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Sample records for high assay utilization

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

    Science.gov (United States)

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

    2008-10-01

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

  2. On-line radiochemical assay for monoamine oxidase utilizing high-performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Nissinen, E.; Linko-Loeppoenen SMae; Maennistoe P4

    1984-12-01

    A fast and sensitive assay for the determination of monoamine oxidase activity was developed. The method is based on the separation and quantitation of /sup 14/C-labeled assay products by high-performance liquid chromatography, which is interfaced directly into a flow-through radioactivity detector. This allows on-line quantitation of the radioactive compounds with picomole sensitivity. The method makes possible the complete separation and detection of the deaminated products of monoamine oxidase A and B substrates benzylamine and 5-hydroxytryptamine, respectively. This assay has been applied to the measurement of monoamine oxidase A and B activities in rat brain.

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

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    Yiyi Sun

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

  4. Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

    Science.gov (United States)

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  5. Utilizing high throughput screening data for predictive toxicology models: protocols and application to MLSCN assays

    Science.gov (United States)

    Guha, Rajarshi; Schürer, Stephan C.

    2008-06-01

    Computational toxicology is emerging as an encouraging alternative to experimental testing. The Molecular Libraries Screening Center Network (MLSCN) as part of the NIH Molecular Libraries Roadmap has recently started generating large and diverse screening datasets, which are publicly available in PubChem. In this report, we investigate various aspects of developing computational models to predict cell toxicity based on cell proliferation screening data generated in the MLSCN. By capturing feature-based information in those datasets, such predictive models would be useful in evaluating cell-based screening results in general (for example from reporter assays) and could be used as an aid to identify and eliminate potentially undesired compounds. Specifically we present the results of random forest ensemble models developed using different cell proliferation datasets and highlight protocols to take into account their extremely imbalanced nature. Depending on the nature of the datasets and the descriptors employed we were able to achieve percentage correct classification rates between 70% and 85% on the prediction set, though the accuracy rate dropped significantly when the models were applied to in vivo data. In this context we also compare the MLSCN cell proliferation results with animal acute toxicity data to investigate to what extent animal toxicity can be correlated and potentially predicted by proliferation results. Finally, we present a visualization technique that allows one to compare a new dataset to the training set of the models to decide whether the new dataset may be reliably predicted.

  6. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  7. Utility of a novel lipoarabinomannan assay for the diagnosis of tuberculous meningitis in a resource-poor high-HIV prevalence setting

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    Ndung'u Thumbi

    2009-11-01

    Full Text Available Abstract Background In Africa, tuberculous meningitis (TBM is an important opportunistic infection in HIV-positive patients. Current diagnostic tools for TBM perform sub-optimally. In particular, the rapid diagnosis of TBM is challenging because smear microscopy has a low yield and PCR is not widely available in resource-poor settings. Methods We evaluated the performance outcome of a novel standardized lipoarabinomannan (LAM antigen-detection assay, using archived cerebrospinal fluid samples, in 50 African TBM suspects of whom 68% were HIV-positive. Results Of the 50 participants 14, 23 and 13 patients had definite, probable and non-TBM, respectively. In the non-TB group there were 5 HIV positive patients who were lost to follow-up and in whom concomitant infection with Mycobacterium tuberculosis could not be definitively excluded. The test sensitivities and specificities were as follows: LAM assay 64% and 69% (cut-point 0.22, smear microscopy 0% and 100% and PCR 93% and 77%, respectively. Conclusion In this preliminary proof-of-concept study, a rapid diagnosis of TBM could be achieved using LAM antigen detection. Although specificity was sub-optimal, the estimates provided here may be unreliable because of a classification bias inherent in the study design where it was not possible to exclude TBM in the presumed non-TBM cases owing to a lack of clinical follow-up. As PCR is largely unavailable, the LAM assay may well prove to be a useful adjunct for the rapid diagnosis of TBM in high HIV-incidence settings. These preliminary results justify further enquiry and prospective studies are now required to definitively establish the place of this technology for the diagnosis of TBM.

  8. Clinical utility of the PCA3 urine assay in European men scheduled for repeat biopsy.

    NARCIS (Netherlands)

    Haese, A.; Taille, A de la; Poppel, H van; Marberger, M.; Stenzl, A.; Mulders, P.F.A.; Huland, H.; Abbou, C.C.; Remzi, M.; Tinzl, M.; Feyerabend, S.; Stillebroer, A.B.; Gils, M.P.M.Q.; Schalken, J.A.

    2008-01-01

    BACKGROUND: The Prostate CAncer gene 3 (PCA3) assay has shown promise as an aid in prostate cancer (pCA) diagnosis in identifying men with a high probability of a positive (repeat) biopsy. OBJECTIVE: This study evaluated the clinical utility of the PROGENSA PCA3 assay. DESIGN, SETTING, AND PARTICIPA

  9. Evaluation of two high through-put (HTP) androgen receptor based assays: Utility of data for prioritization for further testing versus prediction of adverse effects.

    Science.gov (United States)

    The androgen signaling pathway plays a critical role in sexual differentiation during development in mammals and is one of the better understood pathways in human development. Thus it was chosen as a model pathway to evaluate the potential of HTP in vitro assays as risk assessmen...

  10. Delivery of High-Quality Biomarker Assays

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    Brian N. Swanson

    2002-01-01

    Full Text Available Biomarker measurements now support key decisions throughout the drug development process, from lead optimization to regulatory approvals. They are essential for documenting exposure-response relationships, specificity and potency toward the molecular target, untoward effects, and therapeutic applications. In a broader sense, biomarkers constitute the basis of clinical pathology and laboratory medicine. The utility of biomarkers is limited by their specificity and sensitivity toward the drug or disease process and by their overall variability. Understanding and controlling sources of variability is not only imperative for delivering high-quality assay results, but ultimately for controlling the size and expense of research studies. Variability in biomarker measurements is affected by: biological and environmental factors (e.g., gender, age, posture, diet and biorhythms, sample collection factors (e.g., preservatives, transport and storage conditions, and collection technique, and analytical factors (e.g., purity of reference material, pipetting precision, and antibody specificity. The quality standards for biomarker assays used in support of nonclinical safety studies fall under GLP (FDA regulations, whereas, those assays used to support human diagnostics and healthcare are established by CLIA (CMS regulations and accrediting organizations such as the College of American Pathologists. While most research applications of biomarkers are not regulated, biomarker laboratories in all settings are adopting similar laboratory practices in order to deliver high-quality data. Because of the escalation in demand for biomarker measurements, the highly-parallel (multi-plexed assay platforms that have fueled the rise of genomics will likely evolve into the analytical engines that drive the biomarker laboratories of tomorrow.

  11. Smartphone-enabled filterless fluorescence assay utilizing the pyrene excimer

    Science.gov (United States)

    Goertz, John P.; White, Ian M.

    2015-03-01

    Fluorescence microscopy offers a number of advantages for cell- and biomarker-based diagnostics with regards to ease of use and interpretation, sensitivity, and specificity. However, its use in low-resource settings is often hindered by the need for bulky microscopes with expensive excitation and filter setups. While many advances have been made towards utilizing smartphones as microscopes, there remains a reliance on complex attachments to facilitate fluorescence microscopy. Here, we report progress towards a filter-less fluorescent assay utilizing ultraviolet light, an unmodified smartphone, and pyrene-labeled aptamers. The pyrene monomer is excited at a wavelength of 350 nm and emits at approximately 390 nm; when two pyrene molecules are brought into close proximity, however, they form an excimer which emits at approximately 490 nm. We have engineered pyrene-conjugated DNA sequences such that the fluorophores, normally in monomeric configuration, are brought into proximity upon binding of the DNA to its target. The large Stokes shift between excitation and emission of the excimer allows us to detect such biorecognition events with an unfiltered smartphone camera, enabling the use of this assay in low-resource settings where portability and easeof- use are paramount.

  12. Latex immunoagglutination assay for bovine viral diarrhea virus utilizing forward light scattering in a microfluidic device

    Science.gov (United States)

    Heinze, Brian C.; Song, Jae-Young; Han, Jin-Hee; Yoon, Jeong-Yeol

    2008-02-01

    We have investigated the utilization of particle agglutination assays using forward light scattering measurements in a microfluidic device towards detecting viral particles. The model viral target was bovine viral diarrhea virus (BVDV). Highly carboxylated polystyrene microspheres (510 nm) were coated with anti-BVDV monoclonal antibodies. This solution was in turn used to detect live modified BVDV. This assay was first performed in a two well slide for proof of concept and then in a simple y-channel microfluidic device with optical fibers arranged in a close proximity setup. Particle immunoagglutination was detected through static light scattering measurements taken at 45° to incident light. In the microfluidic device, modified live BVDV was detected with a detection limit of 0.5 TCID 50 mL -1.

  13. A highly scalable peptide-based assay system for proteomics.

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    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  14. Utility of Pooled HIV RNA RT-PCR Assay in Diagnosing Acute HIV Infections

    Institute of Scientific and Technical Information of China (English)

    张麒; 蒋岩; 刘全忠

    2004-01-01

    Abstract: The P24 antigen test, HIV RNA PCR test,HIV isolation/culture and fourth-generation HIV uniform Ag/Ab assay are being utilized in diagnosing acute HIV infection in different labs. Many factors limit the use of screening for acute HIV in high-risk populations, in blood donors and during voluntary HIV testing, including, cost, technique, sensitivity and specificity. In this review we explore a new NAAT method which involves HIV RNA RT-PCR on pooled samples. This technique is able to screen for acute infections in a large testing volume and may he used as a screening method in high-risk populations and blood donors.

  15. Utilization of cardiac troponin assays in adult and pediatric populations: Guideline recommendations vs. reality.

    Science.gov (United States)

    Saenger, Amy K; Haymond, Shannon

    2015-12-01

    We hypothesized significant gaps remained for cardiac troponin (cTn) utilization in the United States, despite an emerging evidence base and guideline recommendations. We tested this hypothesis and investigated differences and trends between the use of cTn in adult versus pediatric hospitals. Individuals were identified by a targeted distribution through personal contact and professional society email lists. Participants completed an online survey (Qualtrics) from 07/15/13 to 07/26/13. The 31-item questionnaire used skipped logic and collected data about the respondent and their institutional cTn clinical practices. Data tabulation and analysis were conducted using Qualtrics and Microsoft Excel. A total of 159 unique laboratories responded to the survey, representing primarily adult (81%) versus pediatric (19%) institutions. 59% of laboratories utilize the guideline recommended 99th percentile as the upper reference limit (URL) for cTn, with large variability in reporting practices among users of the same assay. 73% of laboratories reported simultaneous ordering of other cardiac biomarkers with cTn, a majority which included CK-MB. Interpretive comments were used with cTn in 71 laboratories with a significant amount of heterogeneity. Pediatric hospitals reported a lower frequency of cTn orders and were less likely to consider elevated cTn a critical value. Gaps in current utilization and reporting of cTn exist, along with practices inconsistent with clinical guideline recommendations. Implementation of the 99th percentile and serial sampling protocols will be critical to adoption of high-sensitivity cTn assays. Differences in chest pain etiology are the most likely reason for the notable differences between cTn use in adults versus pediatric hospitals. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

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    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.

  17. Utility of line probe assay for the early detection of multidrug-resistant pulmonary tuberculosis

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    K Madhuri

    2015-01-01

    Full Text Available Background: Despite endorsement of the line probe assay (LPA for the diagnosis of drug-resistant pulmonary tuberculosis patients, there is limited data available on the performance of LPAs in India, especially from high burden states like Maharashtra, for the early diagnosis and detection of drug resistance, in order to initiate timely and appropriate treatment. Objective: To evaluate the utility of the line probe assay (LPA for the early diagnosis of drug-resistant pulmonary tuberculosis as compared to the ′Gold standard′ 1% proportion method (PM. Materials and Methods: A total of 687 patients suspected of pulmonary tuberculosis were screened. One hundred samples (95 sputum and 5 BAL, positive for Acid Fast Bacilli (AFB by Ziehl Neelson (ZN smears, were included in the study. Digested and decontaminated specimens were subjected directly to the LPA (Genotype MTBDR@ plus assay and were processed in parallel using the conventional culture on the Lowenstein-Jensen (LJ medium followed by drug susceptibility testing (DST using the PM. Results: All the 100 samples gave interpretable results on LPA with a turnaround time of 24-48 hours as opposed to six to eight weeks taken by the 1% proportion method. Sensitivity for the detection of rifampicin, isoniazid, and multidrug resistance (MDR was 98.1, 92.1, and 95%, respectively, with a specificity of 97.8% for rifampicin and 98.33% for MDR detection. It also had the additional advantage of allowing a study of mutation patterns. Conclusions: High performance characteristics and a short turnaround time makes LPA an excellent diagnostic tool, for an early and accurate diagnosis, in a high MDR- TB-prevalent region, as reflected from our data.

  18. Mining High Utility Itemsets with Regular Occurrence

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    Komate Amphawan

    2016-09-01

    Full Text Available High utility itemset mining (HUIM plays an important role in the data mining community and in a wide range of applications. For example, in retail business it is used for finding sets of sold products that give high profit, low cost, etc. These itemsets can help improve marketing strategies, make promotions/ advertisements, etc. However, since HUIM only considers utility values of items/itemsets, it may not be sufficient to observe product-buying behavior of customers such as information related to “regular purchases of sets of products having a high profit margin”. To address this issue, the occurrence behavior of itemsets (in the term of regularity simultaneously with their utility values was investigated. Then, the problem of mining high utility itemsets with regular occurrence (MHUIR to find sets of co-occurrence items with high utility values and regular occurrence in a database was considered. An efficient single-pass algorithm, called MHUIRA, was introduced. A new modified utility-list structure, called NUL, was designed to efficiently maintain utility values and occurrence information and to increase the efficiency of computing the utility of itemsets. Experimental studies on real and synthetic datasets and complexity analyses are provided to show the efficiency of MHUIRA combined with NUL in terms of time and space usage for mining interesting itemsets based on regularity and utility constraints.

  19. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Science.gov (United States)

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

    2016-01-01

    Summary 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 an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

  20. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics 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; 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; 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; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

    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 an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  1. Spectrophotometric Enzyme Assays for High-Throughput Screening

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    Jean-Louis Reymond

    2004-01-01

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

  2. High content screening as high quality assay for biological evaluation of photosensitizers in vitro.

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    Gisela M F Vaz

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

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

    Science.gov (United States)

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

  4. Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

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    Hampus Sunner

    2015-09-01

    Full Text Available Research on glucuronoyl esterases (GEs has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.

  5. Evaluation of a BED-SIDE Platelet Function Assay : Performance and Clinical Utility.

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    Lau Wei

    2002-01-01

    Full Text Available Platelets have a pivotal role in the initial defense against insult to the vasculature and are also recognized of critical importance in the acute care settings of percutaneous coronary intervention and cardiopulmonary bypass. In these environments both platelet count and function may be markedly compromised. Unfortunately, current assays to evaluate the parameters of platelet count and function are of limited utility for bed-side testing. Moreover, it is suggested that there may be significant inter patient variation in response to antiplatelet therapy that may be exacerbated by other agents (e.g. heparin that are routinely administered during cardiac intervention. Here we describe a practical, rapid and user-friendly whole blood platelet function assay that has been developed for use in bed-side settings. Platelet agonists were formulated with an anticoagulant and lyophilized in blood collection tubes standardised to receive a l mL fresh whole blood sample. In the presence of an agonist, platelets are activated and interact (aggregate. Using traditional cell counting principles, non-aggregated platelets are counted whereas aggregated platelets are not. The percentage (% of functional platelets in reference to a baseline tube may then be determined. Results are available within four minutes. Platelet aggregation in whole blood demonstrated good correlation with turbidometric aggregometry for both ADP (r=0.91 and collagen (r=0.88. Moreover, in clinical settings where antiplatelet agents were administered, this rapid, bed-side, platelet function assay demonstrated utility in monitoring patient response to these therapies. This novel bed-side assay of platelet function is extremely suitable for the clinical environment with a rapid turn-around time. In addition, it provides a full haematology profile, including platelet count, and should permit enhancement of transfusion and interventional decisions.

  6. Advance Mining of Temporal High Utility Itemset

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    Swati Soni

    2012-04-01

    Full Text Available The stock market domain is a dynamic and unpredictable environment. Traditional techniques, such as fundamental and technical analysis can provide investors with some tools for managing their stocks and predicting their prices. However, these techniques cannot discover all the possible relations between stocks and thus there is a need for a different approach that will provide a deeper kind of analysis. Data mining can be used extensively in the financial markets and help in stock-price forecasting. Therefore, we propose in this paper a portfolio management solution with business intelligence characteristics. We know that the temporal high utility itemsets are the itemsets with support larger than a pre-specified threshold in current time window of data stream. Discovery of temporal high utility itemsets is an important process for mining interesting patterns like association rules from data streams. We proposed the novel algorithm for temporal association mining with utility approach. This make us to find the temporal high utility itemset which can generate less candidate itemsets.

  7. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Science.gov (United States)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  8. Utility of T-Cell Interferon-γ Release Assays for Etiological Diagnosis of Classic Fever of Unknown Origin in a High Tuberculosis Endemic Area--a pilot prospective cohort.

    Science.gov (United States)

    Shi, Xiaochun; Zhang, Lifan; Zhang, Yueqiu; Zhou, Baotong; Liu, Xiaoqing

    2016-01-01

    Tuberculosis (TB), especially extrapulmonary TB is still the leading cause of fever of unknown origin (FUO) in China. However, diagnosis of TB still remains a challenge. The aim of this study was to evaluate the diagnostic value of T-SPOT.TB for etiological diagnosis of classic FUO in adult patients in a high TB endemic area. We prospectively enrolled patients presenting with classic FUO in a tertiary referral hospital in Beijing, China, to investigate the diagnostic sensitivity, specificity, predictive values and likelihood ratio of T-SPOT.TB. Clinical assessment and T-SPOT.TB were performed. Test results were compared with the final confirmed clinical diagnosis. 387 hospitalized patients (male n = 194, female n = 193; median age 46 (range 29-59) yrs) with classic FUO were prospectively enrolled into this study. These FUOs were caused by infection (n = 158, 40.8%), connective tissue disease (n = 82, 21.2%), malignancy (n = 41, 10.6%) and miscellaneous other causes (n = 31, 8.0%), and no cause was determined in 75 (19.4%) patients. 68 cases were diagnosed as active TB eventually. The sensitivity of T-SPOT.TB for the diagnosis of active TB was 70.6% (95%CI 58.9-80.1%), while specificity was 84.4% (95%CI 79.4-88.4%), positive predictive value was 55.8% (95%CI 45.3-65.8%), negative predictive value was 91.2% (95%CI 86.7-94.2%). Among these 68 active TB patients, 12 cases were culture or histology confirmed (11 cases with positive T-SPOT.TB, sensitivity was 91.7%) and 56 cases were clinically diagnosed (37 cases with positive T-SPOT.TB, sensitivity was 66.1%); 14 cases were pulmonary TB (13 cases with positive T-SPOT.TB, sensitivity was 92.9%) and 54 cases were extrapulmonary TB (35 cases with positive T-SPOT.TB, sensitivity was 64.8%). For patients presenting with classic FUO in this TB endemic setting, T-SPOT.TB appears valuable for excluding active TB, with a high negative predictive value.

