Aids, Robots, and Automation.

    Science.gov (United States)

    Andreyewsky, Alexander

    1981-01-01

    Examines electronic aids to translation both as ways to automate it and as an approach to solve problems resulting from shortage of qualified translators. Describes the limitations of robotic MT (Machine Translation) systems, viewing MAT (Machine-Aided Translation) as the only practical solution and the best vehicle for further automation. (MES)

  13. Opening up Library Automation Software

    Science.gov (United States)

    Breeding, Marshall

    2009-01-01

    Throughout the history of library automation, the author has seen a steady advancement toward more open systems. In the early days of library automation, when proprietary systems dominated, the need for standards was paramount since other means of inter-operability and data exchange weren't possible. Today's focus on Application Programming…

  14. Global Distribution of Businesses Marketing Stem Cell-Based Interventions.

    Science.gov (United States)

    Berger, Israel; Ahmad, Amina; Bansal, Akhil; Kapoor, Tanvir; Sipp, Douglas; Rasko, John E J

    2016-08-01

    A structured search reveals that online marketing of stem-cell-based interventions is skewed toward developed economies including the United States, Ireland, Australia, and Germany. Websites made broad, imprecise therapeutic claims and frequently failed to detail procedures. Widespread marketing poses challenges to regulators, bioethicists, and those seeking realistic hope from therapies. PMID:27494673

  15. Interpreting coagulation assays.

    Science.gov (United States)

    Green, David

    2010-09-01

    The interpretation of coagulation assays requires knowledge of the principal clotting pathways. The activated partial thromboplastin time is sensitive to all hemostatic factors except FVII, whereas the prothrombin time reflects levels of prothrombin and FV, FVII, and FX. Using the two tests in concert is helpful in identifying hemophilia, the coagulopathy of liver disease, and disseminated intravascular coagulation. In addition, the activated partial thromboplastin time and prothrombin time are used for monitoring anticoagulant therapy with heparin and warfarin, respectively. Measurement of D-dimer is informative in patients suspected of having thrombotic disorders and determining the risk of thrombosis recurrence. Mixing tests distinguish clotting factor deficiencies from circulating anticoagulants such as heparin, the lupus anticoagulant, and antibodies directed against specific clotting factors. The modified Bethesda assay detects and provides an indication of the strength of FVIII inhibitors. However, interpreting the results of coagulation assays is not always straightforward, and expert consultation is occasionally required to resolve difficult clinical situations. PMID:20855988

  16. Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A.

    Science.gov (United States)

    Bowyer, Annette E; Van Veen, Joost J; Goodeve, Anne C; Kitchen, Steve; Makris, Michael

    2013-12-01

    The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.

  17. Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

    OpenAIRE

    Cornaglia, Matteo; Krishnamani, Gopalan; Mouchiroud, Laurent; Sorrentino, Vincenzo; Lehnert, Thomas; Auwerx, Johan; Gijs, Martin A. M.

    2016-01-01

    Background While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions – e.g. neurodegenerative diseases – often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field. ...

  18. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  19. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  20. Premarket Evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay

    OpenAIRE

    Stellrecht, K A; Espino, A. A.; Maceira, V. P.; Nattanmai, S. M.; Butt, S. A.; Wroblewski, D.; Hannett, G. E.; Musser, K. A.

    2014-01-01

    Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. diffici...

  1. Automated Motivic Analysis

    DEFF Research Database (Denmark)

    Lartillot, Olivier

    2016-01-01

    Motivic analysis provides very detailed understanding of musical composi- tions, but is also particularly difficult to formalize and systematize. A computational automation of the discovery of motivic patterns cannot be reduced to a mere extraction of all possible sequences of descriptions....... The systematic approach inexorably leads to a proliferation of redundant structures that needs to be addressed properly. Global filtering techniques cause a drastic elimination of interesting structures that damages the quality of the analysis. On the other hand, a selection of closed patterns allows...... for lossless compression. The structural complexity resulting from successive repetitions of patterns can be controlled through a simple modelling of cycles. Generally, motivic patterns cannot always be defined solely as sequences of descriptions in a fixed set of dimensions: throughout the descriptions...

  2. Robust automated knowledge capture.

    Energy Technology Data Exchange (ETDEWEB)

    Stevens-Adams, Susan Marie; Abbott, Robert G.; Forsythe, James Chris; Trumbo, Michael Christopher Stefan; Haass, Michael Joseph; Hendrickson, Stacey M. Langfitt

    2011-10-01

    This report summarizes research conducted through the Sandia National Laboratories Robust Automated Knowledge Capture Laboratory Directed Research and Development project. The objective of this project was to advance scientific understanding of the influence of individual cognitive attributes on decision making. The project has developed a quantitative model known as RumRunner that has proven effective in predicting the propensity of an individual to shift strategies on the basis of task and experience related parameters. Three separate studies are described which have validated the basic RumRunner model. This work provides a basis for better understanding human decision making in high consequent national security applications, and in particular, the individual characteristics that underlie adaptive thinking.

  3. Automated Electrostatics Environmental Chamber

    Science.gov (United States)

    Calle, Carlos; Lewis, Dean C.; Buchanan, Randy K.; Buchanan, Aubri

    2005-01-01

    The Mars Electrostatics Chamber (MEC) is an environmental chamber designed primarily to create atmospheric conditions like those at the surface of Mars to support experiments on electrostatic effects in the Martian environment. The chamber is equipped with a vacuum system, a cryogenic cooling system, an atmospheric-gas replenishing and analysis system, and a computerized control system that can be programmed by the user and that provides both automation and options for manual control. The control system can be set to maintain steady Mars-like conditions or to impose temperature and pressure variations of a Mars diurnal cycle at any given season and latitude. In addition, the MEC can be used in other areas of research because it can create steady or varying atmospheric conditions anywhere within the wide temperature, pressure, and composition ranges between the extremes of Mars-like and Earth-like conditions.

  4. Automated Standard Hazard Tool

    Science.gov (United States)

    Stebler, Shane

    2014-01-01

    The current system used to generate standard hazard reports is considered cumbersome and iterative. This study defines a structure for this system's process in a clear, algorithmic way so that standard hazard reports and basic hazard analysis may be completed using a centralized, web-based computer application. To accomplish this task, a test server is used to host a prototype of the tool during development. The prototype is configured to easily integrate into NASA's current server systems with minimal alteration. Additionally, the tool is easily updated and provides NASA with a system that may grow to accommodate future requirements and possibly, different applications. Results of this project's success are outlined in positive, subjective reviews complete by payload providers and NASA Safety and Mission Assurance personnel. Ideally, this prototype will increase interest in the concept of standard hazard automation and lead to the full-scale production of a user-ready application.

  5. Automated synthetic scene generation

    Science.gov (United States)

    Givens, Ryan N.

    Physics-based simulations generate synthetic imagery to help organizations anticipate system performance of proposed remote sensing systems. However, manually constructing synthetic scenes which are sophisticated enough to capture the complexity of real-world sites can take days to months depending on the size of the site and desired fidelity of the scene. This research, sponsored by the Air Force Research Laboratory's Sensors Directorate, successfully developed an automated approach to fuse high-resolution RGB imagery, lidar data, and hyperspectral imagery and then extract the necessary scene components. The method greatly reduces the time and money required to generate realistic synthetic scenes and developed new approaches to improve material identification using information from all three of the input datasets.

  6. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    Science.gov (United States)

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  7. Automated Essay Scoring

    Directory of Open Access Journals (Sweden)

    Semire DIKLI

    2006-01-01

    Full Text Available Automated Essay Scoring Semire DIKLI Florida State University Tallahassee, FL, USA ABSTRACT 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, 2004. AES is defined as the computer technology that evaluates and scores the written prose (Shermis & Barrera, 2002; Shermis & Burstein, 2003; Shermis, Raymat, & Barrera, 2003. Revision and feedback are essential aspects of the writing process. Students need to receive feedback in order to increase their writing quality. However, responding to student papers can be a burden for teachers. Particularly if they have large number of students and if they assign frequent writing assignments, providing individual feedback to student essays might be quite time consuming. AES systems can be very useful because they can provide the student with a score as well as feedback within seconds (Page, 2003. Four types of AES systems, which are widely used by testing companies, universities, and public schools: Project Essay Grader (PEG, Intelligent Essay Assessor (IEA, E-rater, and IntelliMetric. AES is a developing technology. Many AES systems are used to overcome time, cost, and generalizability issues in writing assessment. The accuracy and reliability of these systems have been proven to be high. The search for excellence in machine scoring of essays is continuing and numerous studies are being conducted to improve the effectiveness of the AES systems.

  8. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  9. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis Skovsgaard; Kirkby, Nikolai S; Bestle, Morten H;

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  10. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  11. Automating the radiographic NDT process

    International Nuclear Information System (INIS)

    Automation, the removal of the human element in inspection, has not been generally applied to film radiographic NDT. The justication for automating is not only productivity but also reliability of results. Film remains in the automated system of the future because of its extremely high image content, approximately 8 x 109 bits per 14 x 17. The equivalent to 2200 computer floppy discs. Parts handling systems and robotics applied for manufacturing and some NDT modalities, should now be applied to film radiographic NDT systems. Automatic film handling can be achieved with the daylight NDT film handling system. Automatic film processing is becoming the standard in industry and can be coupled to the daylight system. Robots offer the opportunity to automate fully the exposure step. Finally, computer aided interpretation appears on the horizon. A unit which laser scans a 14 x 17 (inch) film in 6 - 8 seconds can digitize film information for further manipulation and possible automatic interrogations (computer aided interpretation). The system called FDRS (for Film Digital Radiography System) is moving toward 50 micron (*approx* 16 lines/mm) resolution. This is believed to meet the need of the majority of image content needs. We expect the automated system to appear first in parts (modules) as certain operations are automated. The future will see it all come together in an automated film radiographic NDT system (author)

  12. Precision multidimensional assay for high-throughput microRNA drug discovery

    OpenAIRE

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modul...

  13. Automated Fluid Interface System (AFIS)

    Science.gov (United States)

    1990-01-01

    Automated remote fluid servicing will be necessary for future space missions, as future satellites will be designed for on-orbit consumable replenishment. In order to develop an on-orbit remote servicing capability, a standard interface between a tanker and the receiving satellite is needed. The objective of the Automated Fluid Interface System (AFIS) program is to design, fabricate, and functionally demonstrate compliance with all design requirements for an automated fluid interface system. A description and documentation of the Fairchild AFIS design is provided.

  14. Comparison of automated and manual determination of HER2 status in breast cancer for diagnostic use: a comparative methodological study using the Ventana BenchMark automated staining system and manual tests.

    Science.gov (United States)

    Bánkfalvi, Agnes; Boecker, Werner; Reiner, Angelika

    2004-10-01

    This study was performed to test the validity of manual and automated HER2 tests in one hundred routinely formalin-fixed and paraffin-embedded diagnostic breast carcinoma tissues. Immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) assays for HER2 were separately carried out in two institutes of pathology specialised in diagnostics of breast diseases. Manual immunostaining was performed by the Dako-HercepTest. Automated IHC and FISH were carried out in the Ventana BenchMark platform by using the Pathway-CB11 antibody and the INFORM(R) HER2 probe, respectively. Positivity rates varied between HercepTest (26%), automated CB11 IHC (23%) and automated FISH (22%). Overall concordance between positive (2+, 3+) and negative (0; 1+) results of manual and automated IHC was 97%, between automated FISH and IHC 92%, and between automated FISH and HercepTest 89%. The frequency of 2+ IHC scores was 13% using the BenchMark and 14% with the HercepTest; 6/12 and 8/14 of the respective cases were not amplified by FISH. Automated FISH was not interpretable in 11 of 100 specimens. In the 89 informative cases, automated IHC resulted in increased specificity (92% vs. 88%), increased positive predictive value (73% vs. 64%) and increased efficiency (92% vs. 89%). We conclude that automation improves the accuracy of HER2 detection in diagnostic breast carcinoma tissues and provides a new approach for the global standardization of clinical HER2 tests.

  15. Stem Cell-Based Therapies for Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Lei Hao

    2014-01-01

    Full Text Available In recent years, stem cell-based approaches have attracted more attention from scientists and clinicians due to their possible therapeutical effect on stroke. Animal studies have demonstrated that the beneficial effects of stem cells including embryonic stem cells (ESCs, inducible pluripotent stem cells (iPSCs, neural stem cells (NSCs, and mesenchymal stem cell (MSCs might be due to cell replacement, neuroprotection, endogenous neurogenesis, angiogenesis, and modulation on inflammation and immune response. Although several clinical studies have shown the high efficiency and safety of stem cell in stroke management, mainly MSCs, some issues regarding to cell homing, survival, tracking, safety, and optimal cell transplantation protocol, such as cell dose and time window, should be addressed. Undoubtably, stem cell-based gene therapy represents a novel potential therapeutic strategy for stroke in future.

  16. Cell-Based Strategies for Meniscus Tissue Engineering

    OpenAIRE

    Wei Niu; Weimin Guo; Shufeng Han; Yun Zhu; Shuyun Liu; Quanyi Guo

    2016-01-01

    Meniscus injuries remain a significant challenge due to the poor healing potential of the inner avascular zone. Following a series of studies and clinical trials, tissue engineering is considered a promising prospect for meniscus repair and regeneration. As one of the key factors in tissue engineering, cells are believed to be highly beneficial in generating bionic meniscus structures to replace injured ones in patients. Therefore, cell-based strategies for meniscus tissue engineering play a ...

  17. Stem Cell-Based Therapeutics to Improve Wound Healing

    OpenAIRE

    Hu, Michael S.; Tripp Leavitt; Samir Malhotra; Dominik Duscher; Pollhammer, Michael S.; Walmsley, Graham G.; Zeshaan N. Maan; Alexander T. M. Cheung; Manfred Schmidt; Georg M. Huemer; Longaker, Michael T.; Peter Lorenz, H.

    2015-01-01

    Issues surrounding wound healing have garnered deep scientific interest as well as booming financial markets invested in novel wound therapies. Much progress has been made in the field, but it is unsurprising to find that recent successes reveal new challenges to be addressed. With regard to wound healing, large tissue deficits, recalcitrant wounds, and pathological scar formation remain but a few of our most pressing challenges. Stem cell-based therapies have been heralded as a promising mea...