  9. Utility of T-Cell Interferon-γ Release Assays for Etiological Diagnosis of Classic Fever of Unknown Origin in a High Tuberculosis Endemic Area--a pilot prospective cohort.

    Directory of Open Access Journals (Sweden)

    Xiaochun Shi

    Full Text Available Tuberculosis (TB, especially extrapulmonary TB is still the leading cause of fever of unknown origin (FUO in China. However, diagnosis of TB still remains a challenge. The aim of this study was to evaluate the diagnostic value of T-SPOT.TB for etiological diagnosis of classic FUO in adult patients in a high TB endemic area.We prospectively enrolled patients presenting with classic FUO in a tertiary referral hospital in Beijing, China, to investigate the diagnostic sensitivity, specificity, predictive values and likelihood ratio of T-SPOT.TB. Clinical assessment and T-SPOT.TB were performed. Test results were compared with the final confirmed clinical diagnosis.387 hospitalized patients (male n = 194, female n = 193; median age 46 (range 29-59 yrs with classic FUO were prospectively enrolled into this study. These FUOs were caused by infection (n = 158, 40.8%, connective tissue disease (n = 82, 21.2%, malignancy (n = 41, 10.6% and miscellaneous other causes (n = 31, 8.0%, and no cause was determined in 75 (19.4% patients. 68 cases were diagnosed as active TB eventually. The sensitivity of T-SPOT.TB for the diagnosis of active TB was 70.6% (95%CI 58.9-80.1%, while specificity was 84.4% (95%CI 79.4-88.4%, positive predictive value was 55.8% (95%CI 45.3-65.8%, negative predictive value was 91.2% (95%CI 86.7-94.2%. Among these 68 active TB patients, 12 cases were culture or histology confirmed (11 cases with positive T-SPOT.TB, sensitivity was 91.7% and 56 cases were clinically diagnosed (37 cases with positive T-SPOT.TB, sensitivity was 66.1%; 14 cases were pulmonary TB (13 cases with positive T-SPOT.TB, sensitivity was 92.9% and 54 cases were extrapulmonary TB (35 cases with positive T-SPOT.TB, sensitivity was 64.8%.For patients presenting with classic FUO in this TB endemic setting, T-SPOT.TB appears valuable for excluding active TB, with a high negative predictive value.

  10. Utility of immunochromatographic assay as a rapid point of care test for screening of antenatal syphilis

    Directory of Open Access Journals (Sweden)

    Bineeta Kashyap

    2015-01-01

    Full Text Available Background and Objectives: Syphilis is one of the most common preventable causes of adverse effects during pregnancy. Antenatal screening prevents the delay between diagnosis and treatment there by reducing the risk of congenital syphilis. The objective of this study was to evaluate the utility of an immunochromatographic assay as a point of care test for antenatal screening of syphilis. Materials and Methods: Sera of 200 antenatal mothers were evaluated for serodiagnosis of syphilis by the venereal disease research laboratory (VDRL, Treponema pallidum hemagglutination assay (TPHA and SD BIOLINE Syphilis 3.0 test. The performance of SD BIOLINE Syphilis 3.0 test was compared with VDRL as screening assay and TPHA as a confirmatory test. Results: The antenatal prevalence of syphilis was found to be 2% by both VDRL and TPHA. The sensitivity, specificity, positive predictive value, and the negative predictive value of SD BIOLINE Syphilis 3.0 test were 75%, 100%, 100%, and 99.45%, respectively. Conclusions: Antenatal screening and treatment of maternal syphilis are cost-effective health interventions even under the low prevalence of infection. SD BIOLINE Syphilis 3.0 test, although having less sensitivity than the existing testing strategy, can have a tremendous impact on the disease burden if used prudently for the screening of antenatal mothers in peripheral health settings.

  11. Utility of immunochromatographic assay as a rapid point of care test for screening of antenatal syphilis.

    Science.gov (United States)

    Kashyap, Bineeta; Sagar, Tanu; Kaur, Iqbal R

    2015-01-01

    Syphilis is one of the most common preventable causes of adverse effects during pregnancy. Antenatal screening prevents the delay between diagnosis and treatment there by reducing the risk of congenital syphilis. The objective of this study was to evaluate the utility of an immunochromatographic assay as a point of care test for antenatal screening of syphilis. Sera of 200 antenatal mothers were evaluated for serodiagnosis of syphilis by the venereal disease research laboratory (VDRL), Treponema pallidum hemagglutination assay (TPHA) and SD BIOLINE Syphilis 3.0 test. The performance of SD BIOLINE Syphilis 3.0 test was compared with VDRL as screening assay and TPHA as a confirmatory test. The antenatal prevalence of syphilis was found to be 2% by both VDRL and TPHA. The sensitivity, specificity, positive predictive value, and the negative predictive value of SD BIOLINE Syphilis 3.0 test were 75%, 100%, 100%, and 99.45%, respectively. Antenatal screening and treatment of maternal syphilis are cost-effective health interventions even under the low prevalence of infection. SD BIOLINE Syphilis 3.0 test, although having less sensitivity than the existing testing strategy, can have a tremendous impact on the disease burden if used prudently for the screening of antenatal mothers in peripheral health settings.

  12. Droplet microfluidics for high-throughput biological assays.

    Science.gov (United States)

    Guo, Mira T; Rotem, Assaf; Heyman, John A; Weitz, David A

    2012-06-21

    Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10(8) samples to be screened in one day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.

  13. Clean and Highly Efficient Utilization of Coal

    Institute of Scientific and Technical Information of China (English)

    WANG Jianguo; YANG Li

    2011-01-01

    @@ Clean and highly efficient utilization of coal is an important scientific and technological issue.As the petroleum resource decreases but its consumption increases, all of the countries in the world have to face the big issue of sustainable development of energy and economy and protection of environment.Therefore, study on clean coal technology (CCT) has attracted much attention and become one of important themes of energy research.

  14. Optimization of oestradiol assays to improve utility in an in vitro fertilization setting.

    Science.gov (United States)

    Peavey, M; Akbas, N; Gibbons, W; Zarutskie, P; Devaraj, S

    2017-01-01

    Background The measurement of oestradiol is an integral component for the management of ovarian stimulation for in vitro fertilization. Automated immunoassays offer fast assay times and high throughput, with less sensitivity and specificity. The aim of this study is to optimize the oestradiol assay in patients undergoing ovarian stimulation for in vitro fertilization via comparison of oestradiol values obtained using two immunoassays compared with mass spectrometry. Methods Patients undergoing ovarian stimulation were prospectively recruited. Serum samples were analysed with ADVIA Centaur® CP Immunoassay, Abbott Architect i1000® immunoassay and AB Sciex 5500 liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Per cent bias was determined for each system to report the average tendency of the values to be larger or smaller than the LC-MS/MS value. Linear regression of total follicular volume and oestradiol was computed. Results The ADVIA Centaur® CP assay had a positive bias of 20% compared with LC-MS/MS, while the Architect i1000® had a non-significant, negative bias of 0.3%. With regression fit, a clear, positive relationship was seen between follicular volume and oestradiol. The Architect i1000® assay had a greater correlation (R(2 )= 0.46) compared with Centaur® CP (R(2 )= 0.36), when oestradiol values were >1000 pg/mL (3670 pmol/L). Conclusions The Abbott Architect i1000® oestradiol assay exhibits greater agreement with LC-MS/MS and exhibited better correlation to follicular volume when oestradiol values are >1000 pg/mL (3670 pmol/L), prompting a change in the clinic's oestradiol platform. Attention to assay quality assurance via LC-MS/MS can improve the oestradiol accuracy and permit more informed clinical decisions for improved patient outcomes.

  15. Development of CYP11B1 and CYP11B2 assays utilizing homogenates of adrenal glands: Utility of monkey as a surrogate for human.

    Science.gov (United States)

    Cerny, Matthew A; Csengery, Alexander; Schmenk, Jennifer; Frederick, Kosea

    2015-11-01

    Elevated levels of aldosterone are associated with arterial hypertension, congestive heart failure, chronic kidney disease, and obesity. Aldosterone is produced predominantly in the zona glomerulosa of the cortex of the adrenal gland by the enzyme aldosterone synthase (CYP11B2). Treatment of the above indications by decreasing production of aldosterone is thought to be of therapeutic benefit by lessening the deleterious effects of aldosterone mediated through both the mineralocorticoid receptor and also through so called non-genomic pathways. However, inhibition of the highly similar enzyme, CYP11B1, which is responsible for the production of cortisol, must be avoided in the development of clinically useful aldosterone synthase inhibitors due to the resulting impairment of the cortisol-induced stress response. In efforts to assess the interactions of compounds with the CYP11B enzymes, a variety of cell-based inhibitor screening assays for both CYP11B1 and CYP11B2 have been reported. Herein we report details of assays employing both cynomolgus monkey adrenal homogenate (CAH) and human adrenal homogenate (HAH) as sources of CYP11B1 and CYP11B2 enzymes. Utilizing both CAH and HAH, we have characterized the kinetics of the CYP11B1-mediated conversion of 11-deoxycortisol to cortisol and the CYP11B2-mediated oxidation of corticosterone to aldosterone. Inhibition assays for both CYP11B1 and CYP11B2 were subsequently developed. Based on a comparison of human and monkey amino acid sequences, kinetics data, and inhibition values derived from the HAH and CAH assays, evidence is provided in support of using cynomolgus monkey tissue-derived cell homogenates as suitable surrogates for the human enzymes.

  16. Microbial thiocyanate utilization under highly alkaline conditions.

    Science.gov (United States)

    Sorokin, D Y; Tourova, T P; Lysenko, A M; Kuenen, J G

    2001-02-01

    Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase

  17. Utility of hERG assays as surrogate markers of delayed cardiac repolarization and QT safety.

    Science.gov (United States)

    Gintant, Gary A; Su, Zhi; Martin, Ruth L; Cox, Bryan F

    2006-01-01

    HERG (human-ether-a-go-go-related gene) encodes for a cardiac potassium channel that plays a critical role in defining ventricular repolarization. Noncardiovascular drugs associated with a rare but potentially lethal ventricular arrhythmia (Torsades de Pointes) have been linked to delayed cardiac repolarization and block of hERG current. This brief overview will discuss the role of hERG current in cardiac electrophysiology, its involvement in drug-induced delayed repolarization, and approaches used to define drug effects on hERG current. In addition, examples of hERG blocking drugs acting differently (i.e., overt and covert hERG blockade due to multichannel block) together with the utility and limitations of hERG assays as tools to predict the risk of delayed repolarization and proarrhythmia are discussed.

  18. Multipath colourimetric assay for copper(II) ions utilizing MarR functionalized gold nanoparticles

    Science.gov (United States)

    Wang, Yulong; Wang, Limin; Su, Zhenhe; Xue, Juanjuan; Dong, Jinbo; Zhang, Cunzheng; Hua, Xiude; Wang, Minghua; Liu, Fengquan

    2017-02-01

    We use the multiple antibiotic resistance regulator (MarR), as a highly selective biorecognition elements in a multipath colourimetric sensing strategy for the fast detection of Cu2+ in water samples. The colourimetric assay is based on the aggregation of MarR-coated gold nanoparticles in the presence of Cu2+ ions, which induces a red-to-purple colour change of the solution. The colour variation in the gold nanoparticle aggregation process can be used for qualitative and quantitative detection of Cu2+ by the naked eye, and with UV–vis and smartphone-based approaches. The three analysis techniques used in the multipath colourimetric assay complement each other and provide greater flexibility for differing requirements and conditions, making the assay highly applicable for Cu2+ detection. Under optimal conditions, the Cu2+ concentration was quantified in less than 5 min with limits of detection for the naked eye, UV–vis and smartphone-based approaches of 1 μM, 405 nM and 61 nM, respectively. Moreover, the sensing system exhibited excellent selectivity and practical application for Cu2+ detection in real water samples. Thus, our strategy has great potential for application in on-site monitoring of Cu2+, and the unique response of MarR towards copper ions may provide a new approach to Cu2+ sensing.

  19. Multipath colourimetric assay for copper(II) ions utilizing MarR functionalized gold nanoparticles

    Science.gov (United States)

    Wang, Yulong; Wang, Limin; Su, Zhenhe; Xue, Juanjuan; Dong, Jinbo; Zhang, Cunzheng; Hua, Xiude; Wang, Minghua; Liu, Fengquan

    2017-01-01

    We use the multiple antibiotic resistance regulator (MarR), as a highly selective biorecognition elements in a multipath colourimetric sensing strategy for the fast detection of Cu2+ in water samples. The colourimetric assay is based on the aggregation of MarR-coated gold nanoparticles in the presence of Cu2+ ions, which induces a red-to-purple colour change of the solution. The colour variation in the gold nanoparticle aggregation process can be used for qualitative and quantitative detection of Cu2+ by the naked eye, and with UV–vis and smartphone-based approaches. The three analysis techniques used in the multipath colourimetric assay complement each other and provide greater flexibility for differing requirements and conditions, making the assay highly applicable for Cu2+ detection. Under optimal conditions, the Cu2+ concentration was quantified in less than 5 min with limits of detection for the naked eye, UV–vis and smartphone-based approaches of 1 μM, 405 nM and 61 nM, respectively. Moreover, the sensing system exhibited excellent selectivity and practical application for Cu2+ detection in real water samples. Thus, our strategy has great potential for application in on-site monitoring of Cu2+, and the unique response of MarR towards copper ions may provide a new approach to Cu2+ sensing. PMID:28155905

  20. High slot utilization systems for electric machines

    Science.gov (United States)

    Hsu, John S

    2009-06-23

    Two new High Slot Utilization (HSU) Systems for electric machines enable the use of form wound coils that have the highest fill factor and the best use of magnetic materials. The epoxy/resin/curing treatment ensures the mechanical strength of the assembly of teeth, core, and coils. In addition, the first HSU system allows the coil layers to be moved inside the slots for the assembly purpose. The second system uses the slided-in teeth instead of the plugged-in teeth. The power density of the electric machine that uses either system can reach its highest limit.

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

    Science.gov (United States)

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

    2015-03-01

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

  2. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  3. A high-throughput assay of NK cell activity in whole blood and its clinical application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: kylee117@yuhs.ac [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: jkim63@yuhs.ac [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  4. 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.

  5. Cardiac troponins and high-sensitivity cardiac troponin assays.

    Science.gov (United States)

    Conrad, Michael J; Jarolim, Petr

    2014-03-01

    Measurement of circulating cardiac troponins I and T has become integral to the diagnosis of myocardial infarction. This article discusses the structure and function of the troponin complex and the release of cardiac troponin molecules from the injured cardiomyocyte into the circulation. An overview of current cardiac troponin assays and their classification according to sensitivity is presented. The diagnostic criteria, role, and usefulness of cardiac troponin for myocardial infarction are discussed. In addition, several examples are given of the usefulness of high-sensitivity cardiac troponin assays for short-term and long-term prediction of adverse events.

  6. Fluorescence polarization assays in high-throughput screening and drug discovery: a review

    Science.gov (United States)

    Hall, Matthew D.; Yasgar, Adam; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

    2016-06-01

    The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.

  7. Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

    Science.gov (United States)

    Zhang, Jia; Li, Kai; Pardinas, Jose R; Sommer, Steve S; Yao, Kai-Tai

    2005-02-01

    DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

  8. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

    Science.gov (United States)

    Klaus, Joseph P; Botten, Jason

    2016-03-02

    Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

  9. A system for performing high throughput assays of synaptic function.

    Directory of Open Access Journals (Sweden)

    Chris M Hempel

    Full Text Available Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

  10. Use of a high resolution melt real-time polymerase chain reaction (PCR) assay for the environmental monitoring of Vibrio cholerae

    CSIR Research Space (South Africa)

    Le Rouw, Wouter J

    2011-10-01

    Full Text Available A real-time polymerase chain reaction (PCR) assay utilizing high resolution melt (HRM) curve analysis was developed and tested for the monitoring of Vibrio cholerae in water samples. The assay utilized previously published primers that are specific...

  11. Non-destructive assay of drum package radioactive wastes utilizing tomographic gamma scanning

    Energy Technology Data Exchange (ETDEWEB)

    Ausbrooks, K. L. [Univ. of Tennessee, Knoxville, TN (United States)

    1996-05-01

    A methodology for nondestructive assay of drum packaged radioactive waste materials is investigated using Emission Computed Tomography procedures. A requirement of this method is accurate gamma attenuation correction. This is accomplished by the use of a constant density distribution for the drum content, thereby requiring the need for a homogeneous medium. The current predominant NDA technique is the use of the Segmented Gamma Scanner. Tomographic Gamma Scanning improves upon this method by providing a low resolution three-dimensional image of the source distribution, yielding both spatial and activity information. Reconstruction of the source distribution is accomplished by utilization of algebraic techniques with a nine by six voxel model with detector information gathered over scanning intervals of ninety degrees. Construction of a linear system to describe the scenario was accomplished using a point-source response function methodology, where a 54 x 120 matrix contained the projected detector responses for each source-detector geometry. Entries in this matrix were calculated using the point-kernal shielding code QAD-CGGP. Validation was performed using the MCNP photon transport code. Solutions to the linear system were determined using the Non-Negative Least Squares (NNLS) algorithm and the LSMOD algorithm. A series of four scans were performed, each reconstructing the source distribution of a mock-up waste package containing a single 73 mCi 137Cs point source. For each scan, the source was located in a different location. Results of the reconstruction routines accurately predict the location and activity of the source. The range of activity calculated using the NNLS routine is 0.2681 mCi with an average value of 77.7995 mCi. The range of values calculated using LSMOD is 5.1843 mCi with an average of 72.8018 mCi.

  12. Enzyme inhibition assay for pyruvate dehydrogenase complex: Clinical utility for the diagnosis of primary biliary cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Katsuhisa Omagri; Hiroaki Hazama; Shigeru Kohno

    2005-01-01

    Primary biliary cirrhosis (PBC) is usually diagnosed by the presence of characteristic histopathological features of the liver and/or antimitochondrial antibodies (AMA) in the serum traditionally detected by immunofluorescence.Recently, new and more accurate serological assays for the detection of AMA, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and enzyme inhibition assay, have been developed. Of these,the enzyme inhibition assay for the detection of antipyruvate dehydrogenase complex (PDC) antibodies offers certain advantages such as objectivity, rapidity,simplicity, and low cost. Since this assay has almost 100% specificity, it may have particular applicability in screening the at-risk segment of the population in developing countries. Moreover, this assay could be also used for monitoring the disease course in PBC. Almost all sera of PBC-suspected patients can be confirmed for PBC or non-PBC by the combination results of immunoblotting and enzyme inhibition assay without histopathological examination. For the development of a "complete" or "gold standard" diagnostic assay for PBC, similar assays of the enzyme inhibition for anti2-oxoglutarate dehydrogenase complex (OGDC) and anti-branched chain oxo-acid dehydrogenase complex (BCOADC) antibodies will be needed in future.

  13. High throughput heme assay by detection of chemiluminescence of reconstituted horseradish peroxidase.

    Science.gov (United States)

    Takahashi, Shigekazu; Masuda, Tatsuru

    2009-06-01

    In living organisms, heme is an essential molecule for various biological functions. Recent studies also suggest that heme functions as organelle-derived signal that regulates fundamental cell processes. Furthermore, estimation of heme is widely used for studying various blood disorders. In this regard, development of a rapid, sensitive, and high throughput heme assay has been sought. The most frequently used method of measuring heme by pyridine hemochrome is time, labor, and material intensive, and therefore limiting in its utility for large scale, high throughput analysis. Recently, we reported alternative method that is sensitive and specific to heme, which is based on the ability of horseradish peroxidase (HRP) apo-enzyme to reconstitute with heme to form an active holo-enzyme. Here, we developed high throughput heme assay by performing reactions on multi-well plate with highly sensitive chemiluminescence detection reagents. Detection of chemiluminescence in charged coupled device (CCD)-based gel doc apparatus enables simultaneous measurement of multiple samples. Furthermore, the high sensitivity of this assay allowed a direct measurement of heme in solvent extracts after dilution. This assay is sensitive, quick, provides a large dynamic range, and is well suited for large-scale analysis of heme extracted from minute amount of samples.

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

    KAUST Repository

    Soufan, Othman

    2015-12-14

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

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

    Directory of Open Access Journals (Sweden)

    Othman Soufan

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

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

    Science.gov (United States)

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B

    2015-01-01

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

  17. High throughput comet assay to study genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Naouale El Yamani

    2015-06-01

    Full Text Available The unique physicochemical properties of engineered nanomaterials (NMs have accelerated their use in diverse industrial and domestic products. Although their presence in consumer products represents a major concern for public health safety, their potential impact on human health is poorly understood. There is therefore an urgent need to clarify the toxic effects of NMs and to elucidate the mechanisms involved. In view of the large number of NMs currently being used, high throughput (HTP screening technologies are clearly needed for efficient assessment of toxicity. The comet assay is the most used method in nanogenotoxicity studies and has great potential for increasing throughput as it is fast, versatile and robust; simple technical modifications of the assay make it possible to test many compounds (NMs in a single experiment. The standard gel of 70-100 μL contains thousands of cells, of which only a tiny fraction are actually scored. Reducing the gel to a volume of 5 μL, with just a few hundred cells, allows twelve gels to be set on a standard slide, or 96 as a standard 8x12 array. For the 12 gel format, standard slides precoated with agarose are placed on a metal template and gels are set on the positions marked on the template. The HTP comet assay, incorporating digestion of DNA with formamidopyrimidine DNA glycosylase (FPG to detect oxidised purines, has recently been applied to study the potential induction of genotoxicity by NMs via reactive oxygen. In the NanoTEST project we investigated the genotoxic potential of several well-characterized metal and polymeric nanoparticles with the comet assay. All in vitro studies were harmonized; i.e. NMs were from the same batch, and identical dispersion protocols, exposure time, concentration range, culture conditions, and time-courses were used. As a kidney model, Cos-1 fibroblast-like kidney cells were treated with different concentrations of iron oxide NMs, and cells embedded in minigels (12

  18. High-throughput comet assay using 96 minigels.

    Science.gov (United States)

    Gutzkow, Kristine B; Langleite, Torgrim M; Meier, Silja; Graupner, Anne; Collins, Andrew R; Brunborg, Gunnar

    2013-05-01

    The single-cell gel electrophoresis--the comet assay--has proved to be a sensitive and relatively simple method that is much used in research for the analysis of specific types of DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number of samples processed per experiment, has been rather poor, and both research and toxicological testing should profit from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by one person, with less time needed for processing, less use of chemicals and requiring fewer cells per sample. Controlled electrophoresis, including circulation of the electrophoresis solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method described in this paper should greatly increase the overall capacity, versatility and robustness of the comet assay.

  19. High-gravity brewing utilizing factorial design

    Directory of Open Access Journals (Sweden)

    R. B. Almeida

    2000-06-01

    Full Text Available A number of factors can influence the behavior of yeast during fermentation. Some of these factors (initial wort concentration, initial pH and percentage of corn syrup in the composition of the wort were studied in order to determine their influence on the productivity of fermentation. Fermentations were carried out at 25ºC utilizing a 2³ factorial design of these factors. The results showed that the percentage of corn syrup had no influence on process productivity, whereas initial pH and especially initial wort concentration did. It can be concluded that using pH and initial wort concentration values higher than those utilized in this work (5.5 and 20ºP, respectively will result in a higher productivity.

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

    KAUST Repository

    Soufan, Othman

    2016-11-10

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

  1. The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

    Science.gov (United States)

    Bourque, D K; Peñaherrera, M S; Yuen, R K C; Van Allen, M I; McFadden, D E; Robinson, W P

    2011-02-01

    An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

  2. High-gravity brewing utilizing factorial design

    OpenAIRE

    Almeida,R. B.; J. B. Almeida e Silva; Lima,U. A.; Assis,A. N.

    2000-01-01

    A number of factors can influence the behavior of yeast during fermentation. Some of these factors (initial wort concentration, initial pH and percentage of corn syrup in the composition of the wort) were studied in order to determine their influence on the productivity of fermentation. Fermentations were carried out at 25ºC utilizing a 2³ factorial design of these factors. The results showed that the percentage of corn syrup had no influence on process productivity, whereas initial pH and es...

  3. A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-05-05

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

  5. High-throughput DNA droplet assays using picoliter reactor volumes.

    Science.gov (United States)

    Srisa-Art, Monpichar; deMello, Andrew J; Edel, Joshua B

    2007-09-01

    The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

  6. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

    Science.gov (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David

    2014-11-01

    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

  7. A novel assay to diagnose hereditary angioedema utilizing inhibition of bradykinin-forming enzymes

    DEFF Research Database (Denmark)

    Joseph, Kusumam; Bains, Sonia; Tholanikunnel, Baby G

    2015-01-01

    BACKGROUND: Hereditary angioedema types I and II are caused by a functional deficiency of C1 inhibitor (C1-INH) leading to overproduction of bradykinin. The current functional diagnostic assays employ inhibition of activated C1s, however, an alternative, more physiologic method, is desirable...

  8. A photonic crystal biosensor assay for ferritin utilizing iron-oxide nanoparticles.

    Science.gov (United States)

    Peterson, Ross D; Cunningham, Brian T; Andrade, Juan E

    2014-06-15

    Iron deficiency anemia afflicts 1 in 3 individuals, mostly women and children worldwide. A novel application using iron-oxide nanoparticles (IONPs) and a photonic crystal (PC) optical biosensor as an immunodiagnostic platform for detection of serum ferritin, a biomarker for iron deficiency, is presented. Human liver ferritin (450 kDa), clinical serum controls, and three commercially available ferritin ELISA tests were used to evaluate the PC biosensor assay in terms of inter- and intra-assay variability, spike-recovery (%), limit of detection (LOD), and matrix effects on binding. For the PC biosensor, signal response from label-free, sandwich with secondary antibody (pAb), and pAb functionalized with iron-oxide nanoparticles (FpAb) assays were detected using the Biomolecular Interaction Detection (BIND) system. Bland-Altman analysis was used to evaluate agreement between expected values for ferritin in control sera and each of the detection platforms. Inter- and intra-assay variability of the PC biosensor were both detection sensitivity of PC biosensors for determination of biomarkers of nutritional status.

  9. High-performance liquid chromatographic assay for benzocaine and p-aminobenzoic acid including preliminary stability data.

    Science.gov (United States)

    Narang, P K; Bird, G; Crouthamel, W G

    1980-12-01

    A high-performance liquid chromatographic assay was developed that separates and quantitates benzocaine and its primary degradation product, p-aminobenzoic acid. This method is rapid, sensitive, and specific. Preliminary stability data obtained with this method demonstrate its utility for this purpose.

  10. Rate, characteristics, and factors associated with high emergency department utilization

    NARCIS (Netherlands)

    M.C. van der Linden (M. Christien); C.L. van den Brand (Crispijn); N. van der Linden (Naomi); A.H. Rambach (Anna H.J.H.); C. Brumsen (Caro)

    2014-01-01

    textabstractBackground: Patients with high emergency department (ED) utilization account for a disproportionate number of ED visits. The existing research on high ED utilization has raised doubts about the homogeneity of the frequent ED user. Attention to differences among the subgroups of frequent

  11. Utility of Preoperative CA125 Assay in the Management Planning of Women Diagnosed with Uterine Cancer

    Directory of Open Access Journals (Sweden)

    N. Povolotskaya

    2014-01-01

    grade did not (P=0.5. Overall, at five-year follow-up, while there were no deaths among the women with preoperative serum CA125 less than 12 U/mL, eleven of the twenty-three deaths (47.82% in the study occurred in women with a preoperative CA125 more than 28 U/mL. Conclusions. A preoperative CA125 assay for women with uterine cancer is a relatively inexpensive, reproducible, and objective test which provides valuable information regarding the risk of metastatic disease and overall likelihood of long term survival. Patients with a low likelihood of metastatic/nodal disease (favourable tissue type and CA125 level < 28 U/mL and significant comorbidities may benefit from avoiding an extended complete staging procedure. Alternatively, a high level of CA125 may prompt further imaging and multidisciplinary discussions to plan for individualised management and consideration for recruitment to clinical trials.

  12. High-throughput protein analysis integrating bioinformatics and experimental assays.

    Science.gov (United States)

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins.

  13. Multicenter comparison study of both analytical and clinical performance across 4 Roche HCV RNA assays utilizing different platforms.

    Science.gov (United States)

    Vermehren, Johannes; Stelzl, Evelyn; Maasoumy, Benjamin; Michel-Treil, Veronique; Berkowski, Caterina; Marins, Ed G; Paxinos, Ellen E; Marino, Enrique; Wedemeyer, Heiner; Sarrazin, Christoph; Kessler, Harald H

    2017-01-25

    Antiviral treatment efficacy for chronic HCV infection is determined based on measurement of HCV RNA at specific time points throughout therapy by highly sensitive and accurate HCV RNA assays. This study evaluated the performance of two recently-developed real-time PCR HCV RNA assays, the cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and the cobas HCV for use on the cobas 4800 system (cobas 4800 HCV) in comparison to two established assays, the COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 (CAP/CTM v2) and the COBAS TaqMan HCV Test, version 2.0 for use with the High Pure System (HPS/CTM v2). Limit of detection (LOD) and linearity at lower concentrations (5-1000 IU/mL) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1-4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1-4.cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/mL difference between expected and observed results) with LOD of 8.2 IU/mL and 11.7 IU/mL, respectively, for GT1. The new assays showed excellent agreement with CAP/CTM v2 and HPS/CTM v2 results in samples with quantifiable viral load. Concordance across the 6 million IU/mL cutoff was high among all four assays (90-94%). In conclusion, both cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear, and correlate well with established Roche assays used in clinical practice.

  14. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

    Science.gov (United States)

    Ghosh, Inca; Sun, Luo; Evans, Thomas C; Xu, Ming-Qun

    2004-10-01

    Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

  15. Clinical utility of the cryptococcal antigen lateral flow assay in a diagnostic mycology laboratory.

    Directory of Open Access Journals (Sweden)

    Brendan J McMullan

    Full Text Available BACKGROUND: Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA to detect cryptococcal antigen (CRAG is reportedly more rapid and convenient than standard latex agglutination (LA, but has not yet been evaluated in a diagnostic laboratory setting. METHODS: One hundred and six serum, 42 cerebrospinal fluid (CSF, and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients. RESULTS: Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56 from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI 93.6-100% compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7-96.1%. Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56 this did not reach statistical significance (p = 0.063. Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients. Agreement between titres obtained by both methods (n = 38 samples was imperfect; correlation between log-transformed titres (r was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively. CONCLUSIONS: Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.

  16. Magnetic nanoparticles for high-sensitivity detection on nucleic acids via superconducting-quantum-interference-device-based immunomagnetic reduction assay

    Science.gov (United States)

    Yang, S. Y.; Chieh, J. J.; Wang, W. C.; Yu, C. Y.; Hing, N. S.; Horng, H. E.; Hong, Chin-Yih; Yang, H. C.; Chang, C. F.; Lin, H. Y.

    2011-03-01

    In this work, we investigate the feasibility of detecting quantitatively DNA molecules utilizing the technology named after the immunomagnetic reduction (IMR) assay. Magnetic nanoparticles dispersed in a phosphate buffer saline solution were bio-functionalized with probing single-strand DNA. A superconducting quantum interference device (SQUID) ac magnetosusceptometer was employed to detect IMR signals related to the concentration of the target DNA. The results reveal that use of IMR assay had merits such as a high convenience level, e.g. wash-free processes and high sensitivity, down to pM, for DNA detection.

  17. High throughput RNAi assay optimization using adherent cell cytometry

    Directory of Open Access Journals (Sweden)

    Pradhan Leena

    2011-04-01

    Full Text Available Abstract Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC. Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM, or non-targeting labeled siRNA, siGLO Red (5 or 50 nM using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19. Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

  18. Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays.

    Science.gov (United States)

    Belouski, Shelley Sims; Wilkinson, Julie; Thomas, John; Kelley, Keith; Wang, Shen-Wu; Suggs, Sid; Ferbas, John

    2010-01-01

    The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry. Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur. We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate.

  19. Development and characterization of a high density SNP genotyping assay for cattle.

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    Lakshmi K Matukumalli

    Full Text Available The success of genome-wide association (GWA studies for the detection of sequence variation affecting complex traits in human has spurred interest in the use of large-scale high-density single nucleotide polymorphism (SNP genotyping for the identification of quantitative trait loci (QTL and for marker-assisted selection in model and agricultural species. A cost-effective and efficient approach for the development of a custom genotyping assay interrogating 54,001 SNP loci to support GWA applications in cattle is described. A novel algorithm for achieving a compressed inter-marker interval distribution proved remarkably successful, with median interval of 37 kb and maximum predicted gap of <350 kb. The assay was tested on a panel of 576 animals from 21 cattle breeds and six outgroup species and revealed that from 39,765 to 46,492 SNP are polymorphic within individual breeds (average minor allele frequency (MAF ranging from 0.24 to 0.27. The assay also identified 79 putative copy number variants in cattle. Utility for GWA was demonstrated by localizing known variation for coat color and the presence/absence of horns to their correct genomic locations. The combination of SNP selection and the novel spacing algorithm allows an efficient approach for the development of high-density genotyping platforms in species having full or even moderate quality draft sequence. Aspects of the approach can be exploited in species which lack an available genome sequence. The BovineSNP50 assay described here is commercially available from Illumina and provides a robust platform for mapping disease genes and QTL in cattle.

  20. High-utilizing Crohn's disease patients under psychosomatic therapy*

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    Jantschek Günther

    2008-10-01

    Full Text Available Abstract Objective Few studies have been published on health care utilization in Crohn's disease and the influence of psychological treatment on high utilizers. Methods The present sub study of a prospective multi center investigation conducted in 87 of 488 consecutive Crohn's disease (CD patients was designed to investigate the influence of the course of Crohn's disease on health care utilization (hospital days (HD and sick leave days (SLD collected by German insurance companies and to examine the conditions of high-utilizing patients. Predictors of health care utilization should be selected. Based on a standardized somatic treatment, high health care utilizing patients of the psychotherapy and control groups should be compared before and after a one-year treatment. Results Multivariate regression analysis identified disease activity at randomization as an important predictor of the clinical course (r2 = 0.28, p 2 = 0.15, p = 0.09. The patients' level of anxiety, depression and lack of control at randomization predicted their health-related quality of life at the end of the study (r2 = 0.51, p 2 = 0.22, p Among high utilizers, a significantly greater drop in HD (p Conclusion The course of Crohn's disease is influenced by psychological as well as somatic factors; especially depression seems important here. A significant drop of health care utilization demonstrates the benefit of psychological treatment in the subgroup of high-utilizing CD patients. Further studies are needed to replicate the findings of the clinical outcome in this CD subgroup.

  1. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Directory of Open Access Journals (Sweden)

    Grace Ka Yan Chan

    Full Text Available In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1 significant underestimation of compound potency and efficacy, and 2 non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  2. The utility of uric acid assay in dogs as an indicator of functional hepatic mass

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    J. M. Hill

    2011-04-01

    Full Text Available Uric acid was used as a test for liver disease before the advent of enzymology. Three old studies criticised uric acid as a test of liver function. Uric acid, as an end-product of purine metabolism in the liver, deserved re-evaluation as a liver function test. Serumtotal bile acids are widely accepted as the most reliable liver function test. This study compared the ability of serumuric acid concentration to assess liver function with that of serumpre-prandial bile acids in dogs. In addition, due to the renal excretion of uric acid the 2 assays were also compared in a renal disease group. Using a control group of healthy dogs, a group of dogs with congenital vascular liver disease, a group of dogs with non-vascular parenchymal liver diseases and a renal disease group, the ability of uric acid and pre-prandial bile acids was compared to detect reduced functional hepatic mass overall and in the vascular or parenchymal liver disease groups separately. Sensitivities, specificities and predictive value parameters were calculated for each test. The medians of uric acid concentration did not differ significantly between any of the groups, whereas pre-prandial bile acids medians were significantly higher in the liver disease groups compared with the normal and renal disease group of dogs. The sensitivity of uric acid in detecting liver disease overall was 65% while the specificity of uric acid in detecting liver disease overall was 59 %. The sensitivity and specificity of uric acid in detecting congenital vascular liver disease was 68%and 59 %, respectively. The sensitivity and specificity of uric acid in detecting parenchymal liver disease was 63%and 60 %, respectively. The overall positive and negative predictive values for uric acid in detecting liver disease were poor and the data in this study indicated uric acid to be an unreliable test of liver function. In dogs suffering from renal compromise serum uric acid concentrations may increase into the

  3. Clinical value of high-sensitivity cardiac troponin assays after acute ischemic stroke

    Directory of Open Access Journals (Sweden)

    Yi-rui CAO

    2016-11-01

    Full Text Available In view of a variety of cardiovascular events complicated by acute ischemic stroke, the importance of monitoring myocardial ischemic symptoms and signs, electrocardiogram, echocardiogram and myocardial injury markers has been gradually recognized by clinicians. Cardiac troponin (cTn by conventional assay has been a unique marker of myocardial injury for its extremely high specificity. However, with the utilization of high-sensitivity cardiac troponin (hs-cTn, cTn is no longer exclusive of a disease, but was given more significance in the diagnosis and application value. Therefore, we described the clinical significance of alterations of serum hs-cTn concentration after ischemic stroke. DOI: 10.3969/j.issn.1672-6731.2016.11.015

  4. Considerations for the design and reporting of enzyme assays in high-throughput screening applications

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    Michael G. Acker

    2014-05-01

    Full Text Available This review describes the key steps and methods which are used to develop enzyme assays suitable for high-throughput screening (HTS applications. The goals of HTS enzyme assays are defined relative to lower-throughput bench top assays and important aspects which go into constructing robust and sensitive enzyme assays are described. Methods that have been applied to common enzyme classes are reviewed and pitfalls related to assay artifacts are discussed. We also suggest a reporting format to describe the steps in HTS enzyme assays.

  5. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Science.gov (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  6. Utility of a Noninvasive 2-Gene Molecular Assay for Cutaneous Melanoma and Effect on the Decision to Biopsy.