  18. Safety issues in cell-based intervention trials

    OpenAIRE

    Dawson, Liza; Bateman-House, Alison S; Mueller Agnew, Dawn; Bok, Hilary; Brock, Dan W.; Chakravarti, Aravinda; Greene, Mark; King, Patricia A.; O'Brien, Stephen J.; Sachs, David H.; Schill, Kathryn E; Siegel, Andrew; Solter, Davor; Suter, Sonia M; Verfaillie, Catherine

    2003-01-01

    We report on the deliberations of an interdisciplinary group of experts in science, law, and philosophy who convened to discuss novel ethical and policy challenges in stem cell research. In this report we discuss the ethical and policy implications of safety concerns in the transition from basic laboratory research to clinical applications of cell-based therapies derived from stem cells. Although many features of this transition from lab to clinic are common to other therapies, three aspects ...

  19. 78 FR 19499 - Request for Information: The National Toxicology Program Requests Information On Assays and...

    Science.gov (United States)

    2013-04-01

    ... cell- based assays or alternative (non-rodent) animal models, that might be used to prioritize compounds for in vivo neurotoxicity testing. DATES: The deadline for receipt of information is May 1, 2013... program established in 1978 (43 FR 53060) to coordinate toxicology research and testing across...

  20. Stem cell-based therapy in neural repair.

    Science.gov (United States)

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Dan-Yen; Chiu, Ing-Ming

    2013-01-01

    Cell-based therapy could aid in alleviating symptoms or even reversing the progression of neurodegenerative diseases and nerve injuries. Fibroblast growth factor 1 (FGF1) has been shown to maintain the survival of neurons and induce neurite outgrowth. Accumulating evidence suggests that combination of FGF1 and cell-based therapy is promising for future therapeutic application. Neural stem cells (NSCs), with the characteristics of self-renewal and multipotency, can be isolated from embryonic stem cells, embryonic ectoderm, and developing or adult brain tissues. For NSC clinical application, several critical problems remain to be resolved: (1) the source of NSCs should be personalized; (2) the isolation methods and protocols of human NSCs should be standardized; (3) the clinical efficacy of NSC transplants must be evaluated in more adequate animal models; and (4) the mechanism of intrinsic brain repair needs to be better characterized. In addition, the ideal imaging technique for tracking NSCs would be safe and yield high temporal and spatial resolution, good sensitivity and specificity. Here, we discuss recent progress and future development of cell-based therapy, such as NSCs, induced pluripotent stem cells, and induced neurons, in neurodegenerative diseases and peripheral nerve injuries. PMID:23806879

  1. National Automated Conformity Inspection Process

    Data.gov (United States)

    Department of Transportation — The National Automated Conformity Inspection Process (NACIP) Application is intended to expedite the workflow process as it pertains to the FAA Form 81 0-10 Request...

  2. Evolution of Home Automation Technology

    Directory of Open Access Journals (Sweden)

    Mohd. Rihan

    2009-01-01

    Full Text Available In modern society home and office automation has becomeincreasingly important, providing ways to interconnectvarious home appliances. This interconnection results infaster transfer of information within home/offices leading tobetter home management and improved user experience.Home Automation, in essence, is a technology thatintegrates various electrical systems of a home to provideenhanced comfort and security. Users are grantedconvenient and complete control over all the electrical homeappliances and they are relieved from the tasks thatpreviously required manual control. This paper tracks thedevelopment of home automation technology over the lasttwo decades. Various home automation technologies havebeen explained briefly, giving a chronological account of theevolution of one of the most talked about technologies ofrecent times.

  3. Automating the Purple Crow Lidar

    Science.gov (United States)

    Hicks, Shannon; Sica, R. J.; Argall, P. S.

    2016-06-01

    The Purple Crow LiDAR (PCL) was built to measure short and long term coupling between the lower, middle, and upper atmosphere. The initial component of my MSc. project is to automate two key elements of the PCL: the rotating liquid mercury mirror and the Zaber alignment mirror. In addition to the automation of the Zaber alignment mirror, it is also necessary to describe the mirror's movement and positioning errors. Its properties will then be added into the alignment software. Once the alignment software has been completed, we will compare the new alignment method with the previous manual procedure. This is the first among several projects that will culminate in a fully-automated lidar. Eventually, we will be able to work remotely, thereby increasing the amount of data we collect. This paper will describe the motivation for automation, the methods we propose, preliminary results for the Zaber alignment error analysis, and future work.

  4. Home automation with Intel Galileo

    CERN Document Server

    Dundar, Onur

    2015-01-01

    This book is for anyone who wants to learn Intel Galileo for home automation and cross-platform software development. No knowledge of programming with Intel Galileo is assumed, but knowledge of the C programming language is essential.

  5. Towards automated traceability maintenance.

    Science.gov (United States)

    Mäder, Patrick; Gotel, Orlena

    2012-10-01

    Traceability relations support stakeholders in understanding the dependencies between artifacts created during the development of a software system and thus enable many development-related tasks. To ensure that the anticipated benefits of these tasks can be realized, it is necessary to have an up-to-date set of traceability relations between the established artifacts. This goal requires the creation of traceability relations during the initial development process. Furthermore, the goal also requires the maintenance of traceability relations over time as the software system evolves in order to prevent their decay. In this paper, an approach is discussed that supports the (semi-) automated update of traceability relations between requirements, analysis and design models of software systems expressed in the UML. This is made possible by analyzing change events that have been captured while working within a third-party UML modeling tool. Within the captured flow of events, development activities comprised of several events are recognized. These are matched with predefined rules that direct the update of impacted traceability relations. The overall approach is supported by a prototype tool and empirical results on the effectiveness of tool-supported traceability maintenance are provided. PMID:23471308

  6. Automated Gas Distribution System

    Science.gov (United States)

    Starke, Allen; Clark, Henry

    2012-10-01

    The cyclotron of Texas A&M University is one of the few and prized cyclotrons in the country. Behind the scenes of the cyclotron is a confusing, and dangerous setup of the ion sources that supplies the cyclotron with particles for acceleration. To use this machine there is a time consuming, and even wasteful step by step process of switching gases, purging, and other important features that must be done manually to keep the system functioning properly, while also trying to maintain the safety of the working environment. Developing a new gas distribution system to the ion source prevents many of the problems generated by the older manually setup process. This developed system can be controlled manually in an easier fashion than before, but like most of the technology and machines in the cyclotron now, is mainly operated based on software programming developed through graphical coding environment Labview. The automated gas distribution system provides multi-ports for a selection of different gases to decrease the amount of gas wasted through switching gases, and a port for the vacuum to decrease the amount of time spent purging the manifold. The Labview software makes the operation of the cyclotron and ion sources easier, and safer for anyone to use.

  7. Automated Template Quantification for DNA Sequencing Facilities

    Science.gov (United States)

    Ivanetich, Kathryn M.; Yan, Wilson; Wunderlich, Kathleen M.; Weston, Jennifer; Walkup, Ward G.; Simeon, Christian

    2005-01-01

    The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n = 198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/μL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by ≥ 20% from the requested concentration (500 ng/μL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/μL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown. PMID:16461949

  8. Automation of the Limulus amoebocyte lysate test by using the Abbott MS-2 microbiology system.

    OpenAIRE

    Jorgensen, J H; Alexander, G A

    1981-01-01

    A rapid, automated method for the performance of the Limulus amoebocyte lysate endotoxin assay has been developed by using the Abbott MS-2 Microbiology System. This instrument automatically determines sequential changes in the optical density of up to 176 samples at 1- or 5-min increments during a 1-h assay period. Graphic representation of optical density changes can be viewed on a cathode-ray tube or reproduced by using a hard-copy printer. Limulus amoebocyte lysate preparations that were o...

  9. Aprendizaje automático

    OpenAIRE

    Moreno, Antonio

    2006-01-01

    En este libro se introducen los conceptos básicos en una de las ramas más estudiadas actualmente dentro de la inteligencia artificial: el aprendizaje automático. Se estudian temas como el aprendizaje inductivo, el razonamiento analógico, el aprendizaje basado en explicaciones, las redes neuronales, los algoritmos genéticos, el razonamiento basado en casos o las aproximaciones teóricas al aprendizaje automático.

  10. 2015 Chinese Intelligent Automation Conference

    CERN Document Server

    Li, Hongbo

    2015-01-01

    Proceedings of the 2015 Chinese Intelligent Automation Conference presents selected research papers from the CIAC’15, held in Fuzhou, China. The topics include adaptive control, fuzzy control, neural network based control, knowledge based control, hybrid intelligent control, learning control, evolutionary mechanism based control, multi-sensor integration, failure diagnosis, reconfigurable control, etc. Engineers and researchers from academia, industry and the government can gain valuable insights into interdisciplinary solutions in the field of intelligent automation.

  11. Technology modernization assessment flexible automation

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, D.W.; Boyd, D.R.; Hansen, N.H.; Hansen, M.A.; Yount, J.A.

    1990-12-01

    The objectives of this report are: to present technology assessment guidelines to be considered in conjunction with defense regulations before an automation project is developed to give examples showing how assessment guidelines may be applied to a current project to present several potential areas where automation might be applied successfully in the depot system. Depots perform primarily repair and remanufacturing operations, with limited small batch manufacturing runs. While certain activities (such as Management Information Systems and warehousing) are directly applicable to either environment, the majority of applications will require combining existing and emerging technologies in different ways, with the special needs of depot remanufacturing environment. Industry generally enjoys the ability to make revisions to its product lines seasonally, followed by batch runs of thousands or more. Depot batch runs are in the tens, at best the hundreds, of parts with a potential for large variation in product mix; reconfiguration may be required on a week-to-week basis. This need for a higher degree of flexibility suggests a higher level of operator interaction, and, in turn, control systems that go beyond the state of the art for less flexible automation and industry in general. This report investigates the benefits and barriers to automation and concludes that, while significant benefits do exist for automation, depots must be prepared to carefully investigate the technical feasibility of each opportunity and the life-cycle costs associated with implementation. Implementation is suggested in two ways: (1) develop an implementation plan for automation technologies based on results of small demonstration automation projects; (2) use phased implementation for both these and later stage automation projects to allow major technical and administrative risk issues to be addressed. 10 refs., 2 figs., 2 tabs. (JF)

  12. Stem cell-based reporter assays for the identification of carcinogenic properties of (non)-genotoxic chemicals

    OpenAIRE

    Gonçalves, Cátia Sofia Alão, 1988-

    2013-01-01

    Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2013 Nos dias que correm, existem a geração contínua de novos produtos químicos, quer seja para o desenvolvimento de drogas, quer para aplicação na indústria alimentar, cosmética ou mesmo na agricultura. Portanto, antes que um composto possa ser colocado à venda no mercado, é necessário realizar vários testes, para que se possa excluir aquelas propriedades nocivas para a saúde humana, in...

  13. Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes

    Directory of Open Access Journals (Sweden)

    Vezzoni Paolo

    2004-03-01

    Full Text Available Abstract Background Primary hepatocytes, one of the most widely used cell types for toxicological studies, have a very limited life span and must be freshly derived from mice or even humans. Attempts to use stable cell lines maintaining the enzymatic pattern of liver cells have been so far unsatisfactory. Stress proteins (heat shock proteins, HSPs have been proposed as general markers of cellular injury and their use for environmental monitoring has been suggested. The aim of this work is to develop a bi-transgenic hepatocyte cell line in order to evaluate the ability of various organic and inorganic chemicals to induce the expression of the HSP70 driven reporter gene. We previously described transgenic mice (Hsp70/hGH secreting high levels of human Growth Hormone (hGH following exposure to toxic compounds in vivo and in vitro in primary cultures derived from different organs. In addition, we also reported another transgenic model (AT/cytoMet allowing the reproducible immortalization of untransformed hepatocytes retaining in vitro complex liver functions. Results The transgenic mouse line Hsp70/hGH was crossed with the AT/cytoMet transgenic strain permitting the reproducible immortalization of untransformed hepatocytes. From double transgenic animals we derived several stable hepatic cell lines (MMH-GH which showed a highly-differentiated phenotype as judged from the retention of epithelial cell polarity and the profile of gene expression, including hepatocyte-enriched transcription factors and detoxifying enzymes. In these cell lines, stresses induced by exposure to inorganic [Sodium Arsenite (NaAsO2 and Cadmium Chloride (CdCl2], and organic [Benzo(aPyrene (BaP, PentaChloroPhenol (PCP, TetraChloroHydroQuinone (TCHQ, 1-Chloro-2,4-DiNitro-Benzene (CDNB] compounds, specifically induced hGH release in the culture medium. Conclusions MMH-GH, an innovative model to evaluate the toxic potential of chemical and physical xenobiotics, provides a simple biological system that may reduce the need for animal experimentation and/or continuously deriving fresh hepatocytes.

  14. Donor-specific cell-based assays in studying sensitivity to low-dose radiation: a population-based perspective

    Directory of Open Access Journals (Sweden)

    Dora eIl'yasova

    2014-11-01

    Full Text Available Currently, a linear no-threshold model is used to estimate health risks associated with exposure to low-dose radiation, a prevalent exposure in the general population, because the direct estimation from epidemiological studies suffers from uncertainty. This model has been criticized based on unique biology of low-dose radiation. Whether the departure from linearity is toward increased or decreased risk is intensely debated. We present an approach based on individual radiosensitivity testing and discuss how individual radiosensitivity can be assessed with the goal to develop a quantifiable measure of cellular response that can be conducted via high-throughput population testing.

  15. Comparative study of in vitro cell based assays versus in vivo toxicity tests to monitor environmental hazard of pesticides

    OpenAIRE

    Alañón Ribas, Maria del Pilar

    2006-01-01

    Carbofuran and chlorpyrifos are two well-known pesticides widely investigated, and its effects on different organisms have been previously reported in separate studies. For this reason were considered to be good model subtances, relevant from the environmental perspective. On the other hand, we selected this kind of compounds because they are used in many tones annually in agriculture and horticulture and they are significant especially in greenhouse-based production of vegetables and fruits ...

  16. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller;

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......-65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings...