    Science.gov (United States)

    Ferris, Laura K; Jansen, Burkhard; Ho, Jonhan; Busam, Klaus J; Gross, Kenneth; Hansen, Doyle D; Alsobrook, John P; Yao, Zuxu; Peck, Gary L; Gerami, Pedram

    2017-07-01

    Expression of long intergenic non-protein coding RNA 518 (LINC00518) and preferentially expressed antigen in melanoma (PRAME) genes, obtained via noninvasive adhesive patch biopsy, is a sensitive and specific method for detection of cutaneous melanoma. However, the utility of this test in biopsy decisions made by dermatologists has not been evaluated. To determine the utility of the pigmented lesion assay (PLA) for LINC00518/PRAME expression in decisions to biopsy a series of pigmented skin lesions. In this secure web-based, multiple-reader-multiple-case study, 45 board-certified dermatologists each evaluated 60 clinical and dermoscopic images of clinically atypical pigmented lesions, first without and then with PLA gene expression information and were asked whether the lesions should be biopsied. Data were collected from March 24, 2014, through November 13, 2015. Participants were given a report for each lesion, which included the results of an assay for expression of LINC00518/PRAME and a PLA score with data on the predictive values of the information provided. Biopsy sensitivity and specificity with vs without PLA data. Forty-five dermatologists (29 male and 16 female) performed the evaluation. After incorporating the PLA into their decision as to whether to biopsy a pigmented lesion suggestive of melanoma, dermatologists improved their mean biopsy sensitivity from 95.0% to 98.6% (P = .01); specificity increased from 32.1% to 56.9% (P biopsy specificity while maintaining or improving sensitivity. This result may increase the number of early melanomas biopsied and reduce the number of benign lesions biopsied, thereby improving patient outcomes and reducing health care costs.

  7. A high throughput single nucleotide polymorphism multiplex assay for parentage assignment in New Zealand sheep.

    Directory of Open Access Journals (Sweden)

    Shannon M Clarke

    Full Text Available Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development--firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage are assigned. An 84 "parentage SNP panel" was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams was absent, highlighting the SNP test's suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.

  8. Making sense of high sensitivity troponin assays and their role in clinical care.

    Science.gov (United States)

    Daniels, Lori B

    2014-04-01

    Cardiac troponin assays have an established and undisputed role in the diagnosis and risk stratification of patients with acute myocardial infarction. As troponin assays gets more sensitive and more precise, the number of potential uses has rapidly expanded, but the use of this test has also become more complicated and controversial. Highly sensitive troponin assays can now detect troponin levels in most individuals, but accurate interpretation of these levels requires a clear understanding of the assay in the context of the clinical scenario. This paper provides a practical and up-to-date overview of the uses of highly sensitive troponin assays for diagnosis, prognosis, and risk stratification in clinical practice.

  9. Development of a high-throughput replicon assay for the identification of respiratory syncytial virus inhibitors.

    Science.gov (United States)

    Tiong-Yip, Choi-Lai; Plant, Helen; Sharpe, Paul; Fan, Jun; Rich, Kirsty; Gorseth, Elise; Yu, Qin

    2014-01-01

    Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.

  10. [Plasma assay of methadone enantiomers with high performance liquid chromatography].

    Science.gov (United States)

    Batista, R; Badré-Sentenac, S; Bardin, C; Chast, F

    2004-05-01

    6-Dimethylamino-4,4-diphenylheptane-3-one (methadone) is a synthetic opioid. Presence of an assymetrical carbon in its structure explains existence of two enantiomers. Levogyral enantiomer or R-methadone exhibits an 25 fold superior analgesic activity to the dextrogyral enantiomer S-methadone. In order to run separately a plasma assay of these two enantiomers, a chromatographic chiral separation method coupled with an ultraviolet detection has been performed. It allows selective assay of each enantiomer. This method, although an analytical interference with d-propoxyphene, is sensitive, reproductible, specific and shows a convenient resolution for the analysis of both the stereospecific and racemic forms. This method can be applied for pharmacokinetic study of the drug in patients treated by methadone.

  11. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    Science.gov (United States)

    Lago-Cachón, D.; Rivas, M.; Martínez-García, J. C.; Oliveira-Rodríguez, M.; Blanco-López, M. C.; García, J. A.

    2017-02-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies.

  12. Systematic evaluation of nanomaterial toxicity: utility of standardized materials and rapid assays.

    Science.gov (United States)

    Harper, Stacey L; Carriere, Jason Lee; Miller, John M; Hutchison, James Evan; Maddux, Bettye L S; Tanguay, Robert L

    2011-06-28

    The challenge of optimizing both performance and safety in nanomaterials hinges on our ability to resolve which structural features lead to desired properties. It has been difficult to draw meaningful conclusions about biological impacts from many studies of nanomaterials due to the lack of nanomaterial characterization, unknown purity, and/or alteration of the nanomaterials by the biological environment. To investigate the relative influence of core size, surface chemistry, and charge on nanomaterial toxicity, we tested the biological response of whole animals exposed to a matrix of nine structurally diverse, precision-engineered gold nanoparticles (AuNPs) of high purity and known composition. Members of the matrix include three core sizes and four unique surface coatings that include positively and negatively charged headgroups. Mortality, malformations, uptake, and elimination of AuNPs were all dependent on these parameters, showing the need for tightly controlled experimental design and nanomaterial characterization. Results presented herein illustrate the value of an integrated approach to identify design rules that minimize potential hazard.

  13. Systematic Evaluation of Nanomaterial Toxicity: Utility of Standardized Materials and Rapid Assays

    Energy Technology Data Exchange (ETDEWEB)

    Harper, Stacey; Carriere, Jason L.; Miller, John M.; Hutchison, James E.; Maddux, Bettye L.; Tanguay, Robert

    2011-06-28

    The challenge of optimizing both performance and safety in nanomaterials hinges on our ability to resolve which structural features lead to desired properties. It has been difficult to draw meaningful conclusions about biological impacts from many studies of nanomaterials due to the lack of nanomaterial characterization, unknown purity, and/or alteration of the nanomaterials by the biological environment. To investigate the relative influence of core size, surface chemistry, and charge on nanomaterial toxicity, we tested the biological response of whole animals exposed to a matrix of nine structurally diverse, precision-engineered gold nanoparticles (AuNPs) of high purity and known composition. Members of the matrix include three core sizes and four unique surface coatings that include positively and negatively charged headgroups. Mortality, malformations, uptake, and elimination of AuNPs were all dependent on these parameters, showing the need for tightly controlled experimental design and nanomaterial characterization. Results presented herein illustrate the value of an integrated approach to identify design rules that minimize potential hazard.

  14. Utilizing direct skin feeding assays for development of vaccines that interrupt malaria transmission: A systematic review of methods and case study.

    Science.gov (United States)

    Brickley, Elizabeth B; Coulibaly, Mamadou; Gabriel, Erin E; Healy, Sara A; Hume, Jen C C; Sagara, Issaka; Traore, Sekou F; Doumbo, Ogobara; Duffy, Patrick E

    2016-11-21

    Shifting the malaria priorities from a paradigm of control and elimination to a goal of global eradication calls for renewed attention to the interruption of malaria transmission. Sustained progress toward eradication will require both improved understanding of infectious reservoirs and efficient development of novel transmission-blocking interventions, such as rapidly acting and highly efficacious therapeutics and vaccines. Here, we review the direct skin feeding assay (DSF), which has been proposed as a valuable tool for measuring the in natura transmission of malaria parasites from human hosts to mosquito vectors across heterogeneous populations. To capture the methodological breadth of this assay's use, we first systematically review and qualitatively synthesize previously published investigations using DSFs to study malaria transmission in humans. Then, using a recent Phase 1 trial in Mali of the Pfs25H-EPA/Alhydrogel® vaccine candidate (NCT01867463) designed to interrupt Plasmodium falciparum transmission as a case study, we describe the potential opportunities and current limitations of utilizing the endpoints measured by DSF in making early clinical decisions for individually randomized transmission-interrupting intervention candidates. Using simulations based on the data collected in the clinical trial, we demonstrate that the capacity of the DSF to serve as an evaluative tool is limited by the statistical power constraints of the "effective sample size" (i.e. the number of subjects that are capable of transmitting at the time of feeding). Altogether, our findings suggest DSFs have great potential utility for assessing the public health impacts of emerging antimalarial tools, but additional research is needed to address issues of scalability and to establish correlation with community-wide clinical endpoints as well as complementary in vitro measures, such as standard membrane feeding assays.

  15. Network Bandwidth Utilization Forecast Model on High Bandwidth Network

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Wucherl; Sim, Alex

    2014-07-07

    With the increasing number of geographically distributed scientific collaborations and the scale of the data size growth, it has become more challenging for users to achieve the best possible network performance on a shared network. We have developed a forecast model to predict expected bandwidth utilization for high-bandwidth wide area network. The forecast model can improve the efficiency of resource utilization and scheduling data movements on high-bandwidth network to accommodate ever increasing data volume for large-scale scientific data applications. Univariate model is developed with STL and ARIMA on SNMP path utilization data. Compared with traditional approach such as Box-Jenkins methodology, our forecast model reduces computation time by 83.2percent. It also shows resilience against abrupt network usage change. The accuracy of the forecast model is within the standard deviation of the monitored measurements.

  16. 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.

  17. An Incremental High-Utility Mining Algorithm with Transaction Insertion

    Directory of Open Access Journals (Sweden)

    Jerry Chun-Wei Lin

    2015-01-01

    Full Text Available Association-rule mining is commonly used to discover useful and meaningful patterns from a very large database. It only considers the occurrence frequencies of items to reveal the relationships among itemsets. Traditional association-rule mining is, however, not suitable in real-world applications since the purchased items from a customer may have various factors, such as profit or quantity. High-utility mining was designed to solve the limitations of association-rule mining by considering both the quantity and profit measures. Most algorithms of high-utility mining are designed to handle the static database. Fewer researches handle the dynamic high-utility mining with transaction insertion, thus requiring the computations of database rescan and combination explosion of pattern-growth mechanism. In this paper, an efficient incremental algorithm with transaction insertion is designed to reduce computations without candidate generation based on the utility-list structures. The enumeration tree and the relationships between 2-itemsets are also adopted in the proposed algorithm to speed up the computations. Several experiments are conducted to show the performance of the proposed algorithm in terms of runtime, memory consumption, and number of generated patterns.

  18. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    Science.gov (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

  19. High throughput assay for evaluation of reactive carbonyl scavenging capacity

    Directory of Open Access Journals (Sweden)

    N. Vidal

    2014-01-01

    Full Text Available Many carbonyl species from either lipid peroxidation or glycoxidation are extremely reactive and can disrupt the function of proteins and enzymes. 4-hydroxynonenal and methylglyoxal are the most abundant and toxic lipid-derived reactive carbonyl species. The presence of these toxics leads to carbonyl stress and cause a significant amount of macromolecular damages in several diseases. Much evidence indicates trapping of reactive carbonyl intermediates may be a useful strategy for inhibiting or decreasing carbonyl stress-associated pathologies. There is no rapid and convenient analytical method available for the assessment of direct carbonyl scavenging capacity, and a very limited number of carbonyl scavengers have been identified to date, their therapeutic potential being highlighted only recently. In this context, we have developed a new and rapid sensitive fluorimetric method for the assessment of reactive carbonyl scavengers without involvement glycoxidation systems. Efficacy of various thiol- and non-thiol-carbonyl scavenger pharmacophores was tested both using this screening assay adapted to 96-well microplates and in cultured cells. The scavenging effects on the formation of Advanced Glycation End-product of Bovine Serum Albumin formed with methylglyoxal, 4-hydroxynonenal and glucose-glycated as molecular models were also examined. Low molecular mass thiols with an α-amino-β-mercaptoethane structure showed the highest degree of inhibitory activity toward both α,β-unsaturated aldehydes and dicarbonyls. Cysteine and cysteamine have the best scavenging ability toward methylglyoxal. WR-1065 which is currently approved for clinical use as a protective agent against radiation and renal toxicity was identified as the best inhibitor of 4-hydroxynonenal.

  20. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    Science.gov (United States)

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  1. The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.

    Directory of Open Access Journals (Sweden)

    Hansook Kim Chong

    Full Text Available Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.

  2. Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases.

    Science.gov (United States)

    Riester, Daniel; Hildmann, Christian; Haus, Patricia; Galetovic, Antonia; Schober, Andreas; Schwienhorst, Andreas; Meyer-Almes, Franz-Josef

    2009-07-01

    Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.

  3. High-sensitivity cardiac troponin assays: answers to frequently asked questions.

    Science.gov (United States)

    Chenevier-Gobeaux, Camille; Bonnefoy-Cudraz, Éric; Charpentier, Sandrine; Dehoux, Monique; Lefevre, Guillaume; Meune, Christophe; Ray, Patrick

    2015-02-01

    Cardiac troponin (cTn) assays have quickly gained in analytical sensitivity to become what are termed 'high-sensitivity cardiac troponin' (hs-cTn) assays, bringing a flurry of dense yet incomplete literature data. The net result is that cTn assays are not yet standardized and there are still no consensus-built data on how to use and interpret cTn assay results. To address these issues, the authors take cues and clues from multiple disciplines to bring responses to frequently asked questions. In brief, the effective use of hs-cTn hinges on knowing: specific assay characteristics, particularly precision at the 99th percentile of a reference population; factors of variation at the 99th percentile value; and the high-individuality of hs-cTn assays, for which the notion of individual kinetics is more informative than straight reference to 'normal' values. The significance of patterns of change between two assay measurements has not yet been documented for every hs-cTn assay. Clinicians need to work hand-in-hand with medical biologists to better understand how to use hs-cTn assays in routine practice.

  4. Development of a thyroperoxidase inhibition assay for high-throughput screening.

    Science.gov (United States)

    Paul, Katie B; Hedge, Joan M; Rotroff, Daniel M; Hornung, Michael W; Crofton, Kevin M; Simmons, Steven O

    2014-03-17

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.

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

    KAUST Repository

    Soufan, Othman

    2016-01-01

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

  6. A sensitive high throughput ELISA for human eosinophil peroxidase: a specific assay to quantify eosinophil degranulation from patient-derived sources.

    Science.gov (United States)

    Ochkur, Sergei I; Kim, John Dongil; Protheroe, Cheryl A; Colbert, Dana; Condjella, Rachel M; Bersoux, Sophie; Helmers, Richard A; Moqbel, Redwan; Lacy, Paige; Kelly, Elizabeth A; Jarjour, Nizar N; Kern, Robert; Peters, Anju; Schleimer, Robert P; Furuta, Glenn T; Nair, Parameswaran; Lee, James J; Lee, Nancy A

    2012-10-31

    Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.

  7. Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection.

    Science.gov (United States)

    Ni, Weiyi; Le Guiner, Caroline; Moullier, Philippe; Snyder, Richard O

    2012-01-01

    Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

  8. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

    Science.gov (United States)

    Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies.

  9. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

  10. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    Science.gov (United States)

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  11. Using the BioAssay Ontology for analyzing high-throughput screening data.

    Science.gov (United States)

    Zander Balderud, Linda; Murray, David; Larsson, Niklas; Vempati, Uma; Schürer, Stephan C; Bjäreland, Marcus; Engkvist, Ola

    2015-03-01

    High-throughput screening (HTS) is the main starting point for hit identification in drug discovery programs. This has led to a rapid increase of available screening data both within pharmaceutical companies and the public domain. We have used the BioAssay Ontology (BAO) 2.0 for assay annotation within AstraZeneca to enable comparison with external HTS methods. The annotated assays have been analyzed to identify technology gaps, evaluate new methods, verify active hits, and compare compound activity between in-house and PubChem assays. As an example, the binding of a fluorescent ligand to formyl peptide receptor 1 (FPR1, involved in inflammation, for example) in an in-house HTS was measured by fluorescence intensity. In total, 155 active compounds were also tested in an external ligand binding flow cytometry assay, a method not used for in-house HTS detection. Twelve percent of the 155 compounds were found active in both assays. By the annotation of assay protocols using BAO terms, internal and external assays can easily be identified and method comparison facilitated. They can be used to evaluate the effectiveness of different assay methods, design appropriate confirmatory and counterassays, and analyze the activity of compounds for identification of technology artifacts.

  12. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  13. Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

    Science.gov (United States)

    O'Clock, George D

    2016-08-01

    Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

  14. Mass Spectrometry-based Assay for High Throughput and High Sensitivity Biomarker Verification

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xuejiang; Tang, Keqi

    2017-06-14

    Searching for disease specific biomarkers has become a major undertaking in the biomedical research field as the effective diagnosis, prognosis and treatment of many complex human diseases are largely determined by the availability and the quality of the biomarkers. A successful biomarker as an indicator to a specific biological or pathological process is usually selected from a large group of candidates by a strict verification and validation process. To be clinically useful, the validated biomarkers must be detectable and quantifiable by the selected testing techniques in their related tissues or body fluids. Due to its easy accessibility, protein biomarkers would ideally be identified in blood plasma or serum. However, most disease related protein biomarkers in blood exist at very low concentrations (<1ng/mL) and are “masked” by many none significant species at orders of magnitude higher concentrations. The extreme requirements of measurement sensitivity, dynamic range and specificity make the method development extremely challenging. The current clinical protein biomarker measurement primarily relies on antibody based immunoassays, such as ELISA. Although the technique is sensitive and highly specific, the development of high quality protein antibody is both expensive and time consuming. The limited capability of assay multiplexing also makes the measurement an extremely low throughput one rendering it impractical when hundreds to thousands potential biomarkers need to be quantitatively measured across multiple samples. Mass spectrometry (MS)-based assays have recently shown to be a viable alternative for high throughput and quantitative candidate protein biomarker verification. Among them, the triple quadrupole MS based assay is the most promising one. When it is coupled with liquid chromatography (LC) separation and electrospray ionization (ESI) source, a triple quadrupole mass spectrometer operating in a special selected reaction monitoring (SRM) mode

  15. Diagnosis and classification of von Willebrand disease: a review of the differential utility of various functional von Willebrand factor assays.

    Science.gov (United States)

    Favaloro, Emmanuel J

    2011-10-01

    von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. VWD is diagnosed following a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant, von Willebrand factor (VWF) protein ('antigen') and VWF function which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focussed on other functional VWF assays, such as collagen binding and so-called 'VWF activity' assays, as possible replacements to the VWF:RCo, or as supplementary tests of VWF 'function'. Additional laboratory testing can comprise a battery of confirmatory and VWD-type assisting assays, including VWF:multimer and von Willebrand factor VIII binding. This review aims to update knowledge of current VWD diagnostics with a particular emphasis on 'functional' VWF assays.

  16. Study Utility Vehicle Makassar City Transport a HighErgonomics

    Directory of Open Access Journals (Sweden)

    Ahmad Hanafie

    2015-09-01

    Full Text Available The development of technology during this was to meet the man, but it should be men must be spoilt, But if it turns out that all that did not make people feel safe, comfortable, healthy and easy, but the planning process, decision-making and developments have experienced a deviation orientation. Public transport Transportation in the Makassar city should be made with implementing aspects promotes ergonomic comfort, but it does not apply in means of transportation to the public. Issues for public vehicles on access up and down not in accordance with The aim of the research vehicle users. is to phrases dimensions body which have an effect on to utility vehicle, to examine the public vehicles that high-promotes ergonomic comfort. The method assessment is the measurement dimensions body to the passengers as well as the use questionnaires and analyzed in a holistic approach ergonomics. Results of research high security tools to public vehicles that high-security vehicle users generally by body dimensions as a powerful than Knee-and-a-half was knee, long your feet, and your elbow kelantai. While utilities yangbernilai ergonomics was the first and second around 24.76 cm and 49.53 cm, wide around 24.25 cm and was hangar 104, 78 cm.

  17. High temperature polymer fuel cells. Heat utilization and co tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Jens, Oluf Jensen; Qingfeng, Li; Ronghuan, He; Gang, Xiao; Ji-An, Gao; Bjerrum, N.J. [Denmark Technical Univ., Department of Chemistry, Lyngby (Denmark)

    2003-09-01

    This paper will report recent results from our group on polymer fuel cells (PEMFC) based on the temperature resistant polymer poly-benzimidazole (PBI), which allow working temperatures up to 200 C. The membrane has a water drag number near zero and need no water management at all. The high working temperature allows for utilization of the excess heat for fuel processing. Moreover, it provides an excellent CO tolerance of several percent, and the system needs no purification of hydrogen from a reformer. Continuous service for over 6 months at 150 C has been demonstrated. (authors)

  18. High-throughput Saccharification assay for lignocellulosic materials.

    Science.gov (United States)

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-07-03

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  19. A Rapid and Highly Selective Resonance Scattering Spectral Assay for Trace Carcinoembryonic Antigen

    Institute of Scientific and Technical Information of China (English)

    LI,Jian-Fu; JIANG,Zhi-Liang; DENG,An-Ping

    2008-01-01

    Gold nanoparticles, in size of 10 nm, were used to label monoclonal carcinoembryonic antigen antibody (CE-Aab) to obtain an immunonanogold resonance scattering (RS) spectral probe (AuCEAAb) for the measurement of trace carcinoembryonic antigen (CEA). In pH 6.8 Na2HPO4-NaH2PO4 buffer solution and the presence of polyeth-ylene glycol (PEG)-6000, the AuCEAAb combined with CEA to form the nanogold-labeled immunocomplex clus-ters in average size of 227 nm, which exhibited two scattering peaks at about 320 and 581 nm. The enhanced RS intensity at 581 nm (△I581 nm) is proportional to CEA concentration in the range of 1.0-50.0 ng/mL, with a detec-tion limit (DL) of 0.52 ng/mL. The immunonanogold RS assay was utilized to determine CEA in serum samples with high sensitivity, good selectivity and simplicity.

  20. High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling.

    Science.gov (United States)

    Trubetskoy, O V; Finel, M; Kurkela, M; Fitzgerald, M; Peters, N R; Hoffman, F M; Trubetskoy, V S

    2007-06-01

    Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.

  1. Investigation into the utility of an immunocytochemical assay in body cavity effusions for diagnosis of feline infectious peritonitis.

    Science.gov (United States)

    Felten, Sandra; Matiasek, Kaspar; Gruendl, Stefanie; Sangl, Laura; Wess, Gerhard; Hartmann, Katrin

    2017-04-01

    Objectives Feline coronaviruses (FCoVs) exist as two biotypes, feline enteric coronavirus and feline infectious peritonitis virus. Although feline infectious peritonitis (FIP) is a very common disease, the ante-mortem diagnosis of this disease still remains a challenge. Immunofluorescence staining of FCoV in macrophages in effusion has been considered as the reference standard for the diagnosis, but recently this method has been shown to have lower specificity than previously reported. In addition, this method is not widely available and requires the use of fluorescence microscopes. Therefore, it was the aim of this study to evaluate the diagnostic potential of an immunocytochemical (ICC) assay using body cavity effusion. Methods Effusion samples from 27 cats with immunohistochemically confirmed FIP and 29 cats with suspected FIP but a definitive diagnosis of another disease were examined. ICC specimens were evaluated with respect to positive immunostaining. In addition, effusion samples were stained with haematoxylin and eosin and evaluated cytologically. Results A diagnostic sensitivity of 85.2% was recorded for effusion specimens (95% confidence interval [CI] 66.3-95.8), while the diagnostic specificity was only 72.4% (95% CI 52.8-87.3). Conclusions and relevance Once the clinical disease of FIP develops in a cat, it always leads to death, and most of the cats are euthanased within a few days or weeks. As false-positive results might lead to euthanasia of cats suffering from potentially treatable diseases, the diagnostic specificity of a diagnostic tool is the most important factor in a fatal disease like FIP. Thus, the diagnostic utility of this test proved to be insufficient and positive ICC results should be interpreted with caution. Nevertheless, full-body necropsy could not be performed in 13/29 control cats. It is possible that these cats actually suffered from early-stage FIP and that this fact might have influenced the diagnostic specificity of the ICC

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Applications of high-throughput clonogenic survival assays in high-LET particle microbeams

    Directory of Open Access Journals (Sweden)

    Antonios eGeorgantzoglou

    2016-01-01

    Full Text Available Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-LET particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells’ clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells’ response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell’s capacity to divide at least 4-5 times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions.

  4. Novel method for the high-throughput processing of slides for the comet assay.

    Science.gov (United States)

    Karbaschi, Mahsa; Cooke, Marcus S

    2014-11-26

    Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

  5. High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.

    Science.gov (United States)

    Carmosino, Monica; Rizzo, Federica; Torretta, Silvia; Procino, Giuseppe; Svelto, Maria

    2013-03-18

    The kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na(+) reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension. Taking advantage of an existing Thallium (Tl(+))-based kit, we set up a Tl(+) influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays. So far the screening of NKCC transporters activity has been done by (86)Rb(+) influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

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

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

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

  7. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

    Science.gov (United States)

    Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

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

    Science.gov (United States)

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

    2016-02-01

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

  9. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

  10. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

    Science.gov (United States)

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K

    2017-02-01

    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  11. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  12. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned.

    Science.gov (United States)

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile

    2008-06-01

    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  13. Microscopic observation drug-susceptibility assay vs. Xpert(®) MTB/RIF for the diagnosis of tuberculosis in a rural African setting: a cost-utility analysis.

    Science.gov (United States)

    Wikman-Jorgensen, Philip E; Llenas-García, Jara; Pérez-Porcuna, Tomàs M; Hobbins, Michael; Ehmer, Jochen; Mussa, Manuel A; Ascaso, Carlos

    2017-06-01

    To compare the cost-utility of microscopic observation drug-susceptibility assay (MODS) and Xpert(®) MTB/RIF implementation for tuberculosis (TB) diagnosis in rural northern Mozambique. Stochastic transmission compartmental TB model from the healthcare provider perspective with parameter input from direct measurements, systematic literature reviews and expert opinion. MODS and Xpert(®) MTB/RIF were evaluated as replacement test of smear microscopy (SM) or as an add-on test after a negative SM. Costs were calculated in 2013 USD, effects in disability-adjusted life years (DALY). Willingness to pay threshold (WPT) was established at once the per capita Gross National Income of Mozambique. MODS as an add-on test to negative SM produced an incremental cost-effectiveness ratio (ICER) of 5647.89USD/DALY averted. MODS as a substitute for SM yielded an ICER of 5374.58USD/DALY averted. Xpert(®) MTB/RIF as an add-on test to negative SM yielded ICER of 345.71USD/DALY averted. Xpert(®) MTB/RIF as a substitute for SM obtained an ICER of 122.13USD/DALY averted. TB prevalence and risk of infection were the main factors impacting MODS and Xpert(®) MTB/RIF ICER in the one-way sensitivity analysis. In the probabilistic sensitivity analysis, Xpert(®) MTB/RIF was most likely to have an ICER below the WPT, whereas MODS was not. Our cost-utility analysis favours the implementation of Xpert(®) MTB/RIF as a replacement of SM for all TB suspects in this rural high TB/HIV prevalence African setting. © 2017 John Wiley & Sons Ltd.

  14. Clinical validation of the HPV-risk assay, a novel real-time PCR assay for detection of high-risk human papillomavirus DNA by targeting the E7 region.

    Science.gov (United States)

    Hesselink, A T; Berkhof, J; van der Salm, M L; van Splunter, A P; Geelen, T H; van Kemenade, F J; Bleeker, M G B; Heideman, D A M

    2014-03-01

    The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk human papillomavirus (HPV) types (i.e., HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and -68), and provides additional genotype information for HPV16 and HPV18. This study evaluated the clinical performance and reproducibility of the HPV-Risk assay with cervical scraping specimens and its utility with self-collected (cervico)vaginal specimens. The clinical performance of the HPV-Risk assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) with cervical scraping specimens was evaluated by a noninferiority analysis, relative to high-risk HPV GP5+/6+ PCR, following international guidelines for HPV test requirements for cervical cancer screening. The HPV-Risk assay showed clinical sensitivity for CIN2+ of 97.1% (95% confidence interval [CI], 89.1 to 99.3%; 67/69 samples) and a clinical specificity for CIN2+ of 94.3% (95% CI, 92.5 to 95.7%; 777/824 samples). The clinical sensitivity and specificity were noninferior to those of GP5+/6+ PCR (noninferiority score test, P=0.006 and 0.0003, respectively). Intralaboratory reproducibility over time (99.5% [95% CI, 98.6 to 99.8%]; 544/547 samples, kappa=0.99) and interlaboratory agreement (99.2% [95% CI, 98.6 to 99.8%]; 527/531 samples, kappa=0.98) for the HPV-Risk assay with cervical scraping specimens were high. The agreement of the HPV-Risk assay results for self-collected (cervico)vaginal specimens and clinician-obtained cervical scraping specimens was also high, i.e., 95.9% (95% CI, 85.1 to 99.0%; 47/49 samples, kappa=0.90) for self-collected lavage samples and 91.6% (95% CI, 84.6 to 95.6%; 98/107 samples, kappa=0.82) for self-collected brush samples. In conclusion, the HPV-Risk assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered clinically validated for cervical screening purposes. The

  15. Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.

    Science.gov (United States)

    Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D; Thomas, David D; Gillispie, Gregory D

    2017-03-01

    We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

  16. Outliers affecting cardiac troponin I measurement: comparison of a new high sensitivity assay with a contemporary assay on the Abbott ARCHITECT analyser.

    Science.gov (United States)

    Sawyer, Nicola; Blennerhassett, John; Lambert, Ramon; Sheehan, Paul; Vasikaran, Samuel D

    2014-07-01

    False-positive cardiac troponin (Tn) results caused by outliers have been reported on various analytical platforms. We have compared the precision profile and outlier rate of the Abbott Diagnostics contemporary troponin I (TnI) assay with their high sensitivity (hs) TnI assay. Three studies were conducted over a 10-month period using routine patients' samples. TnI was measured in duplicate using the contemporary TnI assay in Study 1 and Study 2 (n = 7011 and 7089) and the hs-TnI assay in Study 3 (n = 1522). Critical outliers were defined as duplicate results whose absolute difference exceeded a critical difference (CD = z x √2 x SDAnalytical) at a probability level of 0.0005, with one of the results on the opposite side of the decision limit to its partner. The TnI concentration at 10% imprecision (coefficient of variation) for the contemporary TnI assay was 0.034 µg/L (Study 1) and 0.042 µg/L (Study 2), and 0.006 µg/L (6 ng/L) for the hs-TnI assay. The critical outlier rates for the contemporary TnI assay were 0.51% (Study 1) and 0.37% (Study 2) using a cut-off of 0.04 µg/L, and 0% for the hs-TnI assay using gender-specific cut-offs. The significant number of critical outliers detected using the contemporary TnI assay may pose a risk for misclassification of patients. By contrast, no critical outliers were detected using the hs-TnI assay. However, the total outlier rates for both assays were significantly higher than the expected variability of either assay. The cause of these outliers remains unclear. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  17. Creation of reversed phase high-performance liquid chromatographic technique to assay platelet-activating factor

    Institute of Scientific and Technical Information of China (English)

    杨云梅; 曹红翠; 徐哲荣; 陈晓明

    2004-01-01

    Objective: To establish a new assay for platelet-activating factor (PAF), to compare it with bio-assay; and to discuss its significance in some elderly people diseases such as cerebral infarction and coronary heart disease. Methods: To measure PAF levels in 100 controls, 23 elderly patients with cerebral infarction and 65 cases with coronary heart disease by reversed phase high-performance liquid chromatographic technique (rHPLC). Results: rHPLC is more convenient, sensitive, specific, and less confusing, compared with bio-assay. The level of plasma PAF in patients with cerebral infarction was higher than that in the controls (P<0.01), and in patients with coronary heart disease. Conclusion: Detection of PAF with rHPLC is more reliable and more accurate. The new assay has important significance in PAF research.

  18. Creation of reversed phase high-performance liquid chromatographic technique to assay platelet-activating factor

    Institute of Scientific and Technical Information of China (English)

    杨云梅; 曹红翠; 徐哲荣; 陈晓明

    2004-01-01

    Objective: To establish a new assay for platelet-activating factor (PAF), to compare it with bio-assay; and to discuss its significance in some elderly people diseases such as cerebral infarction and coronary heart disease. Methods: To measure PAF levels in 100 controls, 23 elderly patients with cerebral infarction and 65 cases with coronary heart disease by reversed phase high-performance liquid chromatographic technique (rHPLC). Results:rHPLC is more convenient, sensitive,specific, and less confusing, compared with bio-assay. The level of plasma PAF in patients with cerebral infarction was higher than that in the controls (P<0.01), and in patients with coronary heart disease. Conclusion: Detection of PAF with rHPLC is more reliable and more accurate. The new assay has important significance in PAF research.

  19. A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.

    Science.gov (United States)

    Moretti, Rocco; Thorson, Jon S

    2008-06-15

    A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.

  20. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  1. 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.

  2. Validation of an automated immune turbidimetric assay for serum gelsolin and its possible clinical utility in sepsis.

    Science.gov (United States)

    Horváth-Szalai, Zoltán; Kustán, Péter; Szirmay, Balázs; Lakatos, Ágnes; Christensen, Per H; Huber, Tamás; Bugyi, Beáta; Mühl, Diána; Ludány, Andrea; Miseta, Attila; Kovács, Gábor L; Kőszegi, Tamás

    2017-09-05

    Studies showing the potential predictive value of the actin-binding protein gelsolin, in critically ill patients are scarce. Moreover, even up to now a rapid automated measurement of gelsolin has still remained a challenge. Therefore, we developed and validated an automated serum gelsolin immune turbidimetric assay for possible clinical use. Validation of serum gelsolin assay was performed on a Cobas 8000/c502 analyzer (Roche) according to the second edition of Eurachem guidelines. Furthermore, we also studied the diagnostic value of serum gelsolin in sepsis when investigating sera of septic (n = 25), systemic inflammatory response syndrome (SIRS; n = 8) and control patients (n = 14). We compared our previously published Western blot data with those of the new turbidimetric assay. The sample volume was 7 μL and the assay time was 10 minutes. The detection limit was 0.72 mg/L, intra- and inter-assay imprecision remained in most cases less than 5% expressed as CV. Recovery was found to be 84.56%-93.52% and linearity study gave an appropriate correlation coefficient by linear regression analysis (r(2)  = .998). Septic patients exhibited lower (P = .015) first-day serum gelsolin levels than SIRS patients, which confirmed our previous Western blot results. The determined cut-off point for serum gelsolin was 14.05 mg/L (sensitivity: 75%; specificity: 60%) when investigating its diagnostic value in sepsis. Based on the results, our immune turbidimetric measurement offers a rapid and accurate quantitation of gelsolin in human serum samples. Serum gelsolin seems a promising additional diagnostic marker of sepsis which has to be further investigated. © 2017 Wiley Periodicals, Inc.

  3. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

    Science.gov (United States)

    Caballero, Iván; Lafuente, María José; Gamo, Francisco-Javier; Cid, Concepción

    2016-08-01

    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

  4. Characterization of a new highly sensitive immunometric assay for thyroglobulin with reduced interference from autoantibodies.

    Science.gov (United States)

    Broughton, Marianne Nordlund; Nome, Ragnhild; Sandven, Ingvill; Paus, Elisabeth; Bjøro, Trine

    2016-06-01

    Measurements of serum thyroglobulin (Tg) with sensitive immunoassays are of great importance for the management of patients with differentiated thyroid carcinomas. However, interference of circulating autoantibodies to Tg (hTgAb) hampers the usefulness of most assays. We have produced a panel of monoclonal antibodies (mAbs) selected to bind Tg in the presence of Tg autoantibodies and developed a sensitive immunoassay for Tg with minor interference by hTgAbs. The antibodies were characterized by cross-inhibition and immunoassay combination studies, as well as affinity estimation. The within-run and total imprecision of the assay were determined with 2664 samples in 60 separate runs. The most sensitive assay combination with superior protection against autoantibodies consisted of two solid phase mAbs and two tracer mAbs with distinct binding sites. The assay was linear and displayed a wide dynamic range up to 1342 μg/l with a functional sensitivity of 0.1 μg/l and a total imprecision of less than 10 %. There was good agreement between the new high sensitive immunofluorometric assay (IFMA) and two well-established Tg assays from Brahms Kryptor and Roche Diagnostics. Mean difference between the new IFMA and the Kryptor assay was 0.059 μg/l with a 95 % confidence interval of -0.032 to 0.151 μg/l, whereas the mean difference between the new IFMA and the Roche assay was -0.80 μg/l with a 95 % confidence interval of -1.24 to -0.35 μg/l.

  5. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  6. A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

    Science.gov (United States)

    Lai, Yiling; Xiang, Meichun; Liu, Shuchun; Li, Erwei; Che, Yongsheng; Liu, Xingzhong

    2014-04-01

    Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

  7. In vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening

    Science.gov (United States)

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Lysine acetylation is a post-translational modification that is carried out by acetyltransferases. The MYST proteins form the largest and most diverse family of acetyltransferases, which regulate gene expression, DNA repair, and cell cycle homeostasis, among other activities, by acetylating both histone and non-histone proteins. This chapter will describe methods for the preparation and biochemical characterization of MYST family acetyltransferases, including protocols for the preparation of recombinant protein, enzyme assays for measuring steady state parameters and binding assays to measure cofactor and inhibitor binding. We also provide details on adapting these assays for high throughput screening for small molecule MYST inhibitors. This chapter seeks to prepare researchers for some hurdles that they may encounter when studying the MYST proteins so that there may be better opportunity to plan appropriate controls and obtain high quality data. PMID:27372752

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

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Paul Daniel Phillips

    2016-05-01

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

  10. Bio-mass utilization in high pressure cogeneration boiler

    Science.gov (United States)

    Koundinya, Sandeep; Maria Ambrose Raj, Y.; Sreeram, K.; Divakar Shetty A., S.

    2017-07-01

    Coal is widely used all over the world in almost all power plants. The dependence on coal has increased enormously as the demand for electricity has reached its peak. Coal being a non-renewable source is depleting fast. We being the engineers, it's our duty to conserve the natural resources and optimize the coal consumption. In this project, we have tried to optimize the bio-mass utilization in high pressure cogeneration boiler. The project was carried in Seshasayee Paper and Boards Limited, erode related to Boiler No:10 operating at steam pressure of 105 kscg and temperature of 510°C. Available bio-mass fuels in and around the mill premises are bagasse, bagasse pith, cane trash and chipper dust. In this project, we have found out the coal equivalent replacement by the above bio-mass fuel(s) to facilitate deciding on the optimized quantity of coal that can be replaced by biomass without modifying the existing design of the plant. The dominant fuel (coal) which could be displaced with the substitute biomass fuel had been individually (biomass) analyzed.

  11. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    Science.gov (United States)

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks. Copyright © 2012 Wiley Periodicals, Inc.