  17. Early identification of hERG liability in drug discovery programs by automated patch clamp

    Directory of Open Access Journals (Sweden)

    Timm eDanker

    2014-09-01

    Full Text Available Blockade of the cardiac ion channel coded by hERG can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

  18. Contribution of Automated Technologies to Ion Channel Drug Discovery.

    Science.gov (United States)

    Picones, Arturo; Loza-Huerta, Arlet; Segura-Chama, Pedro; Lara-Figueroa, Cesar O

    2016-01-01

    Automated technologies are now resolving the historical relegation that ion channels have endured as targets for the new drug discovery and development global efforts. The richness and adequacy of functional assay methodologies, remarkably fluorescence-based detection of ions fluxes and patch-clamp electrophysiology recording of ionic currents, are now automated and increasingly employed for the analysis of ion channel activity. While the former is currently the most commonly applied, the latter is finally reaching the throughput capacity to be engaged in the primary screening of chemical libraries conformed by hundreds of thousands of compounds. The use of automated instrumentation for the study of ion channel functionality (and dysfunctionality), particularly in the search for novel pharmacological agents with therapeutic purposes, has now reached out beyond the industrial setting, its original natural enclave, and is making its way into a growing number of academic labs and core facilities. The present chapter reviews the increasing contributions accomplished by a variety of different key automated technologies which have revolutionized the strategies to approach the discovery and development of new drugs targeting ion channels. PMID:27038379

  19. Field-based multiplex and quantitative assay platforms for diagnostics

    Science.gov (United States)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  20. In vitro clinical trials: the future of cell-based profiling

    Directory of Open Access Journals (Sweden)

    Nathan T Ross

    2014-05-01

    Full Text Available The drug discovery process classically revolves around a set of biochemical and cellular assays to drive potency optimization and structural-activity relationship (SAR models. Layered on top of these concepts is the inclusion of molecular features that affect final drug use, things like: bioavailability, toxicity, stability, solubility, formulation, route of administration, etc. Paradoxically, most drugs entering clinical trials are only tested in a handful of human genetic backgrounds before they are given to people. Here we review efforts and opine on the use of large scale in vitro cellular and in vivo models that attempt to model human disease and include diversity found in the human genetic population. Because hundreds to thousands of individual assays are needed to scratch the surface of human genetic diversity, sophisticated high throughput automation technologies or pooling & deconvolution strategies are required. Characterization of each model needs to be extensive to enable non-biased informatics based modeling. Such approaches will enable deep understanding of genetic to pharmacological response and result in new methods for patient stratification in the clinic. Oncology medicines and cancer genetics have been paving the way for these approaches and we expect to see continued expansion to other fields such as immunology and neuroscience.

  1. AUTOMATED ANALYSIS OF BREAKERS

    Directory of Open Access Journals (Sweden)

    E. M. Farhadzade

    2014-01-01

    Full Text Available Breakers relate to Electric Power Systems’ equipment, the reliability of which influence, to a great extend, on reliability of Power Plants. In particular, the breakers determine structural reliability of switchgear circuit of Power Stations and network substations. Failure in short-circuit switching off by breaker with further failure of reservation unit or system of long-distance protection lead quite often to system emergency.The problem of breakers’ reliability improvement and the reduction of maintenance expenses is becoming ever more urgent in conditions of systematic increasing of maintenance cost and repair expenses of oil circuit and air-break circuit breakers. The main direction of this problem solution is the improvement of diagnostic control methods and organization of on-condition maintenance. But this demands to use a great amount of statistic information about nameplate data of breakers and their operating conditions, about their failures, testing and repairing, advanced developments (software of computer technologies and specific automated information system (AIS.The new AIS with AISV logo was developed at the department: “Reliability of power equipment” of AzRDSI of Energy. The main features of AISV are:· to provide the security and data base accuracy;· to carry out systematic control of breakers conformity with operating conditions;· to make the estimation of individual  reliability’s value and characteristics of its changing for given combination of characteristics variety;· to provide personnel, who is responsible for technical maintenance of breakers, not only with information but also with methodological support, including recommendations for the given problem solving  and advanced methods for its realization.

  2. Radon assay for SNO+

    International Nuclear Information System (INIS)

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+

  3. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  4. Growth cone collapse assay.

    Science.gov (United States)

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  5. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  6. Detection of anti-salmonella flgk antibodies in chickens by automated capillary immunoassay

    Science.gov (United States)

    Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. However, this technology has not ...

  7. Defining new criteria for selection of cell-based intestinal models using publicly available databases

    Directory of Open Access Journals (Sweden)

    Christensen Jon

    2012-06-01

    Full Text Available Abstract Background The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. Results We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. Conclusions This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models

  8. An efficient strategy for cell-based antibody library selection using an integrated vector system

    Directory of Open Access Journals (Sweden)

    Yoon Hyerim

    2012-09-01

    Full Text Available Abstract Background Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. Results A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. Conclusions This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

  9. Automated determination of angiotensin-converting enzyme in serum.

    Science.gov (United States)

    Peters, R H; Golbach, A J; van den Bergh, F A

    1987-07-01

    This is an adaptation of the Fujirebio "ACEcolor" kit for automated measurement of angiotensin-converting enzyme (EC 3.4.15.1) in serum with the Cobas Fara centrifugal analyzer. The linear range extends to an activity of 110 U/L. Results obtained by the present method and by the manual method were identical, and correlated closely (r = 0.983) with those by Cushman's modified method. The reference interval for 77 adult blood-bank donors was 9-25 U/L (mean 17, SD 4 U/L). Within-run and between-run CVs are 1.7 and 4.0%, respectively. The present method permits rapid, precise, and economical measurement of the enzyme and allows users of a Cobas Fara centrifugal analyzer to introduce a fully automated assay for angiotensin-converting enzyme into their clinical laboratory.

  10. Flexible automated platform for blood group genotyping on DNA microarrays.

    Science.gov (United States)

    Paris, Sandra; Rigal, Dominique; Barlet, Valérie; Verdier, Martine; Coudurier, Nicole; Bailly, Pascal; Brès, Jean-Charles

    2014-05-01

    The poor suitability of standard hemagglutination-based assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%-99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems. PMID:24726279

  11. Dendritic cell-based cancer immunotherapy for colorectal cancer.

    Science.gov (United States)

    Kajihara, Mikio; Takakura, Kazuki; Kanai, Tomoya; Ito, Zensho; Saito, Keisuke; Takami, Shinichiro; Shimodaira, Shigetaka; Okamoto, Masato; Ohkusa, Toshifumi; Koido, Shigeo

    2016-05-01

    Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related mortality worldwide. Although systemic therapy is the standard care for patients with recurrent or metastatic CRC, the prognosis is extremely poor. The optimal sequence of therapy remains unknown. Therefore, alternative strategies, such as immunotherapy, are needed for patients with advanced CRC. This review summarizes evidence from dendritic cell-based cancer immunotherapy strategies that are currently in clinical trials. In addition, we discuss the possibility of antitumor immune responses through immunoinhibitory PD-1/PD-L1 pathway blockade in CRC patients. PMID:27158196

  12. Dendritic cell-based cancer immunotherapy for colorectal cancer.

    Science.gov (United States)

    Kajihara, Mikio; Takakura, Kazuki; Kanai, Tomoya; Ito, Zensho; Saito, Keisuke; Takami, Shinichiro; Shimodaira, Shigetaka; Okamoto, Masato; Ohkusa, Toshifumi; Koido, Shigeo

    2016-05-01

    Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related mortality worldwide. Although systemic therapy is the standard care for patients with recurrent or metastatic CRC, the prognosis is extremely poor. The optimal sequence of therapy remains unknown. Therefore, alternative strategies, such as immunotherapy, are needed for patients with advanced CRC. This review summarizes evidence from dendritic cell-based cancer immunotherapy strategies that are currently in clinical trials. In addition, we discuss the possibility of antitumor immune responses through immunoinhibitory PD-1/PD-L1 pathway blockade in CRC patients.

  13. Programmable automation systems in PSA

    International Nuclear Information System (INIS)

    The Finnish safety authority (STUK) requires plant specific PSAs, and quantitative safety goals are set on different levels. The reliability analysis is more problematic when critical safety functions are realized by applying programmable automation systems. Conventional modeling techniques do not necessarily apply to the analysis of these systems, and the quantification seems to be impossible. However, it is important to analyze contribution of programmable automation systems to the plant safety and PSA is the only method with system analytical view over the safety. This report discusses the applicability of PSA methodology (fault tree analyses, failure modes and effects analyses) in the analysis of programmable automation systems. The problem of how to decompose programmable automation systems for reliability modeling purposes is discussed. In addition to the qualitative analysis and structural reliability modeling issues, the possibility to evaluate failure probabilities of programmable automation systems is considered. One solution to the quantification issue is the use of expert judgements, and the principles to apply expert judgements is discussed in the paper. A framework to apply expert judgements is outlined. Further, the impacts of subjective estimates on the interpretation of PSA results are discussed. (orig.) (13 refs.)

  14. International Conference Automation : Challenges in Automation, Robotics and Measurement Techniques

    CERN Document Server

    Zieliński, Cezary; Kaliczyńska, Małgorzata

    2016-01-01

    This book presents the set of papers accepted for presentation at the International Conference Automation, held in Warsaw, 2-4 March of 2016. It presents the research results presented by top experts in the fields of industrial automation, control, robotics and measurement techniques. Each chapter presents a thorough analysis of a specific technical problem which is usually followed by numerical analysis, simulation, and description of results of implementation of the solution of a real world problem. The presented theoretical results, practical solutions and guidelines will be valuable for both researchers working in the area of engineering sciences and for practitioners solving industrial problems. .

  15. VLSI implementation of moment invariants for automated inspection

    Science.gov (United States)

    Armstrong, G. A.; Simpson, M. L.; Bouldin, D. W.

    This paper describes the design of a very large scale integration (VLSI) application specific integrated circuit (ASIC) for use in automated inspection. The inspection scheme uses Hu and Maitra's algorithms for moment invariants. A prototype design was generated that resolved the long delay time of the multiplier by custom designing adder cells based on the Manchester carry chain. The prototype ASIC is currently being fabricated in 2.0-micron CMOS technology and has been simulated at 20 MHz. The final ASICs will be used in parallel at the board level to achieve the 230 MOPs necessary to perform the moment invariant algorithms in real time on 512 by 512 pixel images with 256 grey scales.

  16. Automated Low-Cost Smartphone-Based Lateral Flow Saliva Test Reader for Drugs-of-Abuse Detection

    OpenAIRE

    Adrian Carrio; Carlos Sampedro; Jose Luis Sanchez-Lopez; Miguel Pimienta; Pascual Campoy

    2015-01-01

    Lateral flow assay tests are nowadays becoming powerful, low-cost diagnostic tools. Obtaining a result is usually subject to visual interpretation of colored areas on the test by a human operator, introducing subjectivity and the possibility of errors in the extraction of the results. While automated test readers providing a result-consistent solution are widely available, they usually lack portability. In this paper, we present a smartphone-based automated reader for drug-of-abuse lateral fl...

  17. Stem cell-based therapies for HIV/AIDS.

    Science.gov (United States)

    Pernet, Olivier; Yadav, Swati Seth; An, Dong Sung

    2016-08-01

    One of the current focuses in HIV/AIDS research is to develop a novel therapeutic strategy that can provide a life-long remission of HIV/AIDS without daily drug treatment and, ultimately, a cure for HIV/AIDS. Hematopoietic stem cell-based anti-HIV gene therapy aims to reconstitute the patient immune system by transplantation of genetically engineered hematopoietic stem cells with anti-HIV genes. Hematopoietic stem cells can self-renew, proliferate and differentiate into mature immune cells. In theory, anti-HIV gene-modified hematopoietic stem cells can continuously provide HIV-resistant immune cells throughout the life of a patient. Therefore, hematopoietic stem cell-based anti-HIV gene therapy has a great potential to provide a life-long remission of HIV/AIDS by a single treatment. Here, we provide a comprehensive review of the recent progress of developing anti-HIV genes, genetic modification of hematopoietic stem progenitor cells, engraftment and reconstitution of anti-HIV gene-modified immune cells, HIV inhibition in in vitro and in vivo animal models, and in human clinical trials. PMID:27151309

  18. Cell-Based Strategies for Meniscus Tissue Engineering

    Science.gov (United States)

    Niu, Wei; Guo, Weimin; Han, Shufeng; Zhu, Yun; Liu, Shuyun; Guo, Quanyi

    2016-01-01

    Meniscus injuries remain a significant challenge due to the poor healing potential of the inner avascular zone. Following a series of studies and clinical trials, tissue engineering is considered a promising prospect for meniscus repair and regeneration. As one of the key factors in tissue engineering, cells are believed to be highly beneficial in generating bionic meniscus structures to replace injured ones in patients. Therefore, cell-based strategies for meniscus tissue engineering play a fundamental role in meniscal regeneration. According to current studies, the main cell-based strategies for meniscus tissue engineering are single cell type strategies; cell coculture strategies also were applied to meniscus tissue engineering. Likewise, on the one side, the zonal recapitulation strategies based on mimicking meniscal differing cells and internal architectures have received wide attentions. On the other side, cell self-assembling strategies without any scaffolds may be a better way to build a bionic meniscus. In this review, we primarily discuss cell seeds for meniscus tissue engineering and their application strategies. We also discuss recent advances and achievements in meniscus repair experiments that further improve our understanding of meniscus tissue engineering. PMID:27274735

  19. Stem Cell-Based Cell Therapy for Glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Meiling Jin

    2014-01-01

    Full Text Available Glomerulonephritis (GN, characterized by immune-mediated inflammatory changes in the glomerular, is a common cause of end stage renal disease. Therapeutic options for glomerulonephritis applicable to all cases mainly include symptomatic treatment and strategies to delay progression. In the attempt to yield innovative interventions fostering the limited capability of regeneration of renal tissue after injury and the uncontrolled pathological process by current treatments, stem cell-based therapy has emerged as novel therapy for its ability to inhibit inflammation and promote regeneration. Many basic and clinical studies have been performed that support the ability of various stem cell populations to ameliorate glomerular injury and improve renal function. However, there is a long way before putting stem cell-based therapy into clinical practice. In the present article, we aim to review works performed with respect to the use of stem cell of different origins in GN, and to discuss the potential mechanism of therapeutic effect and the challenges for clinical application of stem cells.