  12. High-sensitivity cardiac troponin assays: From improved analytical performance to enhanced risk stratification.

    Science.gov (United States)

    Kozinski, Marek; Krintus, Magdalena; Kubica, Jacek; Sypniewska, Grazyna

    2017-05-01

    Implementation of cardiac troponin (cTn) assays has revolutionized the diagnosis, risk stratification, triage and management of patients with suspected myocardial infarction (MI). The Universal Definition of MI brought about a shift in the diagnostics of MI, from an approach primarily based on electrocardiography (ECG) to one primarily based on biomarkers. Currently, detection of a rise and/or fall in concentration or activity of myocardial necrosis biomarkers, preferentially cTns, with at least one value above the 99th percentile upper reference limit (URL), is the essential component for the diagnosis of MI. High-sensitivity cardiac troponin (hs-cTn) assays with their superior analytical performance were designed to further facilitate clinical decision making. The ability of hs-cTn assays to detect measurable cTn concentrations in at least 50% of healthy individuals, along with their improved precision (expressed as coefficient of variation ≤10% at the 99th percentile URL) associated with increased recognition of changing values, leads to enhanced risk stratification of patients with suspected MI, and also enables them to be used as prognostic tools potentially useful in other patient subsets. In this comprehensive review, we aim to integrate updated laboratory and clinical knowledge regarding hs-cTn assays in order to promote their optimal use in daily practice. We primarily focus on the role of hs-cTn assays in patients with suspected MI, discussing recommended diagnostic algorithms and result interpretation. Emphasis is also placed on the release of cTns following myocardial injury, the characteristics of antibodies used in available cTn immunoassays, and analytical performance of hs-cTn assays. In this paper, we also review potential challenges related to the selection of a healthy reference population in determining 99th percentile values, biological variation of hs-cTns, inequality between hs-cTn assays, and outline the current status of c

  13. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

    Science.gov (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...

  15. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    Science.gov (United States)

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  16. The next-generation Hybrid Capture High-Risk HPV DNA assay on a fully automated platform.

    Science.gov (United States)

    Eder, Paul S; Lou, Jianrong; Huff, John; Macioszek, Jerzy

    2009-07-01

    A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble system consists of an analytical subsystem (JE2000) that utilizes a re-engineered Hybrid Capture chemistry (NextGen) to maintain the high level of clinical sensitivity established by the digene High-Risk HPV DNA Test (HC2), while creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens. Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and low-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay. The Limit of Detection (LOD) is HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 10(7) copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6). The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading.

  17. A universal homogeneous assay for high-throughput determination of binding kinetics.

    Science.gov (United States)

    Schiele, Felix; Ayaz, Pelin; Fernández-Montalván, Amaury

    2015-01-01

    There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug-target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR-FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein-protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.

  18. 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

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  19. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Science.gov (United States)

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael

    2011-01-01

    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  20. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    Full Text Available Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  1. Management and High-rank Utilization of Chemical Information

    Science.gov (United States)

    Sasaki, Shinichi

    This is the record of Special Lecture at the 24th Annual Meeting on Information Science and Technology. Firstly, lecturer outlined the production way and a general utilization of database on 13C and H magnetic resonance spectrum, which is produced by his group. In order to make higher the rank of utilization for this kind of factual database, he described three new systems such as CHEMICS, TUTORS and AIPHOS.

  2. Development of a central nervous system axonal myelination assay for high throughput screening.

    Science.gov (United States)

    Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

    2016-04-22

    Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

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

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

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

  4. Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

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    Michele E Murphy

    2016-01-01

    Full Text Available Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform. Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method's specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector.

  5. Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis

    Science.gov (United States)

    DiStefano, Daniel J.; Kraiouchkine, Nikolai; Mallette, Laura; Maliga, Marianne; Kulnis, Gregory; Keller, Paul M.; Clark, H. Fred; Shaw, Alan R.

    2005-01-01

    Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ∼92% of samples (3, 17, 19). PMID:16333070

  6. Evaluation of the PharmGKB knowledge base as a resource for efficiently assessing the clinical validity and utility of pharmacogenetic assays.

    Science.gov (United States)

    Kawamoto, Kensaku; Orlando, Lori A; Voora, Deepak; Lobach, David F; Joy, Scott; Cho, Alex; Ginsburg, Geoffrey S

    2009-11-14

    Prior to clinical use, pharmacogenetic tests should be systematically evaluated for their clinical validity and utility. Here, we evaluated whether the publicly available, online Pharmacogenomics Knowledge Base (PharmGKB) could facilitate such assessments by efficiently identifying relevant peer-reviewed manuscripts. The search targets were 55 manuscripts regarding clinical validity and utility included in systematic reviews of warfarin, antidepressant, and irinotecan pharmacogenetics. When direct inclusion in PharmGKB was the search criterion, recall was 33% and precision was 16%. However, recall increased to 78% when citation within a PharmGKB-identified manuscript was added as a search criterion. These recalled manuscripts accounted for 87% of the study subjects, and domain experts determined that the omission of the remaining manuscripts was unlikely to have changed the conclusions of the reviews. Thus, we conclude that PharmGKB can facilitate the systematic assessment of pharmacogenetic assays through the efficient identification of relevant peer-reviewed manuscripts.

  7. A highly sensitive and selective diagnostic assay based on virus nanoparticles

    Science.gov (United States)

    Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon

    2009-04-01

    Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

  8. High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair

    Directory of Open Access Journals (Sweden)

    Sarika Pathak-Sharma

    2017-07-01

    Full Text Available The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.

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

    Science.gov (United States)

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

    2013-12-01

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

  10. High Purity Germanium Gamma-PHA Assay of Uranium Scrap Cans Used in 321-M Facility

    Science.gov (United States)

    Salaymeh, S. R.; Dewberry, R. A.; Casella, V.

    2001-12-01

    The Analytical Development Section of SRTC was requested by the Facilities Disposition Division (FDD) to determine the holdup of enriched uranium in the 321-M facility as part of an overall deactivation project of the facility. The 321-M facility was used to fabricate enriched uranium fuel assemblies, lithium-aluminum target tubes, neptunium assemblies, and miscellaneous components for the production reactors. The facility also includes the 324-M storage building and the passageway connecting it to 321-M. The results of the holdup assays are essential for determining compliance with the Solid Waste's Waste Acceptance Criteria, Material Control & Accountability, and to meet criticality safety controls. This report describes and documents the use of a portable HPGe detector and EG&G DART system that contains a high voltage power supply, signal processing electronics, a personal computer with Gamma-Vision software, and space to store and manipulate multiple 4096-channel gamma-ray spectra to assay for 235U content. The system was used to assay a large number of scrap cans used to store highly enriched uranium (HEU) chips and filings. This report includes a description of two efficiency calibration configurations and also the results of the assay. A description of the quality control checks is included as well.

  11. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  12. High-Volume Utilization of Cotton in Nonwovens

    Science.gov (United States)

    This article briefly explores possible utilization of cotton in volume in the traditional textile sector and in the growing nontraditional nonwovens sector and covers the challenges and opportunities that may exist on the way. Some new concepts in the development of predominantly cotton-based textil...

  13. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    Science.gov (United States)

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

  14. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors.

    Science.gov (United States)

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B

    2014-04-01

    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  15. High-throughput colorimetric assays for nucleotide sugar formation and glycosyl transfer.

    Science.gov (United States)

    Gantt, Richard W; Thorson, Jon S

    2012-01-01

    Glycosyltransferases are ubiquitous in nature, catalyzing glycosidic bond formation in the context of an enormous range of substrates, which include all major classes of biological molecules. Because this wide range of substrates lacks a shared, distinguishable feature that can be altered by glycosyl transfer, general assays for detection of glycosyltransferase activity have long been largely limited to low-throughput methods. Of those high-throughput assays reported in the literature, many are confined to specific glycosyl transfer reactions with modified aglycon acceptors selected for their unique analytical properties. Herein are described a series of protocols centered on the use of 2-chloro-4-nitrophenyl glycoside donors and the reversibility of glycosyltransferase-catalyzed reactions to enable a colorimetric assay for the formation of sugar nucleotides, coupled reaction systems for the glycodiversification of small molecules, and a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering, and other fundamental sugar nucleotide-dependent investigations. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid.

    Science.gov (United States)

    Gerlo, E; Malfait, R

    1985-09-13

    A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.

  17. Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

    DEFF Research Database (Denmark)

    Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper

    2009-01-01

    of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC...... in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. RESULTS: We generated....... CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools....

  18. Clinical utility of a next generation sequencing panel assay for Marfan and Marfan-like syndromes featuring aortopathy.

    Science.gov (United States)

    Wooderchak-Donahue, Whitney; VanSant-Webb, Chad; Tvrdik, Tatiana; Plant, Parker; Lewis, Tracey; Stocks, Jennifer; Raney, Joshua A; Meyers, Lindsay; Berg, Alizabeth; Rope, Alan F; Yetman, Anji T; Bleyl, Steven B; Mesley, Rebecca; Bull, David A; Collins, R Thomas; Ojeda, Mayra Martinez; Roberts, Amy; Lacro, Ronald; Woerner, Audrey; Stoler, Joan; Bayrak-Toydemir, Pinar

    2015-08-01

    Aortopathy can be defined as aortic dilation, aneurysm, dissection, and tortuosity. Familial aortopathy may occur secondary to fibrillin-1 (FBN1) mutations in the setting of Marfan syndrome, or may occur as a result of other genetic defects with different, but occasionally overlapping, phenotypes. Because of the phenotypic overlap and genetic heterogeneity of disorders featuring aortopathy, we developed a next generation sequencing (NGS) assay and comparative genomic hybridization (CGH) array to detect mutations in 10 genes that cause thoracic aortic aneurysms (TAAs). Here, we report on the clinical and molecular findings in 175 individuals submitted for aortopathy panel testing at ARUP laboratories. Ten genes associated with heritable aortopathies were targeted using hybridization capture prior to sequencing. NGS results were analyzed, and variants were confirmed using Sanger sequencing. Array CGH was used to detect copy-number variation. Of 175 individuals, 18 had a pathogenic mutation and 32 had a variant of uncertain significance (VUS). Most pathogenic mutations (72%) were identified in FBN1. A novel large SMAD3 duplication and FBN1 deletion were identified. Over half who had TAAs or other aortic involvement tested negative for a mutation, suggesting that additional aortopathy genes exist. We anticipate that the clinical sensitivity of at least 10.3% will rise with VUS reclassification and as additional genes are identified and included in the panel. The aortopathy NGS panel aids in the timely molecular diagnosis of individuals with disorders featuring aortopathy and guides proper treatment.

  19. Patterning cell using Si-stencil for high-throughput assay

    KAUST Repository

    Wu, Jinbo

    2011-01-01

    In this communication, we report a newly developed cell pattering methodology by a silicon-based stencil, which exhibited advantages such as easy handling, reusability, hydrophilic surface and mature fabrication technologies. Cell arrays obtained by this method were used to investigate cell growth under a temperature gradient, which demonstrated the possibility of studying cell behavior in a high-throughput assay. This journal is © The Royal Society of Chemistry 2011.

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

    OpenAIRE

    Weatherford Wendy; Stankewicz Casey; Rininsland Frauke; McBranch Duncan

    2005-01-01

    Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small s...

  1. High-throughput enzyme-linked immunoabsorbant assay (ELISA) electrochemiluminescent detection of botulinum toxins in foods for food safety and defence purposes.

    Science.gov (United States)

    Phillips, R W; Abbott, D

    2008-09-01

    Clostridum species produce seven serotypes (A-G) of botulinum toxin, four of which (A, B, E, and F) are normally associated with human illness. To date, the most reliable test for botulinum toxin is the mouse bioassay. The authors' laboratory has been exploring the use of an antibody-based assay similar to an enzyme-linked immunoabsorbant assay (ELISA) but utilizing electrochemiluminescent technology (BioVerify assay) as an alternative to the mouse bioassay for testing food samples. The detection limit of this assay is as low as 10 ng g(-1) depending on the food matrix and the serotype detected. Detection of botulinum toxin between 10 and 200 ng g(-1) is a linear curve allowing for the possibility of performing quantitative as well as qualitative testing of samples. The ease of the assay, limited sample preparation, and low detection limit make the BioVerify assay and instrument an excellent, high-throughput option for detecting botulinum toxins in food matrices.

  2. Rapid and highly informative diagnostic assay for H5N1 influenza viruses.

    Directory of Open Access Journals (Sweden)

    Nader Pourmand

    Full Text Available A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

  3. Systolic Tree Algorithms for Discovering High Utility Item sets from Transactional Database

    Directory of Open Access Journals (Sweden)

    B. Shibi

    2015-10-01

    Full Text Available Utility mining emerges as an important topic in data mining field. Here high utility itemsets mining refers to importance or profitability of an item to users. Efficient mining of high utility itemsets plays an important role in many real-life applications and is an important research issue in data mining area. Number of Algorithms utility pattern growth (UP-Growth and UP-Growth+( A data structure having tree like structure named utility pattern tree (UP-Tree is used for storing the information about high utility item set such that by using only double scanning of database, candidate itemsets can be efficiently generate. But that will lead to high requirement of space and time and so that performance will be less. So a new algorithm is proposed in this paper, for efficient discovering of high utility itemsets from transactional database.

  4. Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

    Science.gov (United States)

    Ramirez, Christina N; Ozawa, Tatsuya; Takagi, Toshimitsu; Antczak, Christophe; Shum, David; Graves, Robert; Holland, Eric C; Djaballah, Hakim

    2011-06-01

    Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.

  5. High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Escribano, David; de Salas, Felipe; Pardo, Isabel; Camarero, Susana

    2017-08-01

    Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.

  6. A PCR-high-resolution melt assay for rapid differentiation of nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

    Science.gov (United States)

    Pickering, Janessa; Binks, Michael J; Beissbarth, Jemima; Hare, Kim M; Kirkham, Lea-Ann S; Smith-Vaughan, Heidi

    2014-02-01

    We have developed a PCR-high-resolution melt (PCR-HRM) assay to discriminate nontypeable Haemophilus influenzae (NTHi) colonies from Haemophilus haemolyticus. This method is rapid and robust, with 96% sensitivity and 92% specificity compared to the hpd#3 assay. PCR-HRM is ideal for high-throughput screening for NTHi surveillance and clinical trials.

  7. Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop.

    Science.gov (United States)

    Karabasanavar, Nagappa S; Singh, S P; Kumar, Deepak; Shebannavar, Sunil N

    2014-02-15

    We describe a highly specific PCR assay for the authentic identification of pork. Accurate detection of tissues derived from pig (Sus scrofa) was accomplished by using newly designed primers targeting porcine mitochondrial displacement (D-loop) region that yielded an unique amplicon of 712 base pairs (bp). Possibility of cross-amplification was precluded by testing as many as 24 animal species (mammals, birds, rodent and fish). Suitability of PCR assay was confirmed in raw (n = 20), cooked (60, 80 and 100 °C), autoclaved (121 °C) and micro-oven processed pork. Sensitivity of detection of pork in other species meat using unique pig-specific PCR was established to be at 0.1%; limit of detection (LOD) of pig DNA was 10 pg (pico grams). The technique can be used for the authentication of raw, processed and adulterated pork and products under the circumstances of food adulteration related disputes or forensic detection of origin of pig species.

  8. Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

    Science.gov (United States)

    Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

    2017-09-01

    Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

  9. Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography.

    Science.gov (United States)

    Haraguchi, K; Kai, M; Kohashi, K; Ohkura, Y

    1980-12-05

    A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

  10. Development of a facile method for high throughput screening with reporter gene assays.

    Science.gov (United States)

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  11. A new non-radioactive deoxyhypusine synthase assay adaptable to high throughput screening.

    Science.gov (United States)

    Park, Myung Hee; Mandal, Ajeet; Mandal, Swati; Wolff, Edith C

    2017-08-17

    Deoxyhypusine synthase (DHS) catalyzes the post-translational modification of eukaryotic translation factor 5A (eIF5A) by the polyamine, spermidine, that converts one specific lysine residue to deoxyhypusine [N (ε) -4-aminobutyl(lysine)], which is subsequently hydroxylated to hypusine [N (ε) -4-amino-2-hydroxybutyl(lysine)]. Hypusine synthesis represents the most critical function of polyamine. As eIF5A has been implicated in various human diseases, identification of specific inhibitors of hypusine modification is of vital importance. DHS catalyzes a complex reaction that occurs in two stages, first, the NAD-dependent cleavage of spermidine to form an enzyme-butylimine intermediate and enzyme-bound NADH, and second, the transfer of the butylimine moiety from the enzyme intermediate to the eIF5A precursor and subsequent reduction of the eIF5A-butylimine intermediate by enzyme-bound NADH to form deoxyhypusine [N (ε) -4-aminobutyl(lysine)]. Our data demonstrate that there is a measurable release of enzyme-bound NADH in the absence of eIF5A precursor and that the DHS activity can be determined by coupling the first phase reaction with the NADH-Glo assay in which the generation of luminescence is dependent on NADH derived from the DHS partial reaction. The conventional DHS assay that measures the incorporation of radioactivity from [1,8-(3)H]spermidine into the eIF5A precursor in the complete reaction cannot be readily adapted for high throughput screening (HTS). In contrast, the non-radioactive DHS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and could be configured for HTS of small molecule libraries for the identification of new inhibitors of DHS. Furthermore, the coupled assay provides new insights into the dynamics of the DHS reaction especially regarding the fate of NADH.

  12. A highly sensitive assay for monitoring the secretory pathway and ER stress.

    Directory of Open Access Journals (Sweden)

    Christian E Badr

    Full Text Available BACKGROUND: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER stress in real-time based on the naturally secreted Gaussia luciferase (Gluc. METHODOLOGY/PRINCIPLE FINDINGS: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP, a well established assay for monitoring of protein processing and ER stress in mammalian cells. CONCLUSIONS/SIGNIFICANCE: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.

  13. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor.

    Science.gov (United States)

    Blankvoort, B M; de Groene, E M; van Meeteren-Kreikamp, A P; Witkamp, R F; Rodenburg, R J; Aarts, J M

    2001-11-01

    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 androgen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (AR-LUX) was validated. An EC50 value of 86 pM was determined for the standard androgen R1881 with a detection limit of 46 pM. Other androgens like dihydrotestosterone, 17beta-trenbolone, and bolasterone also induced luciferase expression, while anti-androgens suppressed these responses. As expected, AR-mediated responses were also elicited by high concentrations of the steroids progesterone, 17beta-estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-levels are reliably reflected in the luciferase induction response, as exemplified by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and forskolin. This feature is especially useful when assessing complex mixtures, e.g., environmental samples or natural compound libraries. From these data it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throughput screening.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal...... requirements. To avoid labeling of the highly variable peptide, we labeled the invariant ß2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually...

  15. Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India

    Directory of Open Access Journals (Sweden)

    Fahmiya Leena Yacoob

    2016-01-01

    Full Text Available In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days as compared to solid and liquid cultures (35.6 days and 13.5 days, resp..

  16. A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

    Directory of Open Access Journals (Sweden)

    Anli Zhang

    2015-06-01

    Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

  17. High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport.

    Science.gov (United States)

    Hanson, Mathew; Jordan, Lorne D; Shipelskiy, Yan; Newton, Salete M; Klebba, Phillip E

    2016-03-01

    The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8-1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria. © 2015 Society for Laboratory Automation and Screening.