  20. Cell-based microfluidic platform for mimicking human olfactory system.

    Science.gov (United States)

    Lee, Seung Hwan; Oh, Eun Hae; Park, Tai Hyun

    2015-12-15

    Various attempts have been made to mimic the human olfactory system using human olfactory receptors (hORs). In particular, OR-expressed cell-based odorant detection systems mimic the smell sensing mechanism of humans, as they exploit endogenous cellular signaling pathways. However, the majority of such cell-based studies have been performed in the liquid phase to maintain cell viability, and liquid odorants were used as detection targets. Here, we present a microfluidic device for the detection of gaseous odorants which more closely mimics the human olfactory system. Cells expressing hOR were cultured on a porous membrane. The membrane was then flipped over and placed between two compartments. The upper compartment is the gaseous part where gaseous odorants are supplied, while the lower compartment is the aqueous part where viable cells are maintained in the liquid medium. Using this simple microfluidic device, we were able to detect gaseous odorant molecules by a fluorescence signal. The fluorescence signal was generated by calcium influx resulting from the interaction between odorant molecules and the hOR. The system allowed detection of gaseous odorant molecules in real-time, and the findings showed that the fluorescence responses increased dose-dependently in the range of 0-2 ppm odorant. In addition, the system can discriminate among gaseous odorant molecules. This microfluidic system closely mimics the human olfactory system in the sense that the submerged cells detect gaseous odorants.

  1. Cell-Based Strategies for Meniscus Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Wei Niu

    2016-01-01

    Full Text Available Meniscus injuries remain a significant challenge due to the poor healing potential of the inner avascular zone. Following a series of studies and clinical trials, tissue engineering is considered a promising prospect for meniscus repair and regeneration. As one of the key factors in tissue engineering, cells are believed to be highly beneficial in generating bionic meniscus structures to replace injured ones in patients. Therefore, cell-based strategies for meniscus tissue engineering play a fundamental role in meniscal regeneration. According to current studies, the main cell-based strategies for meniscus tissue engineering are single cell type strategies; cell coculture strategies also were applied to meniscus tissue engineering. Likewise, on the one side, the zonal recapitulation strategies based on mimicking meniscal differing cells and internal architectures have received wide attentions. On the other side, cell self-assembling strategies without any scaffolds may be a better way to build a bionic meniscus. In this review, we primarily discuss cell seeds for meniscus tissue engineering and their application strategies. We also discuss recent advances and achievements in meniscus repair experiments that further improve our understanding of meniscus tissue engineering.

  2. Automated power management and control

    Science.gov (United States)

    Dolce, James L.

    1991-01-01

    A comprehensive automation design is being developed for Space Station Freedom's electric power system. A joint effort between NASA's Office of Aeronautics and Exploration Technology and NASA's Office of Space Station Freedom, it strives to increase station productivity by applying expert systems and conventional algorithms to automate power system operation. The initial station operation will use ground-based dispatches to perform the necessary command and control tasks. These tasks constitute planning and decision-making activities that strive to eliminate unplanned outages. We perceive an opportunity to help these dispatchers make fast and consistent on-line decisions by automating three key tasks: failure detection and diagnosis, resource scheduling, and security analysis. Expert systems will be used for the diagnostics and for the security analysis; conventional algorithms will be used for the resource scheduling.

  3. Computer automation and artificial intelligence

    International Nuclear Information System (INIS)

    Rapid advances in computing, resulting from micro chip revolution has increased its application manifold particularly for computer automation. Yet the level of automation available, has limited its application to more complex and dynamic systems which require an intelligent computer control. In this paper a review of Artificial intelligence techniques used to augment automation is presented. The current sequential processing approach usually adopted in artificial intelligence has succeeded in emulating the symbolic processing part of intelligence, but the processing power required to get more elusive aspects of intelligence leads towards parallel processing. An overview of parallel processing with emphasis on transputer is also provided. A Fuzzy knowledge based controller for amination drug delivery in muscle relaxant anesthesia on transputer is described. 4 figs. (author)

  4. Unmet needs in automated cytogenetics

    International Nuclear Information System (INIS)

    Though some, at least, of the goals of automation systems for analysis of clinical cytogenetic material seem either at hand, like automatic metaphase finding, or at least likely to be met in the near future, like operator-assisted semi-automatic analysis of banded metaphase spreads, important areas of cytogenetic analsis, most importantly the determination of chromosomal aberration frequencies in populations of cells or in samples of cells from people exposed to environmental mutagens, await practical methods of automation. Important as are the clinical diagnostic applications, it is apparent that increasing concern over the clastogenic effects of the multitude of potentially clastogenic chemical and physical agents to which human populations are being increasingly exposed, and the resulting emergence of extensive cytogenetic testing protocols, makes the development of automation not only economically feasible but almost mandatory. The nature of the problems involved, and acutal of possible approaches to their solution, are discussed

  5. Manual versus automated blood sampling

    DEFF Research Database (Denmark)

    Teilmann, A C; Kalliokoski, Otto; Sørensen, Dorte B;

    2014-01-01

    corticosterone metabolites, and expressed more anxious behavior than did the mice of the other groups. Plasma corticosterone levels of mice subjected to tail blood sampling were also elevated, although less significantly. Mice subjected to automated blood sampling were less affected with regard to the parameters......Facial vein (cheek blood) and caudal vein (tail blood) phlebotomy are two commonly used techniques for obtaining blood samples from laboratory mice, while automated blood sampling through a permanent catheter is a relatively new technique in mice. The present study compared physiological parameters......, glucocorticoid dynamics as well as the behavior of mice sampled repeatedly for 24 h by cheek blood, tail blood or automated blood sampling from the carotid artery. Mice subjected to cheek blood sampling lost significantly more body weight, had elevated levels of plasma corticosterone, excreted more fecal...

  6. Network based automation for SMEs

    DEFF Research Database (Denmark)

    Shahabeddini Parizi, Mohammad; Radziwon, Agnieszka

    2016-01-01

    The implementation of appropriate automation concepts which increase productivity in Small and Medium Sized Enterprises (SMEs) requires a lot of effort, due to their limited resources. Therefore, it is strongly recommended for small firms to open up for the external sources of knowledge, which co......, this paper develops and discusses a set of guidelines for systematic productivity improvement within an innovative collaboration in regards to automation processes in SMEs.......The implementation of appropriate automation concepts which increase productivity in Small and Medium Sized Enterprises (SMEs) requires a lot of effort, due to their limited resources. Therefore, it is strongly recommended for small firms to open up for the external sources of knowledge, which...... could be obtained through network interaction. Based on two extreme cases of SMEs representing low-tech industry and an in-depth analysis of their manufacturing facilities this paper presents how collaboration between firms embedded in a regional ecosystem could result in implementation of new...

  7. Design automation for integrated circuits

    Science.gov (United States)

    Newell, S. B.; de Geus, A. J.; Rohrer, R. A.

    1983-04-01

    Consideration is given to the development status of the use of computers in automated integrated circuit design methods, which promise the minimization of both design time and design error incidence. Integrated circuit design encompasses two major tasks: error specification, in which the goal is a logic diagram that accurately represents the desired electronic function, and physical specification, in which the goal is an exact description of the physical locations of all circuit elements and their interconnections on the chip. Design automation not only saves money by reducing design and fabrication time, but also helps the community of systems and logic designers to work more innovatively. Attention is given to established design automation methodologies, programmable logic arrays, and design shortcuts.

  8. Automated Podcasting System for Universities

    Directory of Open Access Journals (Sweden)

    Ypatios Grigoriadis

    2013-03-01

    Full Text Available This paper presents the results achieved at Graz University of Technology (TU Graz in the field of automating the process of recording and publishing university lectures in a very new way. It outlines cornerstones of the development and integration of an automated recording system such as the lecture hall setup, the recording hardware and software architecture as well as the development of a text-based search for the final product by method of indexing video podcasts. Furthermore, the paper takes a look at didactical aspects, evaluations done in this context and future outlook.

  9. Agile Data: Automating database refactorings

    Directory of Open Access Journals (Sweden)

    Bruno Xavier

    2014-09-01

    Full Text Available This paper discusses an automated approach to database change management throughout the companies’ development workflow. By using automated tools, companies can avoid common issues related to manual database deployments. This work was motivated by analyzing usual problems within organizations, mostly originated from manual interventions that may result in systems disruptions and production incidents. In addition to practices of continuous integration and continuous delivery, the current paper describes a case study in which a suggested pipeline is implemented in order to reduce the deployment times and decrease incidents due to ineffective data controlling.

  10. Design automation, languages, and simulations

    CERN Document Server

    Chen, Wai-Kai

    2003-01-01

    As the complexity of electronic systems continues to increase, the micro-electronic industry depends upon automation and simulations to adapt quickly to market changes and new technologies. Compiled from chapters contributed to CRC's best-selling VLSI Handbook, this volume covers a broad range of topics relevant to design automation, languages, and simulations. These include a collaborative framework that coordinates distributed design activities through the Internet, an overview of the Verilog hardware description language and its use in a design environment, hardware/software co-design, syst

  11. 2013 Chinese Intelligent Automation Conference

    CERN Document Server

    Deng, Zhidong

    2013-01-01

    Proceedings of the 2013 Chinese Intelligent Automation Conference presents selected research papers from the CIAC’13, held in Yangzhou, China. The topics include e.g. adaptive control, fuzzy control, neural network based control, knowledge based control, hybrid intelligent control, learning control, evolutionary mechanism based control, multi-sensor integration, failure diagnosis, and reconfigurable control. Engineers and researchers from academia, industry, and government can gain an inside view of new solutions combining ideas from multiple disciplines in the field of intelligent automation.   Zengqi Sun and Zhidong Deng are professors at the Department of Computer Science, Tsinghua University, China.

  12. 2013 Chinese Intelligent Automation Conference

    CERN Document Server

    Deng, Zhidong

    2013-01-01

    Proceedings of the 2013 Chinese Intelligent Automation Conference presents selected research papers from the CIAC’13, held in Yangzhou, China. The topics include e.g. adaptive control, fuzzy control, neural network based control, knowledge based control, hybrid intelligent control, learning control, evolutionary mechanism based control, multi-sensor integration, failure diagnosis, and reconfigurable control. Engineers and researchers from academia, industry, and government can gain an inside view of new solutions combining ideas from multiple disciplines in the field of intelligent automation. Zengqi Sun and Zhidong Deng are professors at the Department of Computer Science, Tsinghua University, China.

  13. Automated synthesis of sialylated oligosaccharides

    Directory of Open Access Journals (Sweden)

    Davide Esposito

    2012-09-01

    Full Text Available Sialic acid-containing glycans play a major role in cell-surface interactions with external partners such as cells and viruses. Straightforward access to sialosides is required in order to study their biological functions on a molecular level. Here, automated oligosaccharide synthesis was used to facilitate the preparation of this class of biomolecules. Our strategy relies on novel sialyl α-(2→3 and α-(2→6 galactosyl imidates, which, used in combination with the automated platform, provided rapid access to a small library of conjugation-ready sialosides of biological relevance.

  14. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  15. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    International Nuclear Information System (INIS)

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility

  16. Toward designing for trust in database automation

    International Nuclear Information System (INIS)

    Appropriate reliance on system automation is imperative for safe and productive work, especially in safety-critical systems. It is unsafe to rely on automation beyond its designed use; conversely, it can be both unproductive and unsafe to manually perform tasks that are better relegated to automated tools. Operator trust in automated tools mediates reliance, and trust appears to affect how operators use technology. As automated agents become more complex, the question of trust in automation is increasingly important. In order to achieve proper use of automation, we must engender an appropriate degree of trust that is sensitive to changes in operating functions and context. In this paper, we present research concerning trust in automation in the domain of automated tools for relational databases. Lee and See have provided models of trust in automation. One model developed by Lee and See identifies three key categories of information about the automation that lie along a continuum of attributional abstraction. Purpose-, process-and performance-related information serve, both individually and through inferences between them, to describe automation in such a way as to engender r properly-calibrated trust. Thus, one can look at information from different levels of attributional abstraction as a general requirements analysis for information key to appropriate trust in automation. The model of information necessary to engender appropriate trust in automation [1] is a general one. Although it describes categories of information, it does not provide insight on how to determine the specific information elements required for a given automated tool. We have applied the Abstraction Hierarchy (AH) to this problem in the domain of relational databases. The AH serves as a formal description of the automation at several levels of abstraction, ranging from a very abstract purpose-oriented description to a more concrete description of the resources involved in the automated process

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sager J Gosai

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

  19. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Directory of Open Access Journals (Sweden)

    Thomas Zuliani

    Full Text Available Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  20. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Science.gov (United States)

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  1. Automated Ply Inspection (API) for AFP Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The Automated Ply Inspection (API) system autonomously inspects layups created by high speed automated fiber placement (AFP) machines. API comprises a high accuracy...

  2. PathogenMip Assay: A Multiplex Pathogen Detection Assay

    OpenAIRE

    Akhras, Michael S.; Sreedevi Thiyagarajan; Villablanca, Andrea C.; Davis, Ronald W; Pål Nyrén; Nader Pourmand

    2007-01-01

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The ...

  3. Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

    Directory of Open Access Journals (Sweden)

    Irina Gates

    Full Text Available Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT, newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

  4. Towards Automated System Synthesis Using SCIDUCTION

    OpenAIRE

    Jha, Susmit Kumar

    2011-01-01

    Automated synthesis of systems that are correct by construction has been a long-standing goal of computer science. Synthesis is a creative task and requires human intuition and skill. Its complete automation is currently beyond the capacity of programs that do automated reasoning. However, there is a pressing need for tools and techniques that can automate non-intuitive and error-prone synthesis tasks. This thesis proposes a novel synthesis approach to solve such tasks in the synthesis of pro...

  5. Automated activation-analysis system

    International Nuclear Information System (INIS)

    An automated delayed neutron counting and instrumental neutron activation analysis system has been developed at Los Alamos National Laboratory's Omega West Reactor (OWR) to analyze samples for uranium and 31 additional elements with a maximum throughput of 400 samples per day. The system and its mode of operation for a large reconnaissance survey are described

  6. Automation, Performance and International Competition

    DEFF Research Database (Denmark)

    Kromann, Lene; Sørensen, Anders

    This paper presents new evidence on trade‐induced automation in manufacturing firms using unique data combining a retrospective survey that we have assembled with register data for 2005‐2010. In particular, we establish a causal effect where firms that have specialized in product types for which ...

  7. Feasibility Analysis of Crane Automation

    Institute of Scientific and Technical Information of China (English)

    DONG Ming-xiao; MEI Xue-song; JIANG Ge-dong; ZHANG Gui-qing

    2006-01-01

    This paper summarizes the modeling methods, open-loop control and closed-loop control techniques of various forms of cranes, worldwide, and discusses their feasibilities and limitations in engineering. Then the dynamic behaviors of cranes are analyzed. Finally, we propose applied modeling methods and feasible control techniques and demonstrate the feasibilities of crane automation.

  8. Automation of Feynman diagram evaluations

    International Nuclear Information System (INIS)

    A C-program DIANA (DIagram ANAlyser) for the automation of Feynman diagram evaluations is presented. It consists of two parts: the analyzer of diagrams and the interpreter of a special text manipulating language. This language can be used to create a source code for analytical or numerical evaluations and to keep the control of the process in general

  9. Automated Clustering of Similar Amendments

    CERN Document Server

    CERN. Geneva

    2016-01-01

    The Italian Senate is clogged by computer-generated amendments. This talk will describe a simple strategy to cluster them in an automated fashion, so that the appropriate Senate procedures can be used to get rid of them in one sweep.

  10. Automated visual inspection of textile

    DEFF Research Database (Denmark)

    Jensen, Rune Fisker; Carstensen, Jens Michael

    1997-01-01

    A method for automated inspection of two types of textile is presented. The goal of the inspection is to determine defects in the textile. A prototype is constructed for simulating the textile production line. At the prototype the images of the textile are acquired by a high speed line scan camera...

  11. Distribution system analysis and automation

    CERN Document Server

    Gers, Juan

    2013-01-01

    A comprehensive guide to techniques that allow engineers to simulate, analyse and optimise power distribution systems which combined with automation, underpin the emerging concept of the "smart grid". This book is supported by theoretical concepts with real-world applications and MATLAB exercises.

  12. Teacherbot: Interventions in Automated Teaching

    Science.gov (United States)

    Bayne, Sian

    2015-01-01

    Promises of "teacher-light" tuition and of enhanced "efficiency" via the automation of teaching have been with us since the early days of digital education, sometimes embraced by academics and institutions, and sometimes resisted as a set of moves which are damaging to teacher professionalism and to the humanistic values of…

  13. Automation, Labor Productivity and Employment

    DEFF Research Database (Denmark)

    Kromann, Lene; Rose Skaksen, Jan; Sørensen, Anders

    CEBR fremlægger nu den første rapport i AIM-projektet. Rapporten viser, at der er gode muligheder for yderligere automation i en stor del af de danske fremstillingsvirksomheder. For i dag er gennemsnitligt kun omkring 30 % af virksomhedernes produktionsprocesser automatiserede. Navnlig procesområ...

  14. Adaptation : A Partially Automated Approach

    NARCIS (Netherlands)

    Manjing, Tham; Bukhsh, F.A.; Weigand, H.

    2014-01-01

    This paper showcases the possibility of creating an adaptive auditing system. Adaptation in an audit environment need human intervention at some point. Based on a case study this paper focuses on automation of adaptation process. It is divided into solution design and validation parts. The artifact

  15. Automation of Space Inventory Management

    Science.gov (United States)

    Fink, Patrick W.; Ngo, Phong; Wagner, Raymond; Barton, Richard; Gifford, Kevin

    2009-01-01

    This viewgraph presentation describes the utilization of automated space-based inventory management through handheld RFID readers and BioNet Middleware. The contents include: 1) Space-Based INventory Management; 2) Real-Time RFID Location and Tracking; 3) Surface Acoustic Wave (SAW) RFID; and 4) BioNet Middleware.

  16. Automation; The New Industrial Revolution.

    Science.gov (United States)

    Arnstein, George E.

    Automation is a word that describes the workings of computers and the innovations of automatic transfer machines in the factory. As the hallmark of the new industrial revolution, computers displace workers and create a need for new skills and retraining programs. With improved communication between industry and the educational community to…

  17. Improved Computational Model of Grid Cells Based on Column Structure

    Institute of Scientific and Technical Information of China (English)

    Yang Zhou; Dewei Wu; Weilong Li; Jia Du

    2016-01-01

    To simulate the firing pattern of biological grid cells, this paper presents an improved computational model of grid cells based on column structure. In this model, the displacement along different directions is processed by modulus operation, and the obtained remainder is associated with firing rate of grid cell. Compared with the original model, the improved parts include that: the base of modulus operation is changed, and the firing rate in firing field is encoded by Gaussian⁃like function. Simulation validates that the firing pattern generated by the improved computational model is more consistent with biological characteristic than original model. Besides, the firing pattern is badly influenced by the cumulative positioning error, but the computational model can also generate the regularly hexagonal firing pattern when the real⁃time positioning results are modified.

  18. Glial progenitor cell-based treatment of the childhood leukodystrophies

    DEFF Research Database (Denmark)

    Osorio, M Joana; Goldman, Steven A

    2016-01-01

    stem cell-derived human neural or glial progenitor cells may comprise a promising strategy for both structural remyelination and metabolic rescue. A broad variety of pediatric white matter disorders, including the primary hypomyelinating disorders, the lysosomal storage disorders, and the broader group...... genetic editing of pluripotent stem cells. Yet these challenges notwithstanding, the promise of glial progenitor cell-based treatment of the childhood myelin disorders offers hope to the many victims of this otherwise largely untreatable class of disease....... and astrocytes are the major affected cell populations, and are either structurally impaired or metabolically compromised through cell-intrinsic pathology, or are the victims of mis-accumulated toxic byproducts of metabolic derangement. In either case, glial cell replacement using implanted tissue or pluripotent...

  19. A stem cell-based approach to cartilage repair.

    Science.gov (United States)

    Johnson, Kristen; Zhu, Shoutian; Tremblay, Matthew S; Payette, Joshua N; Wang, Jianing; Bouchez, Laure C; Meeusen, Shelly; Althage, Alana; Cho, Charles Y; Wu, Xu; Schultz, Peter G

    2012-05-11

    Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ), and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis. PMID:22491093

  20. Use of recombinant salmonella flagellar hook protein (flgk) for detection of anti-salmonella antibodies in chickens by automated capillary immunoassay

    Science.gov (United States)

    Background: Conventional immunoblot assays are a very useful tool for specific protein identification, but are tedious, labor-intensive and time-consuming. An automated capillary electrophoresis-based immunoblot assay called "Simple Western" has recently been developed that enables the protein sepa...

  1. Implementation of automated testing for 1,25-dihydroxyvitamin D: Return of experience from a core-laboratory.

    Science.gov (United States)

    Miller, Nathalie; Gruson, Damien

    2016-02-01

    Measurement of 1,25(OH)2D, the most biologically active form of vitamin D, circulating levels is relevant in several physiopathological states such as chronic kidney disease, parathyroid dysfunction, sarcoidosis, and vitamin D dependent rickets. Our study determined the performances of a novel automated 1,25(OH)2D immunoassay in a core-laboratory environment. We observed satisfactory analytical performances for this assay and an excellent agreement with a well established LC-MS/MS method. Furthermore, this assay allows a reduced TAT, integration in automated core-laboratories and potential consolidation with other tests of the bone and mineral metabolism. PMID:26519091

  2. Two-tiered keratinocyte assay: IL-18 production by NCTC2544 cells to determine the skin sensitizing capacity and an epidermal equivalent assay to determine sensitizer potency

    OpenAIRE

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke; Madsen, Charlotte Bernhard; Eltze, Tobias; Ezendam, Janine; Galbiati, Valentina; Gremmer, Eric; Krul, Cyrille; Landin, Annette; Landsiedel, Robert; Pieters, Raymond; Rasmussen, Tina; Reinders, Judith; Roggen, Erwin

    2012-01-01

    At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it should be very important, both from ethical and economic point of view, to discriminate allergy and irritation events, and to classify sensitizers according to their potency, without the use of animals.The aim of the EU FP6 Integrated Project Sens-it-iv was to develop and optimize an integrated testing strategy consisting of in vitro, human cell based assays which will closely m...

  3. Cell-based phenotypic screening of mast cell degranulation unveils kinetic perturbations of agents targeting phosphorylation

    Science.gov (United States)

    Qin, Shenlu; Wang, Xumeng; Wu, Huanwen; Xiao, Peng; Cheng, Hongqiang; Zhang, Xue; Ke, Yuehai

    2016-01-01

    Mast cells play an essential role in initiating allergic diseases. The activation of mast cells are controlled by a complicated signal network of reversible phosphorylation, and finding the key regulators involved in this network has been the focus of the pharmaceutical industry. In this work, we used a method named Time-dependent cell responding profile (TCRP) to track the process of mast cell degranulation under various perturbations caused by agents targeting phosphorylation. To test the feasibility of this high-throughput cell-based phenotypic screening method, a variety of biological techniques were used. We further screened 145 inhibitors and clustered them based on the similarities of their TCRPs. Stat3 phosphorylation has been widely reported as a key step in mast cell degranulation. Interestingly, our TCRP results showed that a Stat3 inhibitor JSI124 did not inhibit degranulation like other Stat3 inhibitors, such as Stattic, clearly inhibited degranulation. Regular endpoint assays demonstrated that the distinctive TCRP of JSI124 potentially correlated with the ability to induce apoptosis. Consequently, different agents possibly have disparate functions, which can be conveniently detected by TCRP. From this perspective, our TCRP screening method is reliable and sensitive when it comes to discovering and selecting novel compounds for new drug developments. PMID:27502076

  4. Antioxidant activity of polyphenolic myricetin in vitro cell-free and cell-based systems

    Directory of Open Access Journals (Sweden)

    Abolfazl Barzegar

    2016-06-01

    Full Text Available Myricetin (Myc is one of the most important flavonoids in diet due to its abundance in foods with the highest antioxidant activity. The antioxidant activity of Myc was studied in cell-free and cell-based systems to evaluate the ROS protection efficiency of Myc. The studies were based on the assessment of reducing power of Myc according to ferric ion reduction and intracellular ROS level measurement by assaying the cellular fluorescence intensity using dichlorodihydrofluorescein (DCF probe as an indicator for ROS in cells. Moreover, the antitoxic capability of Myc was assessed using MTT method. Data indicated that intracellular ROS are highly toxic and applying low concentration of Myc not only inhibited cellular ROS production but also was accompanying with the protection of cells against the highly toxic and the lethal effects of peroxide compounds. Because of strong correlation between cellular ROS and their cell toxic properties, the higher antioxidant potency of Myc in cell medium resulted in effectively blocking intracellular ROS and protecting cell death. This property is achieved by the help of high polar solubility and cell membrane permeability of Myc.

  5. Illinois: Library Automation and Connectivity Initiatives.

    Science.gov (United States)

    Lamont, Bridget L.; Bloomberg, Kathleen L.

    1996-01-01

    Discussion of library automation in Illinois focuses on ILLINET, the Illinois Library and Information Network. Topics include automated resource sharing; ILLINET's online catalog; regional library system automation; community networking and public library technology development; telecommunications initiatives; electronic access to state government…

  6. You're a What? Automation Technician

    Science.gov (United States)

    Mullins, John

    2010-01-01

    Many people think of automation as laborsaving technology, but it sure keeps Jim Duffell busy. Defined simply, automation is a technique for making a device run or a process occur with minimal direct human intervention. But the functions and technologies involved in automated manufacturing are complex. Nearly all functions, from orders coming in…

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

    OpenAIRE

    Hua, Yun; Shun, Tong Ying; Strock, Christopher J.; Johnston, Paul A.

    2014-01-01

    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” compon...

  8. Comparison between automated system and PCR-based method for identification and antimicrobial susceptibility profile of clinical Enterococcus spp.

    Science.gov (United States)

    Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina

    2014-01-01

    Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.

  9. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    Science.gov (United States)

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  10. An automated HIV-1 Env-pseudotyped virus production for global HIV vaccine trials.

    Directory of Open Access Journals (Sweden)

    Anke Schultz

    Full Text Available BACKGROUND: Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. METHODOLOGY/PRINCIPAL FINDINGS: The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO(2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP guidelines, including the validation parameters accuracy, precision, robustness and specificity. CONCLUSIONS: An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell

  11. Optimizing ELISAs for precision and robustness using laboratory automation and statistical design of experiments.

    Science.gov (United States)

    Joelsson, Daniel; Moravec, Phil; Troutman, Matthew; Pigeon, Joseph; DePhillips, Pete

    2008-08-20

    Transferring manual ELISAs to automated platforms requires optimizing the assays for each particular robotic platform. These optimization experiments are often time consuming and difficult to perform using a traditional one-factor-at-a-time strategy. In this manuscript we describe the development of an automated process using statistical design of experiments (DOE) to quickly optimize immunoassays for precision and robustness on the Tecan EVO liquid handler. By using fractional factorials and a split-plot design, five incubation time variables and four reagent concentration variables can be optimized in a short period of time.

  12. Syphilis detection: evaluation of serological screening and pilot reverse confirmatory assay algorithm in blood donors.

    Science.gov (United States)

    Sommese, Linda; Paolillo, Rossella; Sabia, Chiara; Costa, Dario; De Pascale, Maria Rosaria; Iannone, Carmela; Esposito, Antonella; Schiano, Concetta; Napoli, Claudio

    2016-07-01

    Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test - the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre. PMID:26068964

  13. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

    Science.gov (United States)

    Chang, A; Dusing, S

    2006-01-01

    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  14. Development of an automated sequential injection on-line solvent extraction-back extraction procedure as demonstrated for the determination of cadmium with detection by electrothermal atomic absorption spectrometry

    DEFF Research Database (Denmark)

    Wang, Jianhua; Hansen, Elo Harald

    2002-01-01

    An automated sequential injection (SI) on-line solvent extraction-back extraction separation/preconcentration procedure is described. Demonstrated for the assay of cadmium by electrothermal atomic absorption spectrometry (ETAAS), the analyte is initially complexed with ammonium...

  15. An Overview of Moonlight Applications Test Automation

    Directory of Open Access Journals (Sweden)

    Appasami Govindasamy

    2010-09-01

    Full Text Available Now-a-days web applications are developed by new technologies like Moonlight, Silverlight, JAVAFX, FLEX, etc. Silverlight is Microsoft's cross platform runtime and development technology for running Web-based multimedia applications in windows platform. Moonlight is an open-source implementation of the Silverlight development platform for Linux and other Unix/X11-based operating systems. It is a new technology in .Net 4.0 to develop rich interactive and attractive platform independent web applications. User Interface Test Automation is very essential for Software industries to reduce test time, cost and man power. Moonlight is new .NET technology to develop rich interactive Internet applications with the collaboration of Novel Corporation. Testing these kinds of applications are not so easy to test, especially the User interface test automation is very difficult. Software test automation has the capability to decrease the overall cost of testing and improve software quality, but most testing organizations have not been able to achieve the full potential of test automation. Many groups that implement test automation programs run into a number of common pitfalls. These problems can lead to test automation plans being completely scrapped, with the tools purchased for test automation becoming expensive. Often teams continue their automation effort, burdened with huge costs in maintaining large suites of automated test scripts. This paper will first discuss some of the key benefits of software test automation, and then examine the most common techniques used to implement software test automation of Moonlight Applications Test Automation. It will then discuss test automation and their potential. Finally, it will do test automation.