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

    Science.gov (United States)

    Slattery, Scott D; Hahn, Klaus M

    2014-12-01

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

  19. A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

    Science.gov (United States)

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.

  20. A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

    Science.gov (United States)

    Kashem, Mohammed A; Wa, Chunling; Wolak, John P; Grafos, Nicholas S; Ryan, Kelli R; Sanville-Ross, Mary L; Fogarty, Kylie E; Rybina, Irina V; Shoultz, Alycia; Molinaro, Teresa; Desai, Sudha N; Rajan, Anusha; Huber, John D; Nelson, Richard M

    2014-06-01

    The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 μM in the SPA that induced dose-dependent lymphopenia in mice.

  1. High yield purification of Plasmodium falciparum merozoites for use in opsonizing antibody assays.

    Science.gov (United States)

    Hill, Danika L; Eriksson, Emily M; Schofield, Louis

    2014-07-17

    Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.  

  2. Highly sensitive automated chemiluminometric assay for measuring free human glandular kallikrein-2.

    Science.gov (United States)

    Klee, G G; Goodmanson, M K; Jacobsen, S J; Young, C Y; Finlay, J A; Rittenhouse, H G; Wolfert, R L; Tindall, D J

    1999-06-01

    Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to alpha1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS(R) analyzer. The detection limit of the assay was 1.5 ng/L, the "functional sensitivity" (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

  3. Utilizing HPC Network Technologies in High Energy Physics Experiments

    CERN Document Server

    AUTHOR|(CDS)2088631; The ATLAS collaboration

    2017-01-01

    Because of their performance characteristics high-performance fabrics like Infiniband or OmniPath are interesting technologies for many local area network applications, including data acquisition systems for high-energy physics experiments like the ATLAS experiment at CERN. This paper analyzes existing APIs for high-performance fabrics and evaluates their suitability for data acquisition systems in terms of performance and domain applicability. The study finds that existing software APIs for high-performance interconnects are focused on applications in high-performance computing with specific workloads and are not compatible with the requirements of data acquisition systems. To evaluate the use of high-performance interconnects in data acquisition systems a custom library, NetIO, is presented and compared against existing technologies. NetIO has a message queue-like interface which matches the ATLAS use case better than traditional HPC APIs like MPI. The architecture of NetIO is based on a interchangeable bac...

  4. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika;

    2011-01-01

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... only 1 micro Litre of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired...

  5. Thyroglobulin measurement using highly sensitive assays in patients with differentiated thyroid cancer:

    DEFF Research Database (Denmark)

    Giovanella, Luca; Clark, Penelope M; Chiovato, Luca

    2014-01-01

    Differentiated thyroid cancer (DTC) is the most common endocrine cancer and its incidence has increased in recent decades. Initial treatment usually consists of total thyroidectomy followed by ablation of thyroid remnants by iodine-131. As thyroid cells are assumed to be the only source...... at low concentrations now allows detection of very low Tg concentrations reflecting minimal amounts of thyroid tissue without the need for TSH stimulation. Use of these highly sensitive Tg assays has not yet been incorporated into clinical guidelines but they will, we believe, be used by physicians...

  6. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

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

    Science.gov (United States)

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

    2016-07-01

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

  8. 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.

  9. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    Science.gov (United States)

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

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

    Science.gov (United States)

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

    2017-01-18

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

  11. Radiation Shielding Utilizing A High Temperature Superconducting Magnet Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This project aims to leverage near-term high-temperature superconducting technologies to assess applicability of magnetic shielding for protecting against exposure...

  12. Markers of human immunodeficiency virus infection in high-risk individuals seronegative by first generation enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Pedersen, C; Lindhardt, B O; Lauritzen, E;

    1989-01-01

    A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme...

  13. High-throughput 1,536-well fluorescence polarization assays for α(1-acid glycoprotein and human serum albumin binding.

    Directory of Open Access Journals (Sweden)

    Adam Yasgar

    Full Text Available Two major plasma proteins in humans are primarily responsible for drug binding, the α(1-acid-glycoprotein (AGP and human serum albumin (HSA. The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC(1280 collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.

  14. A high-throughput clinical assay for testing drug facilitation of exposure therapy.

    Science.gov (United States)

    Rodebaugh, Thomas L; Levinson, Cheri A; Lenze, Eric J

    2013-07-01

    Several studies have demonstrated that D-cycloserine (DCS) facilitates exposure therapy. We developed a standardized test of this facilitation (i.e., a clinical assay), with the goal of testing for facilitation more quickly and inexpensively than a full clinical trial. We developed a standardized brief exposure in which participants with social anxiety disorder gave a videotaped speech. Participants were randomized to receive a single capsule of 250 mg DCS or a matching placebo prior to preparation for the speech. Distress levels were rated during the speech and again, approximately 1 week later, during a speech in an identical situation. Our primary measure of DCS's exposure-facilitating effect was between-session habituation: whether or not the participants showed less distress during the second speech compared to the first. We also measured levels of subjective anxiety and fear of scrutiny. Subjects randomized to receive DCS prior to their first speech were more likely to show between-session habituation than those who received placebo. We also found greater reduction of performance-related fear overall in the DCS group. Our clinical assay was able to detect exposure facilitation effects rapidly and in a highly standardized way, and is estimated to take a fraction of the time and costs of a clinical trial. Given the increasing interest in using medications to enhance learning-based psychotherapy, this high-throughput clinical assay approach may be a favorable method for testing novel mechanisms of action, and clarifying optimal parameters, for therapy facilitation. © 2013 Wiley Periodicals, Inc.

  15. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

    Directory of Open Access Journals (Sweden)

    Jakob D Wikstrom

    Full Text Available BACKGROUND: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. METHODOLOGY/PRINCIPAL FINDINGS: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. CONCLUSIONS/SIGNIFICANCE: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  16. A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

    Science.gov (United States)

    Vainshtein, Inna; Silveria, Scott; Kaul, Poonam; Rouhani, Riaz; Eglen, Richard M; Wang, John

    2002-12-01

    A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta

  17. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

    Science.gov (United States)

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga

    2011-06-01

    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  18. High-throughput screening assay for new ligands at human melatonin receptors

    Institute of Scientific and Technical Information of China (English)

    Jian-hua YAN; Hao-ran SU; Jean A BOUTIN; M Pierre RENARD; Ming-wei WANG

    2008-01-01

    Aim: Melatonin (MT) is a neurohormone produced and secreted primarily by the pineal gland in a circadian manner, and mainly acta through 2 receptor subtypes: MT1 and MT2 in humans. The diversity in their tissue distribution is in favor of different functions for each receptor subtype. Selective modulators are therefore required to determine the physiological roles of these melatonin receptor sub-types and their implications in pathological processes. Methods: A homogenous MT1/MT2 receptor binding assay was established for high-throughput screening of new ligands at the hMT1 and/or hMT2 receptors. The functional properties (agonists or antagonists) were assessed by a conventional guanosine-5'[γ-35S] triphosphate (GTP-γS) assay. Results: Three hMT, receptor-selective small mol-ecule antagonists and 1 hMT2 receptor-selective small molecule antagonist with novel structural features were identified following a high-throughput screening campaign of 48 240 synthetic and natural compounds. Conclusion: The findings may assist in the expansion of chemical probes to these 2 receptor subtypes.

  19. High output paraffin actuators: Utilization in aerospace mechanisms

    Science.gov (United States)

    Tibbitts, Scott

    1988-01-01

    High Output Paraffin (HOP) thermal actuators were developed to provide an alternative to conventional aerospace actuators: HOP actuators directly convert temperature changes to useful mechanical work. When fabricated with internal resistance heating elements, they provide an electric linear motor. For applications in which slower response times are acceptable or preferred, HOP actuators have distinct advantages over conventional approaches.

  20. High-power Čerenkov microwave oscillators utilizing High-Current nanosecond Electron beams

    Science.gov (United States)

    Korovin, S. D.; Polevin, S. D.; Rostov, V. V.

    1996-12-01

    A short review is given of results obtained at the Institute of High-Current Electronics of the Siberian Branch of the Russian Academy of Sciences on generating high-power microwave radiation. Most of the research was devoted to a study of stimulated Čerenkov radiation from relativistic electron beams. It is shown that the efficiency of a relativistic 3-cm backward wave tube with a nonuniform coupling resistance can reach 35%. High-frequency radiation was discovered in the emission spectrum of the Čerenkov oscillators and it was shown that the nature of the radiation was associated with the stimulated scattering of low-frequency radiation by the relativistic electrons. Radiation with a power of 500 MW was obtained in the 8-mm wavelength range using a two-beam Čerenkov oscillator. High-current pulse-periodic nanosecond accelerators with a charging device utilizing a Tesla transformer were used in the experiments. The possibility was demonstrated of generating high-power microwave radiation with a pulse-repetition frequency of up to 100 Hz. An average power of ˜500 W was achieved from the relativistic oscillators. A relativistic backward wave tube with a high-current electron beam was used to make a prototype nanosecond radar device. Some of the results presented were obtained jointly with the Russian Academy of Sciences Institute of Applied Physics. Questions concerning multiwave Čerenkov interaction are not considered in this paper.

  1. Aqueous biphasic cancer cell migration assay enables robust, high-throughput screening of anti-cancer compounds.

    Science.gov (United States)

    Lemmo, Stephanie; Nasrollahi, Samila; Tavana, Hossein

    2014-03-01

    Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high-throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy-to-implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two-phase system to pattern a monolayer of cells containing a cell-excluded gap that serves as the migration niche. We adapted this technology to a standard 96-well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti-migratory drug screening.

  2. BioAssay Ontology (BAO: a semantic description of bioassays and high-throughput screening results

    Directory of Open Access Journals (Sweden)

    Smith Robin P

    2011-06-01

    Full Text Available Abstract Background High-throughput screening (HTS is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges. Results We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal http://bioportal.bioontology.org/ontologies/44531. Conclusions After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference.

  3. 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

    2016-10-07

    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.

  4. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

    Science.gov (United States)

    Trubetskoy, Olga V; Gibson, Jasmin R; Marks, Bryan D

    2005-02-01

    Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

  5. The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

    Science.gov (United States)

    Gillissen, M A; Yasuda, E; de Jong, G; Levie, S E; Go, D; Spits, H; van Helden, P M; Hazenberg, M D

    2016-07-01

    Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

  6. The Utilization of Classifications in High-Energy Astrophysics Experiments

    Science.gov (United States)

    Atwood, Bill

    2012-03-01

    The history of high-energy gamma observations stretches back several decades. But it was with the launch of the Energetic Gamma Ray Experiment Telescope (EGRET) in 1991 onboard the Compton Gamma Ray Observatory (CGRO) [1], that the field entered a new era of discovery. At the high-energy end of the electromagnetic spectrum, incoming particles of light, photons, interact with matter mainly by producing electron-positron pairs and this process dominates above an energy of 10-30MeV depending on the material. To a high degree the directionality of the incoming gamma ray is reflected in the e+ and e-, and hence the detection of the trajectories of the e+e- pair can be used to infer the direction of the originating photon. Measuring these high-energy charged particles is the domain of high-energy particle physics and so it should be of little surprise that particle physicists played a significant role in the design and construction of EGRET, as well as the design and implementation of analysis methods for the resulting data. Prior to EGRET, only a handful of sources in the sky were known as high-energy gamma-ray emitters. During EGRET's 9-years mission the final catalog included over 270 sources including new types such as Gamma Ray Bursts (GRBs). This set the stage for the next-generation mission, the Gamma ray Large Area Space Telescope (GLAST) [2]. Very early in the EGRET mission, the realization that the high-energy gamma-ray sky was extremely interesting led to a competition to develop the next-generation instruments. The technology used in EGRET was frozen in the late 1970s and by 1992, enormous advances had been made in experimental particle physics. In particular the effort to develop solid state detectors, targeted for use at the Super Conducting Super Collider (SSC), had made the technology of silicon strip detectors (SSDs) commercially viable for use in large area arrays. Given the limitations imposed by the space environment (e.g., operate in a vacuum, scarce

  7. High-performance liquid chromatographic assay for metamizol metabolites in rat plasma: application to pharmacokinetic studies.

    Science.gov (United States)

    Domínguez-Ramírez, Adriana Miriam; Calzadilla, Patricia Carrillo; Cortés-Arroyo, Alma Rosa; Hurtado Y de la Peña, Marcela; López, José Raúl Medina; Gómez-Hernández, Martín; López-Muñoz, Francisco Javier

    2012-12-01

    In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma samples (50-100μl) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were linear within a range of 1-100μg/ml (r(2)≥0.99). The intra-day coefficients of variation (CV) were in the range of 1.3-8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the range of 0.6-9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1μg/ml for all metabolites using a plasma sample of 100μl. Plasma samples were stable at least for 4 weeks at -20°C. This method was found to be suitable for studying metamizol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and morphine, in single dose. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer.

    Science.gov (United States)

    Fitzpatrick, P A; Akrap, N; Söderberg, E M V; Harrison, H; Thomson, G J; Landberg, G

    2017-02-01

    In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.

  9. Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage.

    Science.gov (United States)

    Brasino, Michael; Lee, Ju Hun; Cha, Jennifer N

    2015-02-01

    For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented-by either chemical functionalization or genetic engineering-with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

  10. Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors

    Science.gov (United States)

    Andresen, Liis; Varik, Vallo; Tozawa, Yuzuru; Jimmy, Steffi; Lindberg, Stina; Tenson, Tanel; Hauryliuk, Vasili

    2016-01-01

    The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors – a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 – showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries. PMID:27775002

  11. Bremsstrahlung-induced highly penetrating probes for nondestructive assay and defect analysis

    CERN Document Server

    Selim, F A; Harmon, J F; Kwofie, J; Spaulding, R; Erickson, G; Roney, T

    2002-01-01

    Nondestructive assay and defect analysis probes based on bremsstrahlung-induced processes have been developed to identify elements and probe defects in large volume samples. Bremsstrahlung beams from (electron accelerators) with end-point energies both above and below neutron emission threshold have been used. Below neutron emission threshold these beams (from 6 MeV small pulsed linacs), which exhibit high penetration, create positrons via pair production inside the material and produce X-ray fluorescence (XRF) radiation. Chemical assays of heavy elements in thick samples up to 10 g/cm sup 2 thick are provided by energy dispersive XRF measurements. The pair-produced positrons annihilate within the material, thereby emitting 511 keV gamma radiation. Doppler broadening spectroscopy of the 511 keV radiation can be performed to characterize the material and measure defects in samples of any desired thickness. This technique has successfully measured induced strain due to tensile stress in steel samples of 0.64 cm...

  12. A High-resolution Typing Assay for Uropathogenic Escherichia coli Based on Fimbrial Diversity.

    Science.gov (United States)

    Ren, Yi; Palusiak, Agata; Wang, Wei; Wang, Yi; Li, Xiao; Wei, Huiting; Kong, Qingke; Rozalski, Antoni; Yao, Zhi; Wang, Quan

    2016-01-01

    Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, causing cystitis, pyelonephritis, and renal failure. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTIs. Accurate and rapid discrimination of UPEC lineages is useful for epidemiological surveillance. Fimbriae are necessary for the adherence of UPEC strains to host uroepithelia, and seem to be abundant and diverse in UPEC strains. By analyzing all the possible fimbrial operons in UPEC strains, we found that closely related strains had similar types of chaperone-usher fimbriae, and the diversity of fimbrial genes was higher than that of multilocus sequence typing (MLST) genes. A typing assay based on the polymorphism of four gene sequences (three fimbrial genes and one housekeeping gene) and the diversity of fimbriae present was developed. By comparison with the MLST, whole-genome sequence (WGS) and fumC/fimH typing methods, this was shown to be accurate and have high resolution, and it was also relatively inexpensive and easy to perform. The assay can supply more discriminatory information for UPEC lineages, and have the potential to be applied in epidemiological surveillance of UPEC isolates.

  13. A Microfabricated 96-Well 3D Assay Enabling High-Throughput Quantification of Cellular Invasion Capabilities

    Science.gov (United States)

    Hao, Rui; Wei, Yuanchen; Li, Chaobo; Chen, Feng; Chen, Deyong; Zhao, Xiaoting; Luan, Shaoliang; Fan, Beiyuan; Guo, Wei; Wang, Junbo; Chen, Jian

    2017-01-01

    This paper presents a 96-well microfabricated assay to study three-dimensional (3D) invasion of tumor cells. A 3D cluster of tumor cells was first generated within each well by seeding cells onto a micro-patterned surface consisting of a central fibronectin-coated area that promotes cellular attachment, surrounded by a poly ethylene glycol (PEG) coated area that is resistant to cellular attachment. Following the formation of the 3D cell clusters, a 3D collagen extracellular matrix was formed in each well by thermal-triggered gelation. Invasion of the tumor cells into the extracellular matrix was subsequently initiated and monitored. Two modes of cellular infiltration were observed: A549 cells invaded into the extracellular matrix following the surfaces previously coated with PEG molecules in a pseudo-2D manner, while H1299 cells invaded into the extracellular matrix in a truly 3D manner including multiple directions. Based on the processing of 2D microscopic images, a key parameter, namely, equivalent invasion distance (the area of invaded cells divided by the circumference of the initial cell cluster) was obtained to quantify migration capabilities of these two cell types. These results validate the feasibility of the proposed platform, which may function as a high-throughput 3D cellular invasion assay. PMID:28240272

  14. Highly Sensitive Colorimetric Assay for Determining Fe3+ Based on Gold Nanoparticles Conjugated with Glycol Chitosan

    Directory of Open Access Journals (Sweden)

    Kyungmin Kim

    2017-01-01

    Full Text Available A highly sensitive and simple colorimetric assay for the detection of Fe3+ ions was developed using gold nanoparticles (AuNPs conjugated with glycol chitosan (GC. The Fe3+ ion coordinates with the oxygen atoms of GC in a hexadentate manner (O-Fe3+-O, decreasing the interparticle distance and inducing aggregation. Time-of-flight secondary ion mass spectrometry showed that the bound Fe3+ was coordinated to the oxygen atoms of the ethylene glycol in GC, which resulted in a significant color change from light red to dark midnight blue due to aggregation. Using this GC-AuNP probe, the quantitative determination of Fe3+ in biological, environmental, and pharmaceutical samples could be achieved by the naked eye and spectrophotometric methods. Sensitive response and pronounced color change of the GC-AuNPs in the presence of Fe3+ were optimized at pH 6, 70°C, and 300 mM NaCl concentration. The absorption intensity ratio (A700/A510 linearly correlated to the Fe3+ concentration in the linear range of 0–180 μM. The limits of detection were 11.3, 29.2, and 46.0 nM for tap water, pond water, and iron supplement tablets, respectively. Owing to its facile and sensitive nature, this assay method for Fe3+ ions can be applied to the analysis of drinking water and pharmaceutical samples.

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

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

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

  16. High Energy Cutting and Stripping Utilizing Liquid Nitrogen

    Science.gov (United States)

    Hume, Howard; Noah, Donald E.; Hayes, Paul W.