  16. Automated approach to radioimmunoassays of somatotropin (human growth hormone) and insulin

    International Nuclear Information System (INIS)

    The adaptation of human somatotropin and insulin assays to the automated Centria radioimmunoassay system [Clin. Chem. 21, 1305 (1975)] is reported. For the somatotropin assay, reaction conditions include a borate/bovine serum albumin buffer (pH 8.4) and a 20-h incubation at 40C. Assay results for clinical samples compared favorably (correlation coefficient = 0.930) with values obtained from a reference laboratory. The means determined for 92 patients' samples were 4.3 μg/liter (reference laboratory) and 5.1 μg/liter (Centria). Intra- and inter-run precision ranged from 3.2 to 15.9%. For the insulin assay, a phosphate/bovine serum albumin buffer (pH 7.4) is used, with a 20-h incubation at 40C. Previously analyzed insulin samples from a reference laboratory were determined by the Centria analyzer with excellent correlation (r = 0.965). Means for patients' samples were 43.0 milli (USP) units of insulin per liter (reference laboratory) and 47.5 milliunits of insulin per liter (Centria). In both assays an anionicexchange gel is used in the separation step. The criterion of parallelism, an indication of the validity of a radioimmunoassay, was satisfied in both assays. The Centria radioimmunoassay system offers the advantage of automating all the critical steps of these radioimmunoassays

  17. Automated methods of corrosion measurement

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Bech-Nielsen, Gregers; Reeve, John Ch;

    1997-01-01

    Measurements of corrosion rates and other parameters connected with corrosion processes are important, first as indicators of the corrosion resistance of metallic materials and second because such measurements are based on general and fundamental physical, chemical, and electrochemical relations....... Hence improvements and innovations in methods applied in corrosion research are likeliy to benefit basic disciplines as well. A method for corrosion measurements can only provide reliable data if the beckground of the method is fully understood. Failure of a method to give correct data indicates a need...... to revise assumptions regarding the basis of the method, which sometimes leads to the discovery of as-yet unnoticed phenomena. The present selection of automated methods for corrosion measurements is not motivated simply by the fact that a certain measurement can be performed automatically. Automation...

  18. CCD characterization and measurements automation

    International Nuclear Information System (INIS)

    Modern mosaic cameras have grown both in size and in number of sensors. The required volume of sensor testing and characterization has grown accordingly. For camera projects as large as the LSST, test automation becomes a necessity. A CCD testing and characterization laboratory was built and is in operation for the LSST project. Characterization of LSST study contract sensors has been performed. The characterization process and its automation are discussed, and results are presented. Our system automatically acquires images, populates a database with metadata information, and runs express analysis. This approach is illustrated on 55Fe data analysis. 55Fe data are used to measure gain, charge transfer efficiency and charge diffusion. Examples of express analysis results are presented and discussed.

  19. Automated nanomanipulation for nanodevice construction

    International Nuclear Information System (INIS)

    Nanowire field-effect transistors (nano-FETs) are nanodevices capable of highly sensitive, label-free sensing of molecules. However, significant variations in sensitivity across devices can result from poor control over device parameters, such as nanowire diameter and the number of electrode-bridging nanowires. This paper presents a fabrication approach that uses wafer-scale nanowire contact printing for throughput and uses automated nanomanipulation for precision control of nanowire number and diameter. The process requires only one photolithography mask. Using nanowire contact printing and post-processing (i.e. nanomanipulation inside a scanning electron microscope), we are able to produce devices all with a single-nanowire and similar diameters at a speed of ∼1 min/device with a success rate of 95% (n = 500). This technology represents a seamless integration of wafer-scale microfabrication and automated nanorobotic manipulation for producing nano-FET sensors with consistent response across devices. (paper)

  20. DOLFIN: Automated Finite Element Computing

    CERN Document Server

    Logg, Anders; 10.1145/1731022.1731030

    2011-01-01

    We describe here a library aimed at automating the solution of partial differential equations using the finite element method. By employing novel techniques for automated code generation, the library combines a high level of expressiveness with efficient computation. Finite element variational forms may be expressed in near mathematical notation, from which low-level code is automatically generated, compiled and seamlessly integrated with efficient implementations of computational meshes and high-performance linear algebra. Easy-to-use object-oriented interfaces to the library are provided in the form of a C++ library and a Python module. This paper discusses the mathematical abstractions and methods used in the design of the library and its implementation. A number of examples are presented to demonstrate the use of the library in application code.

  1. Automated illustration of patients instructions.

    Science.gov (United States)

    Bui, Duy; Nakamura, Carlos; Bray, Bruce E; Zeng-Treitler, Qing

    2012-01-01

    A picture can be a powerful communication tool. However, creating pictures to illustrate patient instructions can be a costly and time-consuming task. Building on our prior research in this area, we developed a computer application that automatically converts text to pictures using natural language processing and computer graphics techniques. After iterative testing, the automated illustration system was evaluated using 49 previously unseen cardiology discharge instructions. The completeness of the system-generated illustrations was assessed by three raters using a three-level scale. The average inter-rater agreement for text correctly represented in the pictograph was about 66 percent. Since illustration in this context is intended to enhance rather than replace text, these results support the feasibility of conducting automated illustration.

  2. Quantitative automated microscopy (QuAM elucidates growth factor specific signalling in pain sensitization

    Directory of Open Access Journals (Sweden)

    Levine Jon D

    2010-12-01

    Full Text Available Abstract Background Dorsal root ganglia (DRG-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base. The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. Results We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+- and IB4(--neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked

  3. Automated medical image segmentation techniques

    OpenAIRE

    Sharma Neeraj; Aggarwal Lalit

    2010-01-01

    Accurate segmentation of medical images is a key step in contouring during radiotherapy planning. Computed topography (CT) and Magnetic resonance (MR) imaging are the most widely used radiographic techniques in diagnosis, clinical studies and treatment planning. This review provides details of automated segmentation methods, specifically discussed in the context of CT and MR images. The motive is to discuss the problems encountered in segmentation of CT and MR images, and the relative merits ...

  4. Automated Periodontal Diseases Classification System

    OpenAIRE

    Aliaa A. A. Youssif; Abeer Saad Gawish,; Mohammed Elsaid Moussa

    2012-01-01

    This paper presents an efficient and innovative system for automated classification of periodontal diseases, The strength of our technique lies in the fact that it incorporates knowledge from the patients' clinical data, along with the features automatically extracted from the Haematoxylin and Eosin (H&E) stained microscopic images. Our system uses image processing techniques based on color deconvolution, morphological operations, and watershed transforms for epithelium & connective tissue se...

  5. Home automation in the workplace.

    Science.gov (United States)

    McCormack, J E; Tello, S F

    1994-01-01

    Environmental control units and home automation devices contribute to the independence and potential of individuals with disabilities, both at work and at home. Devices currently exist that can assist people with physical, cognitive, and sensory disabilities to control lighting, appliances, temperature, security, and telephone communications. This article highlights several possible applications for these technologies and discusses emerging technologies that will increase the benefits these devices offer people with disabilities. PMID:24440955

  6. Small Business Innovations (Automated Information)

    Science.gov (United States)

    1992-01-01

    Bruce G. Jackson & Associates Document Director is an automated tool that combines word processing and database management technologies to offer the flexibility and convenience of text processing with the linking capability of database management. Originally developed for NASA, it provides a means to collect and manage information associated with requirements development. The software system was used by NASA in the design of the Assured Crew Return Vehicle, as well as by other government and commercial organizations including the Southwest Research Institute.

  7. Automated Scheduling Via Artificial Intelligence

    Science.gov (United States)

    Biefeld, Eric W.; Cooper, Lynne P.

    1991-01-01

    Artificial-intelligence software that automates scheduling developed in Operations Mission Planner (OMP) research project. Software used in both generation of new schedules and modification of existing schedules in view of changes in tasks and/or available resources. Approach based on iterative refinement. Although project focused upon scheduling of operations of scientific instruments and other equipment aboard spacecraft, also applicable to such terrestrial problems as scheduling production in factory.

  8. System of automated map design

    International Nuclear Information System (INIS)

    Preprint 'System of automated map design' contains information about the program shell for construction of territory map, performing level line drawing of arbitrary two-dimension field (in particular, the radionuclide concentration field). The work schedule and data structures are supplied, as well as data on system performance. The preprint can become useful for experts in radioecology and for all persons involved in territory pollution mapping or multi-purpose geochemical mapping. (author)

  9. Method development in automated mineralogy

    OpenAIRE

    Sandmann, Dirk

    2015-01-01

    The underlying research that resulted in this doctoral dissertation was performed at the Division of Economic Geology and Petrology of the Department of Mineralogy, TU Bergakademie Freiberg between 2011 and 2014. It was the primary aim of this thesis to develop and test novel applications for the technology of ‘Automated Mineralogy’ in the field of economic geology and geometallurgy. A “Mineral Liberation Analyser” (MLA) instrument of FEI Company was used to conduct most analytical studies. T...

  10. GUI test automation with SWTBot

    OpenAIRE

    Mazurkiewicz, Milosz

    2010-01-01

    In this thesis the author presents theoretical background of GUI test automation as well as technologies, tools and methodologies required to fully understand the test program written in SWTBot. Practical part of the thesis was to implement a program testing File Menu options of Pegasus RCP application developed in Nokia Siemens Networks. Concluding this dissertation, in the author’s opinion test programs written using SWTBot are relatively easy to read and intuitive for people familiar w...

  11. Automated minimax design of networks

    DEFF Research Database (Denmark)

    Madsen, Kaj; Schjær-Jacobsen, Hans; Voldby, J

    1975-01-01

    A new gradient algorithm for the solution of nonlinear minimax problems has been developed. The algorithm is well suited for automated minimax design of networks and it is very simple to use. It compares favorably with recent minimax and leastpth algorithms. General convergence problems related...... to minimax design of networks are discussed. Finally, minimax design of equalization networks for reflectiontype microwave amplifiers is carried out by means of the proposed algorithm....

  12. Adaptation: A Partially Automated Approach

    OpenAIRE

    Manjing, Tham; Bukhsh, F.A.; Weigand, H.

    2014-01-01

    This paper showcases the possibility of creating an adaptive auditing system. Adaptation in an audit environment need human intervention at some point. Based on a case study this paper focuses on automation of adaptation process. It is divided into solution design and validation parts. The artifact design is developed around import procedures of M-company. An overview of the artefact is discussed in detail to fully describes the adaptation mechanism with automatic adjustment for compliance re...

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

    Science.gov (United States)

    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.

  14. [Safety monitoring of cell-based medicinal products (CBMPs)].

    Science.gov (United States)

    Funk, Markus B; Frech, Marion; Spranger, Robert; Keller-Stanislawski, Brigitte

    2015-11-01

    Cell-based medicinal products (CBMPs), a category of advanced-therapy medicinal products (ATMPs), are authorised for the European market by the European Commission by means of the centralized marketing authorisation. By conforming to the German Medicinal Products Act (Sec. 4b AMG), national authorisation can be granted by the Paul-Ehrlich-Institut in Germany exclusively for ATMPs not based on a routine manufacturing procedure. In both procedures, quality, efficacy, and safety are evaluated and the risk-benefit balance is assessed. For the centralised procedure, mainly controlled clinical trial data must be submitted, whereas the requirements for national procedures could be modified corresponding to the stage of development of the ATMP. After marketing authorization, the marketing authorization/license holder is obligated to report all serious adverse reactions to the competent authority and to provide periodic safety update reports. If necessary, post-authorization safety studies could be imposed. On the basis of these regulatory measures, the safety of advanced therapies can be monitored and improved.

  15. Neural stem cell-based treatment for neurodegenerative diseases.

    Science.gov (United States)

    Kim, Seung U; Lee, Hong J; Kim, Yun B

    2013-10-01

    Human neurodegenerative diseases such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are caused by a loss of neurons and glia in the brain or spinal cord. Neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and stem cell-based cell therapies for neurodegenerative diseases have been developed. A recent advance in generation of a new class of pluripotent stem cells, induced pluripotent stem cells (iPSCs), derived from patients' own skin fibroblasts, opens doors for a totally new field of personalized medicine. Transplantation of NSCs, neurons or glia generated from stem cells in animal models of neurodegenerative diseases, including PD, HD, ALS and AD, demonstrates clinical improvement and also life extension of these animals. Additional therapeutic benefits in these animals can be provided by stem cell-mediated gene transfer of therapeutic genes such as neurotrophic factors and enzymes. Although further research is still needed, cell and gene therapy based on stem cells, particularly using neurons and glia derived from iPSCs, ESCs or NSCs, will become a routine treatment for patients suffering from neurodegenerative diseases and also stroke and spinal cord injury.

  16. A MULTISCALE, CELL-BASED FRAMEWORK FOR MODELING CANCER DEVELOPMENT

    Energy Technology Data Exchange (ETDEWEB)

    JIANG, YI [Los Alamos National Laboratory

    2007-01-16

    Cancer remains to be one of the leading causes of death due to diseases. We use a systems approach that combines mathematical modeling, numerical simulation, in vivo and in vitro experiments, to develop a predictive model that medical researchers can use to study and treat cancerous tumors. The multiscale, cell-based model includes intracellular regulations, cellular level dynamics and intercellular interactions, and extracellular level chemical dynamics. The intracellular level protein regulations and signaling pathways are described by Boolean networks. The cellular level growth and division dynamics, cellular adhesion and interaction with the extracellular matrix is described by a lattice Monte Carlo model (the Cellular Potts Model). The extracellular dynamics of the signaling molecules and metabolites are described by a system of reaction-diffusion equations. All three levels of the model are integrated through a hybrid parallel scheme into a high-performance simulation tool. The simulation results reproduce experimental data in both avasular tumors and tumor angiogenesis. By combining the model with experimental data to construct biologically accurate simulations of tumors and their vascular systems, this model will enable medical researchers to gain a deeper understanding of the cellular and molecular interactions associated with cancer progression and treatment.

  17. Stem cell-based therapies for acute radiation syndrome

    International Nuclear Information System (INIS)

    Exposure to high doses of ionizing radiation in the event of accidental or intentional incident such as nuclear/radiological terrorism can lead to debilitating injuries to multiple organs resulting in death within days depending on the amount of radiation dose and the quality of radiation. Unfortunately, there is not a single FDA-licensed drug approved against acute radiation injury. The RadStem Center for Medical Countermeasures against Radiation (RadStem CMGR) program at Einstein is developing stem cell-based therapies to treat acute radiation syndrome (ARS). We have demonstrated that intravenous transplantation of bone marrow-derived and adipose-derived stromal cells, consisting of a mixture of mesenchymal, endothelial and myeloid progenitors can mitigate mice exposed to whole body irradiation of 12 Gy or whole abdominal irradiation of up to 20 Gy. We identified a variety of growth and differentiation factors that individually is unable to improve survival of animals exposed to lethal irradiation, but when administered sequentially mitigates radiation injury and improves survival. We termed this phenomenon as synthetic survival and describe a new paradigm whereby the 'synthetic survival' of irradiated tissues can be promoted by systemic administration of growth factors to amplify residual stem cell clonogens post-radiation exposure, followed by a differentiation factor that favors tissue stem cell differentiation. Synthetic survival can be applied to mitigate lethal radiation injury in multiple organs following radiation-induced hematopoeitic, gastrointestinal and pulmonary syndromes. (author)

  18. [Safety monitoring of cell-based medicinal products (CBMPs)].

    Science.gov (United States)

    Funk, Markus B; Frech, Marion; Spranger, Robert; Keller-Stanislawski, Brigitte

    2015-11-01

    Cell-based medicinal products (CBMPs), a category of advanced-therapy medicinal products (ATMPs), are authorised for the European market by the European Commission by means of the centralized marketing authorisation. By conforming to the German Medicinal Products Act (Sec. 4b AMG), national authorisation can be granted by the Paul-Ehrlich-Institut in Germany exclusively for ATMPs not based on a routine manufacturing procedure. In both procedures, quality, efficacy, and safety are evaluated and the risk-benefit balance is assessed. For the centralised procedure, mainly controlled clinical trial data must be submitted, whereas the requirements for national procedures could be modified corresponding to the stage of development of the ATMP. After marketing authorization, the marketing authorization/license holder is obligated to report all serious adverse reactions to the competent authority and to provide periodic safety update reports. If necessary, post-authorization safety studies could be imposed. On the basis of these regulatory measures, the safety of advanced therapies can be monitored and improved. PMID:26391098

  19. Microbial fuel cell based on electroactive sulfate-reducing biofilm

    International Nuclear Information System (INIS)

    Highlights: ► Regulation and management of electricity generation by variation of residence time. ► Design of microbial fuel cell based on electroactive biofilm on zeolite. ► Engineering solution for removing of the obtained elemental sulfur. - abstract: A two chambered laboratory scale microbial fuel cell (MFC) has been developed, based on natural sulfate-reducing bacterium consortium in electroactive biofilm on zeolite. The MFC utilizes potassium ferricyanide in the cathode chamber as an electron acceptor that derives electrons from the obtained in anode chamber H2S. The molecular oxygen is finally used as a terminal electron acceptor at cathode compartment. The generated power density was 0.68 W m−2 with current density of 3.2 A m−2 at 150 Ω electrode resistivity. The hydrogen sulfide itself is produced by microbial dissimilative sulfate reduction process by utilizing various organic substrates. Finally, elemental sulfur was identified as the predominant final oxidation product in the anode chamber. It was removed from MFC through medium circulation and gathering in an external tank. This report reveals dependence relationship between the progress of general electrochemical parameters and bacterial sulfate-reduction rate. The presented MFC design can be used for simultaneous sulfate purification of mining drainage wastewater and generation of renewable electricity

  20. Preclinical safety testing for cell-based products using animals.

    Science.gov (United States)

    McBlane, James W

    2015-09-01

    The objectives of preclinical testing include to show why there might be therapeutic benefit in patients and to provide information on the product's toxicity. For cell-based products, given even once, there may be long term exposure and this could imply, unlike for conventional drugs, that all preclinical studies may be needed prior to first human use. The duration of exposure to cells should be studied in animals to guide toxicity assessments. Distribution of cells after administration by a route resembling that intended in humans should be studied to understand potential risks. Risk of tumour formation with the product may also need to be characterised. To the extent that this information can be generated by in vitro testing, studies in animals may not be needed and limitations on the capability of preclinical data to predict human toxicity are recognised: species-specificity make some cell products act only in humans and a human cell-product might be expected to be rejected by immunocompetent animals. Does this suggest testing in immunosuppressed animals or of development of an animal-cell product supposedly similar to the human cell product? No single answer seems to fit every situation.

  1. Regulations and guidelines governing stem cell based products: Clinical considerations

    Directory of Open Access Journals (Sweden)

    Bobby George

    2011-01-01

    Full Text Available The use of stem cells as medicines is a promising and upcoming area of research as they may be able to help the body to regenerate damaged or lost tissue in a host of diseases like Parkinson′s, multiple sclerosis, heart disease, liver disease, spinal cord damage, cancer and many more. Translating basic stem cell research into routine therapies is a complex multi-step process which entails the challenge related to managing the expected therapeutic benefits with the potential risks while complying with the existing regulations and guidelines. While in the United States (US and European Union (EU regulations are in place, in India, we do not have a well-defined regulatory framework for "stem cell based products (SCBP". There are several areas that need to be addressed as it is quite different from that of pharmaceuticals. These range from establishing batch consistency, product stability to product safety and efficacy through pre-clinical, clinical studies and marketing authorization. This review summarizes the existing regulations/guidelines in US, EU, India, and the associated challenges in developing SCBP with emphasis on clinical aspects.

  2. A novel multi-functional cell-based microphysiometer

    Institute of Scientific and Technical Information of China (English)

    XU Ying; XU Gaixia; LIU Qingjun; CAI Hua; LI Yan; LI Rong; WANG Ping

    2006-01-01

    This paper presents a novel multi-functional microphysiometer for simultaneous measurements of several extracellular ion concentrations and action potential measurement in living cells based on MLAPS (multi-light addressable potentiometric sensor). In the microphysiometer, sorts of sensitive membranes are illuminated in parallel with n light sources at working frequencies, and the response amplitudes of each frequency component can be measured on-line by parallel processing algorithm. In the experiments, the relations of the extracellular environmental H+, Na +, K +, Ca2 + under the effects of western medicines (dilantin, phenobarbital sodium) and Chinese drugs (scutellaria, medlar, hemlock parsley) were analyzed, and the effects of several drugs were evaluated. Moreover, the action potential signals of different cell types (cardiac myocytes and neurons) could be measured and analyzed by LAPS. By detecting these parameters, the system can monitor the real-time process of the cell metabolism and action potential, observe the functional responses of different kinds of membrane-bound receptors, and evaluate the activities of drugs.

  3. From Antenna to Assay

    Science.gov (United States)

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  4. Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions.

    Science.gov (United States)

    Soares, Filipa A C; Chandra, Amit; Thomas, Robert J; Pedersen, Roger A; Vallier, Ludovic; Williams, David J

    2014-03-10

    The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes.

  5. Total Vitamin D Assay Comparison of the Roche Diagnostics “Vitamin D Total” Electrochemiluminescence Protein Binding Assay with the Chromsystems HPLC Method in a Population with both D2 and D3 forms of Vitamin D

    OpenAIRE

    Gemma Patras; Louisa Grundy; Fasila Pallinalakam; Nafiz Hussein; Faten Mustafa; Arwa Salem; Shoukat Khan; Andrew Turner; Afrozul Haq; Laila Abdel-Wareth; Jaishen Rajah

    2013-01-01

    This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (7...

  6. A High-Throughput Yeast Halo Assay for Bioactive Compounds.

    Science.gov (United States)

    Bray, Walter; Lokey, R Scott

    2016-01-01

    When a disk of filter paper is impregnated with a cytotoxic or cytostatic drug and added to solid medium seeded with yeast, a visible clear zone forms around the disk whose size depends on the concentration and potency of the drug. This is the traditional "halo" assay and provides a convenient, if low-throughput, read-out of biological activity that has been the mainstay of antifungal and antibiotic testing for decades. Here, we describe a protocol for a high-throughput version of the halo assay, which uses an array of 384 pins to deliver ∼200 nL of stock solutions from compound plates onto single-well plates seeded with yeast. Using a plate reader in the absorbance mode, the resulting halos can be quantified and the data archived in the form of flat files that can be connected to compound databases with standard software. This assay has the convenience associated with the visual readout of the traditional halo assay but uses far less material and can be automated to screen thousands of compounds per day. PMID:27587777

  7. Fluorescence assay for evaluating microbicidal activity of hand antiseptics.

    Science.gov (United States)

    Lopez-Gigosos, Rosa M; Mariscal, Alberto; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin

    2015-11-01

    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques.

  8. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment. PMID:27208079

  9. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  10. Micro-fluidic module for blood cell separation for gene expression radiobiological assays

    International Nuclear Information System (INIS)

    Advances in molecular techniques have improved discovery of biomarkers associated with radiation exposure. Gene expression techniques have been demonstrated as effective tools for biodosimetry, and different assay platforms with different chemistries are now available. One of the main challenges is to integrate the sample preparation processing of these assays into micro-fluidic platforms to be fully automated for point-of-care medical countermeasures in the case of a radiological event. Most of these assays follow the same workflow processing that comprises first the collection of blood samples followed by cellular and molecular sample preparation. The sample preparation is based on the specific reagents of the assay system and depends also on the different subsets of cells population and the type of biomarkers of interest. In this article, the authors present a module for isolation of white blood cells from peripheral blood as a prerequisite for automation of gene expression assays on a micro-fluidic cartridge. For each sample condition, the gene expression platform can be adapted to suit the requirements of the selected assay chemistry (authors)

  11. Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

    Institute of Scientific and Technical Information of China (English)

    Quan Jiang

    2016-01-01

    Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury, substantially improve functional outcome. We discuss and review state of the art magnetic resonance im-aging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

  12. Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

    Directory of Open Access Journals (Sweden)

    Quan Jiang

    2016-01-01

    Full Text Available Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury, substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

  13. New Application of the Comet Assay: Chromosome–Comet Assay

    OpenAIRE

    Cortés-Gutiérrez, Elva I.; DÁVILA-RODRÍGUEZ, MARTHA I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be use...

  14. Comparison of chemosensitivity tests: clonogenic assay versus MTT assay.

    Directory of Open Access Journals (Sweden)

    Kawada K

    2002-06-01

    Full Text Available When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay as a substitute for the human tumor clonogenic assay (HTCA, we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939 and good in anthracyclines/anthracenedione (r = 0.611. However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan, and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.

  15. Calorimetric assay of minor actinides

    Energy Technology Data Exchange (ETDEWEB)

    Rudy, C.; Bracken, D.; Cremers, T.; Foster, L.A.; Ensslin, N.

    1996-12-31

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques.

  16. Calorimetric assay of minor actinides

    International Nuclear Information System (INIS)

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques

  17. A high-throughput chemically induced inflammation assay in zebrafish

    Directory of Open Access Journals (Sweden)

    Liebel Urban

    2010-12-01

    Full Text Available Abstract Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

  18. Clinical performance of automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance assay in diagnosis of pulmonary tuberculosis in 214 cases%利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术在214例诊断肺结核患者中的临床应用评价

    Institute of Scientific and Technical Information of China (English)

    何贵清; 李涛; 施伎蝉; 宁洪叶; 吴祥兵; 蔡明明; 吴正兴; 胡陈婵; 蒋贤高

    2016-01-01

    目的:评估利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert M TB/RIF)在温州地区诊断肺结核及利福平耐药的临床应用价值。方法纳入可疑肺结核、临床诊断肺结核和可疑耐药肺结核患者214例,采集痰标本同时送检抗酸染色涂片、液体培养和 Xpert M TB/RIF 检测。以临床最终诊断结果为金标准,比较三种方法检测结核分枝杆菌的敏感度、特异度。以液体药物敏感试验结果为金标准,Xpert M TB/RIF 检测利福平耐药的敏感度和特异度。率的比较采取卡方检验。结果以临床最终诊断结果作为金标准,Xpert M TB/RIF 检测结核分枝杆菌的敏感度高于抗酸染色涂片(69.5%比44.1%,χ2=23.31,P <0.01),而与液体培养比较敏感度差异无统计学意义(69.5%比62.1%,χ2=2.15,P>0.05);Xpert M TB/RIF 在痰涂片阳性与阴性的标本中检测结核分枝杆菌的敏感度分别为97.4%和47.5%,在痰涂片阳性与阴性的标本中检测结核分枝杆菌的特异度均为100.0%。以液体药物敏感试验结果为金标准,Xpert M TB/RIF 检测痰标本利福平耐药的敏感度和特异度分别为92.9%和98.8%。结论 Xpert M TB/RIF 能够快速、准确地检测结核分枝杆菌及其利福平耐药性,且敏感度较高,具有很好的应用价值。%Objective To evaluate the clinical application of the automated nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance (Xpert M TB/RIF) in diagnosis of pulmonary tuberculosis in Wenzhou .Methods A total of 214 patients with suspected pulmonary tuberculosis , clinical diagnosed tuberculosis or suspected drug‐resistant tuberculosis were enrolled in this study .The patients′ sputum specimens were collected for acid‐fast smear ,liquid culture and Xpert M TB/RIF assay .The sensitivity

  19. Modeling and control of fuel cell based distributed generation systems

    Science.gov (United States)

    Jung, Jin Woo

    This dissertation presents circuit models and control algorithms of fuel cell based distributed generation systems (DGS) for two DGS topologies. In the first topology, each DGS unit utilizes a battery in parallel to the fuel cell in a standalone AC power plant and a grid-interconnection. In the second topology, a Z-source converter, which employs both the L and C passive components and shoot-through zero vectors instead of the conventional DC/DC boost power converter in order to step up the DC-link voltage, is adopted for a standalone AC power supply. In Topology 1, two applications are studied: a standalone power generation (Single DGS Unit and Two DGS Units) and a grid-interconnection. First, dynamic model of the fuel cell is given based on electrochemical process. Second, two full-bridge DC to DC converters are adopted and their controllers are designed: an unidirectional full-bridge DC to DC boost converter for the fuel cell and a bidirectional full-bridge DC to DC buck/boost converter for the battery. Third, for a three-phase DC to AC inverter without or with a Delta/Y transformer, a discrete-time state space circuit model is given and two discrete-time feedback controllers are designed: voltage controller in the outer loop and current controller in the inner loop. And last, for load sharing of two DGS units and power flow control of two DGS units or the DGS connected to the grid, real and reactive power controllers are proposed. Particularly, for the grid-connected DGS application, a synchronization issue between an islanding mode and a paralleling mode to the grid is investigated, and two case studies are performed. To demonstrate the proposed circuit models and control strategies, simulation test-beds using Matlab/Simulink are constructed for each configuration of the fuel cell based DGS with a three-phase AC 120 V (L-N)/60 Hz/50 kVA and various simulation results are presented. In Topology 2, this dissertation presents system modeling, modified space

  20. Automated in vivo platform for the discovery of functional food treatments of hypercholesterolemia.

    Directory of Open Access Journals (Sweden)

    Robert M Littleton

    Full Text Available The zebrafish is becoming an increasingly popular model system for both automated drug discovery and investigating hypercholesterolemia. Here we combine these aspects and for the first time develop an automated high-content confocal assay for treatments of hypercholesterolemia. We also create two algorithms for automated analysis of cardiodynamic data acquired by high-speed confocal microscopy. The first algorithm computes cardiac parameters solely from the frequency-domain representation of cardiodynamic data while the second uses both frequency- and time-domain data. The combined approach resulted in smaller differences relative to manual measurements. The methods are implemented to test the ability of a methanolic extract of the hawthorn plant (Crataegus laevigata to treat hypercholesterolemia and its peripheral cardiovascular effects. Results demonstrate the utility of these methods and suggest the extract has both antihypercholesterolemic and postitively inotropic properties.

  1. Methods of automated cell analysis and their application in radiation biology

    International Nuclear Information System (INIS)

    The present review is concerned with the methods of automated analysis of biological microobjects and covers two groups into which all the systems of automated analysis can be divided-systems of flow ( flow cytometry) and scanning (image analysis systems) type. Particular emphasis has been placed on their use in radiobiological studies, namely, in the micronucleus test, a cytogenetic assay for monitoring the clastogenic action of ionizing radiation commonly used at present. Examples of suing methods described and actual setups in other biomedical researches are given. Analysis of advantages and disadvantages of the methods of automated cell analysis enables to choose more thoroughly between the systems of flow and scanning type to use them in particular research

  2. The promise of fuel cell-based automobiles

    Indian Academy of Sciences (India)

    A K Shukla; C L Jackson; K Scott

    2003-02-01

    Fuel cell-based automobiles have gained attention in the last few years due to growing public concern about urban air pollution and consequent environmental problems. From an analysis of the power and energy requirements of a modern car, it is estimated that a base sustainable power of $ca$. 50 kW supplemented with short bursts up to 80 kW will suffice in most driving requirements. The energy demand depends greatly on driving characteristics but under normal usage is expected to be 200 Wh/km. The advantages and disadvantages of candidate fuel-cell systems and various fuels are considered together with the issue of whether the fuel should be converted directly in the fuel cell or should be reformed to hydrogen onboard the vehicle. For fuel cell vehicles to compete successfully with conventional internal-combustion engine vehicles, it appears that direct conversion fuel cells using probably hydrogen, but possibly methanol, are the only realistic contenders for road transportation applications. Among the available fuel cell technologies, polymer–electrolyte fuel cells directly fueled with hydrogen appear to be the best option for powering fuel cell vehicles as there is every prospect that these will exceed the performance of the internal-combustion engine vehicles but for their first cost. A target cost of $ 50/kW would be mandatory to make polymer–electrolyte fuel cells competitive with the internal combustion engines and can only be achieved with design changes that would substantially reduce the quantity of materials used. At present, prominent car manufacturers are deploying important research and development efforts to develop fuel cell vehicles and are projecting to start production by 2005.

  3. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    Science.gov (United States)

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  4. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    Science.gov (United States)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  5. Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery

    NARCIS (Netherlands)

    Malergue, Fabrice; van Agthoven, Andreas; Scifo, Caroline; Egan, Dave; Strous, Ger J

    2015-01-01

    Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate

  6. Rapid screening test for primary hyperaldosteronism: ratio of plasma aldosterone to renin concentration determined by fully automated chemiluminescence immunoassays.

    NARCIS (Netherlands)

    Perschel, F.H.; Schemer, R.; Seiler, L.; Reincke, M.; Deinum, J.; Maser-Gluth, C.; Mechelhoff, D.; Tauber, R.; Diederich, S.

    2004-01-01

    BACKGROUND: The ratio of plasma aldosterone concentration to plasma renin activity (PAC/PRA) is the most common screening test for primary hyperaldosteronism (PHA), but it is not standardized among laboratories. We evaluated new automated assays for the simultaneous measurement of PAC and plasma ren

  7. Laboratory automation and LIMS in forensics

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hansen, Anders Johannes; Morling, Niels

    2013-01-01

    Implementation of laboratory automation and LIMS in a forensic laboratory enables the laboratory, to standardize sample processing. Automated liquid handlers can increase throughput and eliminate manual repetitive pipetting operations, known to result in occupational injuries to the technical staff....... Furthermore, implementation of automated liquid handlers reduces the risk of sample misplacement. A LIMS can efficiently control the sample flow through the laboratory and manage the results of the conducted tests for each sample. Integration of automated liquid handlers with a LIMS provides the laboratory...... with the tools required for setting up automated production lines of complex laboratory processes and monitoring the whole process and the results. Combined, this enables processing of a large number of samples. Selection of the best automated solution for an individual laboratory should be based on user...

  8. Time-correlated single photon counting: an advancing technique in a plate reader for assay development and high throughput screening

    Science.gov (United States)

    Näther, Dirk U.; Fenske, Roger; Hurteaux, Reynald; Majno, Sandra; Smith, S. Desmond

    2006-10-01

    A new plate reader (Nanotaurus) has been developed by Edinburgh Instruments that has the principle design features of a confocal microscope and utilises the technique of Time Correlated Single Photon Counting for data acquisition. The advantages of Fluorescence Lifetime Measurements in the nanosecond time scale and analysis methods to recover lifetime parameters are discussed based on experimental data. First working assays using changes of lifetime parameters are presented that clearly demonstrate the advantages of the new instrument for biochemical assays and show strong promise for cell-based assays, by utilising the independence of lifetime parameters from sample volume and concentration.

  9. Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening

    Directory of Open Access Journals (Sweden)

    Clemens eMoeller

    2011-11-01

    Full Text Available The field of automated patch-clamp electrophysiology has emerged from the tension between the pharmaceutical industry’s need for high-throughput compound screening versus its need to be conservative due to regulatory requirements. On the one hand, hERG channel screening was increasingly requested for new chemical entities, as the correlation between blockade of the ion channel coded by hERG and Torsades de Pointes cardiac arrhythmia gained increasing attention. On the other hand, manual patch-clamping, typically quoted as the gold-standard for understanding ion channel function and modulation, was far too slow (and, consequently, too expensive for keeping pace with the numbers of compounds submitted for hERG channel investigations from pharmaceutical R&D departments. In consequence it became more common for some pharmaceutical companies to outsource safety pharmacological investigations, with a focus on hERG channel interactions. This outsourcing has allowed those pharmaceutical companies to build up operational flexibility and greater independence from internal resources, and allowed them to obtain access to the latest technological developments that emerged in automated patch-clamp electrophysiology – much of which arose in specialized biotech companies. Assays for nearly all major cardiac ion channels are now available by automated patch-clamping using heterologous expression systems, and recently, automated action potential recordings from stem-cell derived cardiomyocytes have been demonstrated. Today, most of the large pharmaceutical companies have acquired automated electrophysiology robots and have established various automated cardiac ion channel safety screening assays on these, in addition to outsourcing parts of their needs for safety screening.

  10. Modern trends in automation of industrial complexes

    OpenAIRE

    Svjatnyj, Vladimir A.; Brovkina, Daniella Yu.

    2016-01-01

    This paper is dedicated to the study of the transition of manufacturing to a new stage of its development due to the introduction of the latest computer technologies, that have become common in the service sector, into the automation process. One of the characteristic features of the development of modern industry is the integration of the achievements of the theory and practice of automation, information technology, robotics and "human–automated object" systems. "Industry 4.0" was designed a...

  11. NSC23925, identified in a high-throughput cell-based screen, reverses multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Zhenfeng Duan

    Full Text Available BACKGROUND: Multidrug resistance (MDR is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically. METHODOLOGY/PRINCIPAL FINDINGS: To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1 but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 microM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors. CONCLUSIONS/SIGNIFICANCE: The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.

  12. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

    Directory of Open Access Journals (Sweden)

    Ahmad Arouri

    Full Text Available The feasibility of exploiting secretory phospholipase A2 (sPLA2 enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

  13. Automated Contingency Management for Propulsion Systems

    Data.gov (United States)

    National Aeronautics and Space Administration — Increasing demand for improved reliability and survivability of mission-critical systems is driving the development of health monitoring and Automated Contingency...

  14. Rapid Automated Mission Planning System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed innovation is an automated UAS mission planning system that will rapidly identify emergency (contingency) landing sites, manage contingency routing,...

  15. The light spot test: Measuring anxiety in mice in an automated home-cage environment.

    Science.gov (United States)

    Aarts, Emmeke; Maroteaux, Gregoire; Loos, Maarten; Koopmans, Bastijn; Kovačević, Jovana; Smit, August B; Verhage, Matthijs; Sluis, Sophie van der

    2015-11-01

    Behavioral tests of animals in a controlled experimental setting provide a valuable tool to advance understanding of genotype-phenotype relations, and to study the effects of genetic and environmental manipulations. To optimally benefit from the increasing numbers of genetically engineered mice, reliable high-throughput methods for comprehensive behavioral phenotyping of mice lines have become a necessity. Here, we describe the development and validation of an anxiety test, the light spot test, that allows for unsupervised, automated, high-throughput testing of mice in a home-cage system. This automated behavioral test circumvents bias introduced by pretest handling, and enables recording both baseline behavior and the behavioral test response over a prolonged period of time. We demonstrate that the light spot test induces a behavioral response in C57BL/6J mice. This behavior reverts to baseline when the aversive stimulus is switched off, and is blunted by treatment with the anxiolytic drug Diazepam, demonstrating predictive validity of the assay, and indicating that the observed behavioral response has a significant anxiety component. Also, we investigated the effectiveness of the light spot test as part of sequential testing for different behavioral aspects in the home-cage. Two learning tests, administered prior to the light spot test, affected the light spot test parameters. The light spot test is a novel, automated assay for anxiety-related high-throughput testing of mice in an automated home-cage environment, allowing for both comprehensive behavioral phenotyping of mice, and rapid screening of pharmacological compounds.

  16. Smart Home Automation with Linux

    CERN Document Server

    Goodwin, Steven

    2010-01-01

    Linux users can now control their homes remotely! Are you a Linux user who has ever wanted to turn on the lights in your house, or open and close the curtains, while away on holiday? Want to be able to play the same music in every room, controlled from your laptop or mobile phone? Do you want to do these things without an expensive off-the-shelf kit? In Beginning Linux Home Automation, Steven Goodwin will show you how a house can be fully controlled by its occupants, all using open source software. From appliances to kettles to curtains, control your home remotely! What you'll learn* Control a

  17. Ball Bearing Stacking Automation System

    Directory of Open Access Journals (Sweden)

    Shafeequerrahman S . Ahmed

    2013-01-01

    Full Text Available This document is an effort to introduce the concept of automation in small scale industries and or small workshops that are involved in the manufacturing of small objects such as nuts, bolts and ball bearing in this case. This an electromechanical system which includes certain mechanical parts that involves one base stand on which one vertical metallic frame is mounted and hinged to this vertical stand is an in humanized effort seems inadequate in this era making necessary the use of Electronics, Computer in the manufacturing processes leading to the concept of Automated Manufacturing System (AMS.The ball bearing stack automation is an effort in this regard. In our project we go for stack automation for any object for example a ball bearing, be that is still a manual system there. It will be microcontroller based project control system equipped with microcontroller 89C51 from any manufacturer like Atmel or Philips. This could have been easily implemented if a PLC could be used for manufacturing the staking unit but I adopted the microcontroller based system so that some more modification in the system can be effected at will as to use the same hardware .Although a very small object i.e. ball bearig or small nut and fixture will be tried to be stacked, the system with more precision and more power handling capacity could be built for various requirements of the industry. For increasing more control capacity, we can use another module of this series. When the bearing is ready, it will be sent for packing. This is sensed by an inductive sensor. The output will be proceeds by PLC and microcontroller card which will be driving the assembly in order to put it into pads or flaps. This project will also count the total number of bearings to be packed and will display it on a LCD for real time reference and a provision is made using a higher level language using hyper terminal of the computer

  18. Robotium automated testing for Android

    CERN Document Server

    Zadgaonkar, Hrushikesh

    2013-01-01

    This is a step-by-step, example-oriented tutorial aimed at illustrating the various test scenarios and automation capabilities of Robotium.If you are an Android developer who is learning how to create test cases to test their application, and are looking to get a good grounding in different features in Robotium, this book is ideal for you. It's assumed that you have some experience in Android development, as well be familiar with the Android test framework, as Robotium is a wrapper to Android test framework.

  19. AUTOMATED TESTING OF OPC SERVERS

    CERN Document Server

    Farnham, B

    2011-01-01

    CERN relies on OPC Server implementations from 3rd party device vendors to provide a software interface to their respective hardware. Each time a vendor releases a new OPC Server version it is regression tested internally to verify that existing functionality has not been inadvertently broken during the process of adding new features. In addition bugs and problems must be communicated to the vendors in a reliable and portable way. This presentation covers the automated test approach used at CERN to cover both cases: Scripts are written in a domain specific language specifically created for describing OPC tests and executed by a custom software engine driving the OPC Server implementation.

  20. Automated Orientation of Aerial Images

    DEFF Research Database (Denmark)

    Høhle, Joachim

    2002-01-01

    Methods for automated orientation of aerial images are presented. They are based on the use of templates, which are derived from existing databases, and area-based matching. The characteristics of available database information and the accuracy requirements for map compilation and orthoimage...... production are discussed on the example of Denmark. Details on the developed methods for interior and exterior orientation are described. Practical examples like the measurement of réseau images, updating of topographic databases and renewal of orthoimages are used to prove the feasibility of the developed...