    2005-01-01

    The Aerospace Industry has endeavored for decades to develop hybrid materials that withstand the rigors of mechanized flight both within our atmosphere and beyond. The development of these high performance materials has led to the need for environmentally friendly technologies for material re-work and removal. The NitroJet(TM) is a fluid jet technology that represents an evolution of the widely used, large-scale water jet fluid jet technology. It involves the amalgamation of fluid jet technology and cryogenics technology to create a new capability that is applicable where water jet or abrasive jet (water jet plus entrained abrasive) are not suitable or acceptable because of technical constraints such as process or materials compatibility, environmental concerns and aesthetic or legal requirements. The NitroJet(TM) uses ultra high-pressure nitrogen to cut materials, strip numerous types of coatings such as paint or powder coating, clean surfaces and profile metals. Liquid nitrogen (LN2) is used as the feed stream and is pressurized in two stages. The first stage pressurizes sub cooled LN2 to an intermediate pressure of between 15,000 and 20,000 psi at which point the temperature of the LN2 is about -250 F. The discharge from this stage is then introduced as feed to a dual intensifier system, which boosts the pressure from 15,000 - 20,000 psi up to the maximum operating pressure of 55,000 psi. A temperature of about -220 F is achieved at which point the nitrogen is supercritical. In this condition the nitrogen cuts, strips and abrades much like ultra high-pressure water would but without any residual liquid to collect, remove or be contaminated. Once the nitrogen has performed its function it harmlessly flashes back into the atmosphere as pure nitrogen gas. The system uses heat exchangers to control and modify the temperature of the various intake and discharge nitrogen streams. Since the system is hydraulically operated, discharge pressures can be easily varied over

  17. Accuracy Enhancement of Inertial Sensors Utilizing High Resolution Spectral Analysis

    Directory of Open Access Journals (Sweden)

    Michael Korenberg

    2012-08-01

    Full Text Available In both military and civilian applications, the inertial navigation system (INS and the global positioning system (GPS are two complementary technologies that can be integrated to provide reliable positioning and navigation information for land vehicles. The accuracy enhancement of INS sensors and the integration of INS with GPS are the subjects of widespread research. Wavelet de-noising of INS sensors has had limited success in removing the long-term (low-frequency inertial sensor errors. The primary objective of this research is to develop a novel inertial sensor accuracy enhancement technique that can remove both short-term and long-term error components from inertial sensor measurements prior to INS mechanization and INS/GPS integration. A high resolution spectral analysis technique called the fast orthogonal search (FOS algorithm is used to accurately model the low frequency range of the spectrum, which includes the vehicle motion dynamics and inertial sensor errors. FOS models the spectral components with the most energy first and uses an adaptive threshold to stop adding frequency terms when fitting a term does not reduce the mean squared error more than fitting white noise. The proposed method was developed, tested and validated through road test experiments involving both low-end tactical grade and low cost MEMS-based inertial systems. The results demonstrate that in most cases the position accuracy during GPS outages using FOS de-noised data is superior to the position accuracy using wavelet de-noising.

  18. Lateral flow immunoassay for diagnosis of Trypanosoma cruzi infection with high correlation to the radioimmunoprecipitation assay.

    Science.gov (United States)

    Houghton, Raymond L; Stevens, Yvonne Y; Hjerrild, Kathryn; Guderian, Jeff; Okamoto, Masahiko; Kabir, Mazbahul; Reed, Steven G; Leiby, David A; Morrow, W John W; Lorca, Myriam; Raychaudhuri, Syamal

    2009-04-01

    The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) "gold standard" in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a

  19. High-resolution field shaping utilizing a masked multileaf collimator.

    Science.gov (United States)

    Williams, P C; Cooper, P

    2000-08-01

    Multileaf collimators (MLCs) have become an important tool in the modern radiotherapy department. However, the current limit of resolution (1 cm at isocentre) can be too coarse for acceptable shielding of all fields. A number of mini- and micro-MLCs have been developed, with thinner leaves to achieve approved resolution. Currently however, such devices are limited to modest field sizes and stereotactic applications. This paper proposes a new method of high-resolution beam collimation by use of a tertiary grid collimator situated below the conventional MLC. The width of each slit in the grid is a submultiple of the MLC width. A composite shaped field is thus built up from a series of subfields, with the main MLC defining the length of each strip within each subfield. Presented here are initial findings using a prototype device. The beam uniformity achievable with such a device was examined by measuring transmission profiles through the grid using a diode. Profiles thus measured were then copied and superposed to generate composite beams, from which the uniformity achievable could be assessed. With the average dose across the profile normalized to 100%, hot spots up to 5.0% and troughs of 3% were identified for a composite beam of 2 x 5.0 mm grids, as measured at Dmax for a 6 MV beam. For a beam composed from 4 x 2.5 mm grids, the maximum across the profile was 3.0% above the average, and the minimum 2.5% below. Actual composite profiles were also formed using the integrating properties of film, with the subfield indexing performed using an engineering positioning stage. The beam uniformity for these fields compared well with that achieved in theory using the diode measurements. Finally sine wave patterns were generated to demonstrate the potential improvements in field shaping and conformity using this device as opposed to the conventional MLC alone. The scalloping effect on the field edge commonly seen on MLC fields was appreciably reduced by use of 2 x 5.0 mm

  20. Direct Utilization of Coal Syngas in High Temperature Fuel Cells

    Energy Technology Data Exchange (ETDEWEB)

    Celik, Ismail B. [West Virginia University, Morgantown, WV (United States)

    2014-10-30

    This EPSCoR project had two primary goals: (i) to build infrastructure and work force at WVU to support long-term research in the area of fuel cells and related sciences; (ii) study effects of various impurities found in coal-syngas on performance of Solid Oxide Fuel Cells (SOFC). As detailed in this report the WVU research team has made significant accomplishments in both of these areas. What follows is a brief summary of these accomplishments: State-of-the-art test facilities and diagnostic tools have been built and put into use. These include cell manufacturing, half-cell and full-cell test benches, XPS, XRD, TEM, Raman, EDAX, SEM, EIS, and ESEM equipment, unique in-situ measurement techniques and test benches (Environmental EM, Transient Mass-Spectrometer-MS, and IR Optical Temperature measurements). In addition, computational capabilities have been developed culminating in a multi-scale multi-physics fuel cell simulation code, DREAM-SOFC, as well as a Beowulf cluster with 64 CPU units. We have trained 16 graduate students, 10 postdoctoral fellows, and recruited 4 new young faculty members who have actively participated in the EPSCoR project. All four of these faculty members have already been promoted to the tenured associate professor level. With the help of these faculty and students, we were able to secure 14 research awards/contracts amounting to a total of circa $5.0 Million external funding in closely related areas of research. Using the facilities mentioned above, the effects of PH3, HCl, Cl2, and H2S on cell performance have been studied in detail, mechanisms have been identified, and also remedies have been proposed and demonstrated in the laboratory. For example, it has been determined that PH3 reacts rapidly with Ni to from secondary compounds which may become softer or even melt at high temperature and then induce Ni migration to the surface of the cell changing the material and micro-structural properties of the cell drastically. It is found that

  1. Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

    Science.gov (United States)

    Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

    2015-08-01

    Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

  2. Perfect high throughput screening assay: a crucial technique for drug discovery

    Institute of Scientific and Technical Information of China (English)

    Guan-hua DU

    2005-01-01

    @@ Since being developed approximately 20 years ago, high throughput screening (HTS) has become one of the key techniques used in drug discovery[1]. However, three main problems are recognized with the use of HTS; namely, with the compound library, drug targets, and assay methods. Until now, the compound library has evolved based on the techniques of combinatorial chemistry and modern phytochemistry. Several functional proteins have emerged following the advance of genomics and proteomics. However,although many functional proteins have been discovered recently, they are not, as sometimes claimed, real drug targets;at best, they might be potential drug targets. The ideal targets selected for drug screening should qualify as drug targets[2]. The selection of targets for drug screening is a crucial procedure in drug screening.

  3. High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes.

    Science.gov (United States)

    Tuttle, R H; Hall, J E; Tidwell, R R

    1997-01-24

    Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4'-amidinophenoxy)-3-pentanol and 1,5-di-(4'-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.

  4. [Development of highly sensitive assay for detection of low serum level of PIVKA-II and its clinical usefulness].

    Science.gov (United States)

    Sakizono, K; Oita, T; Kuroda, M; Kasakura, S

    1996-09-01

    PIVKA-II is well known as a tumor marker of hepatocellular carcinoma. We describe the adaptations to render a commercially available PIVKA-II immunoassay kit (Eitest MONO P-II: Eisai Co., Ltd, Tokyo) more sensitive and evaluated clinical usefulness of this highly sensitive assay for early diagnosis of hepatocellular carcinoma. We measured serum PIVKA-II concentrations with the Eitest kit as described for the kit. To measure serum PIVKA-II concentrations below the sensitivity limit of the Eitest kit (0.0625AU/ml), linearity was determined using a series of dilutions of standard antigen diluted in standard diluent, including PIVKA-II concentrations of 0.002, 0.004, 0.008, 0.016, 0.031, 0.0625AU/ml. The assay was found to be liner from 0.004AU/ml. In this way, one can detect as low PIVKA-II concentrations as 0.004AU/ml. The repeatability-assay coefficients of variation (CV%) obtained from sixteen repeated assays of four sera were distributed between 3.63 and 18.75% and the reproducibility-assay coefficients of variation (CV%) obtained from ten repeated assays of four sera were distributed between 6.27 and 14.04%. We determined the serial changes in serum PIVKA-II levels of two patients with hepatocellular carcinoma after the resection of hepatic tumor. In these patients, elevation of serum PIVKA-II level determined by the highly sensitive assay preceded the detection of relapse by imaging diagnostic procedures. In summary, the assay system we developed has good accuracy and reproducibility for assaying low concentrations of PIVKA-II in serum and is suitable for detecting small increases in PIVKA-II concentrations. This assay system may be useful to earlier detect a relapse of hepatocellular carcinoma.

  5. High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

    Directory of Open Access Journals (Sweden)

    Du Yuefen

    2003-05-01

    Full Text Available Abstract Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA. Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

  6. Rapid and High-throughout Identification of Recombinant Bacteria with Mass Spectrometry Assay

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques were used to identify 12 recombinant proteins (10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori). A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities: RC=100%, mean CVA=98.7% (the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively) and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks (9, 16, 9, and 14 for the four stages, respectively) was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

  7. The modified high-density survival assay is the useful tool to predict the effectiveness of fractionated radiation exposure.

    Science.gov (United States)

    Kuwahara, Yoshikazu; Mori, Miyuki; Oikawa, Toshiyuki; Shimura, Tsutomu; Ohtake, Yosuke; Mori, Shiro; Ohkubo, Yasuhito; Fukumoto, Manabu

    2010-01-01

    The high-density survival (HDS) assay was originally elaborated to assess cancer cell responses to therapeutic agents under the influence of intercellular communication. Here, we simplified the original HDS assay and studied its applicability for the detection of cellular radioresistance. We have recently defined clinically relevant radioresistant (CRR) cells, which continue to proliferate with daily exposure to 2 gray (Gy) of X-rays for more than 30 days in vitro. We established human CRR cell lines, HepG2-8960-R from HepG2, and SAS-R1 and -R2 from SAS, respectively. In an attempt to apply the HDS assay to detect radioresistance with clinical relevance, we simplified the original HDS assay by scoring the total number of surviving cells after exposure to X-rays. The modified HDS assay successfully detected radioresistance with clinical relevance. The modified HDS assay detected CRR phenotype, which is not always detectable by clonogenic assay. Therefore, we believe that the modified HDS assay presented in this study is a powerful tool to predict the effectiveness of fractionated radiotherapy against malignant tumors.

  8. Adapting high-throughput screening methods and assays for biocontainment laboratories.

    Science.gov (United States)

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

    2015-01-01

    High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories.

  9. [Characteristics of dry matter production and nitrogen accumulation in barley genotypes with high nitrogen utilization efficiency].

    Science.gov (United States)

    Huang, Yi; Li, Ting-Xuan; Zhang, Xi-Zhou; Ji, Lin

    2014-07-01

    A pot experiment was conducted under low (125 mg x kg-1) and normal (250 mg x kg(-1)) nitrogen treatments. The nitrogen uptake and utilization efficiency of 22 barley cultivars were investigated, and the characteristics of dry matter production and nitrogen accumulation in barley were analyzed. The results showed that nitrogen uptake and utilization efficiency were different for barley under two nitrogen levels. The maximal values of grain yield, nitrogen utilization efficiency for grain and nitrogen harvest index were 2.87, 2.91 and 2.47 times as those of the lowest under the low nitrogen treatment. Grain yield and nitrogen utilization efficiency for grain and nitrogen harvest index of barley genotype with high nitrogen utilization efficiency were significantly greater than low nitrogen utilization efficiency, and the parameters of high nitrogen utilization efficiency genotype were 82.1%, 61.5% and 50.5% higher than low nitrogen utilization efficiency genotype under the low nitrogen treatment. Dry matter mass and nitrogen utilization of high nitrogen utilization efficiency was significantly higher than those of low nitrogen utilization efficiency. A peak of dry matter mass of high nitrogen utilization efficiency occurred during jointing to heading stage, while that of nitrogen accumulation appeared before jointing. Under the low nitrogen treatment, dry matter mass of DH61 and DH121+ was 34.4% and 38.3%, and nitrogen accumulation was 54. 8% and 58.0% higher than DH80, respectively. Dry matter mass and nitrogen accumulation seriously affected yield before jointing stage, and the contribution rates were 47.9% and 54.7% respectively under the low nitrogen treatment. The effect of dry matter and nitrogen accumulation on nitrogen utilization efficiency for grain was the largest during heading to mature stages, followed by sowing to jointing stages, with the contribution rate being 29.5% and 48.7%, 29.0% and 15.8%, respectively. In conclusion, barley genotype with high

  10. High Gain and High Directive of Antenna Arrays Utilizing Dielectric Layer on Bismuth Titanate Ceramics

    Directory of Open Access Journals (Sweden)

    F. H. Wee

    2012-01-01

    Full Text Available A high gain and high directive microstrip patch array antenna formed from dielectric layer stacked on bismuth titanate (BiT ceramics have been investigated, fabricated, and measured. The antennas are designed and constructed with a combination of two-, four-, and six-BiT elements in an array form application on microwave substrate. For gain and directivity enhancement, a layer of dielectric was stacked on the BiT antenna array. We measured the gain and directivity of BiT array antennas with and without the dielectric layer and found that the gain of BiT array antenna with the dielectric layer was enhanced by about 1.4 dBi of directivity and 1.3 dB of gain over the one without the dielectric layer at 2.3 GHz. The impedance bandwidth of the BiT array antenna both with and without the dielectric layer is about 500 MHz and 350 MHz, respectively, which is suitable for the application of the WiMAX 2.3 GHz system. The utilization of BiT ceramics that covers about 90% of antenna led to high radiation efficiency, and small-size antennas were produced. In order to validate the proposed design, theoretical and measured results are provided and discussed.

  11. Large-Area High Aspect Ratio Plasmonic Interference Lithography Utilizing a Single High-k Mode.

    Science.gov (United States)

    Chen, Xi; Yang, Fan; Zhang, Cheng; Zhou, Jing; Guo, L Jay

    2016-04-26

    Plasmonic lithography, which utilizes subwavelength confinement of surface plasmon polartion (SPP) waves, has the capability of breaking the diffraction limit and delivering high resolution. However, all previously reported results suffer from critical issues, such as shallow pattern depth and pattern nonuniformity even over small exposure areas, which limit the application of the technology. In this work, periodic patterns with high aspect ratios and a half-pitch of about 1/6 of the wavelength were achieved with pattern uniformity in square centimeter areas. This was accomplished by designing a special mask and photoresist (PR) system to select a single high spatial frequency mode and incorporating the PR into a waveguide configuration to ensure uniform light exposure over the entire depth of the photoresist layer. In addition to the experimental progress toward large-scale applications of plasmonic interference lithography, the general criteria of designing such an exposure system is also discussed, which can be used for nanoscale fabrication in this fashion for various applications with different requirements for wavelength, pitch, aspect ratio, and structure.

  12. A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays.

    Science.gov (United States)

    Sandén, Emma; Eberstål, Sofia; Visse, Edward; Siesjö, Peter; Darabi, Anna

    2015-01-01

    In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.

  13. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    Science.gov (United States)

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  14. A high-throughput pipeline for designing microarray-based pathogen diagnostic assays

    Directory of Open Access Journals (Sweden)

    Reifman Jaques

    2008-04-01

    Full Text Available Abstract Background We present a methodology for high-throughput design of oligonucleotide fingerprints for microarray-based pathogen diagnostic assays. The oligonucleotide fingerprints, or DNA microarray probes, are designed for identifying target organisms in environmental or clinical samples. The design process is implemented in a high-performance computing software pipeline that incorporates major algorithmic improvements over a previous version to both reduce computation time and improve specificity assessment. Results The algorithmic improvements result in significant reduction in runtimes, with the updated pipeline being nearly up to five-times faster than the previous version. The improvements in specificity assessment, based on multiple specificity criteria, result in robust and consistent evaluation of cross-hybridization with nontarget sequences. In addition, the multiple criteria provide finer control on the number of resulting fingerprints, which helps in obtaining a larger number of fingerprints with high specificity. Simulation tests for Francisella tularensis and Yersinia pestis, using a well-established hybridization model to estimate cross-hybridization with nontarget sequences, show that the improved specificity criteria yield a larger number of fingerprints as compared to using a single specificity criterion. Conclusion The faster runtimes, achieved as the result of algorithmic improvements, are critical for extending the pipeline to process multiple target genomes. The larger numbers of identified fingerprints, obtained by considering broader specificity criteria, are essential for designing probes for hard-to-distinguish target sequences.

  15. Utilizing High Resolution Panoramas as Virtual Field Experiences in Undergraduate Planetary Science Courses

    Science.gov (United States)

    Piatek, J. L.

    2011-03-01

    Tier-scalable exercises utilizing high resolution panoramas allow students to explore terrestrial landscapes and outcrops virtually, and provide good context for interpreting spacecraft images of solar system bodies.

  16. Pharmacokinetics of mitomycin C in dogs: application of a high-performance liquid chromatographic assay.

    Science.gov (United States)

    Barbhaiya, R H; Papp, E A; Van Harken, D R; Smyth, R D

    1984-09-01

    A normal-phase high-performance liquid chromatographic (HPLC) assay was developed for the determination of mitomycin C in plasma and urine. The method involves extraction of mitomycin C from plasma or urine into ethyl acetate-2-propanol-chloroform (70:15:15) with UV detection at 365 nm. Quantitation was performed with an internal standard (porfiromycin) by the peak height ratio method. Excellent correlation was obtained between the HPLC assay and the established microbiological cup-plate bioassay. The pharmacokinetics of mitomycin C were investigated in beagle dogs following a 1-mg/kg iv (22-mg/m2) bolus dose. The plasma mitomycin C concentration versus time data were analyzed by using an open three-compartment model. The average volume of distribution was 1.90 L or 17% of body weight for the central compartment and 7.7 L or 68% of body weight for the terminal elimination phase. The volumes of distribution at steady state, calculated by model-dependent and -independent methods, compared very well with each other and were 6.5 L or 58% of body weight. Total body clearance averaged 112 mL/min, and the mean terminal plasma half-life was 53 min. The 0-24-h urinary excretion of intact mitomycin C accounted for 19% of the dose. The terminal half-life and percent urinary recovery of mitomycin C in dogs is similar to that in humans. Based on these observations, the dog appears to be a good model for studying the disposition of mitomycin C.

  17. A high throughput screening assay system for the identification of small molecule inhibitors of gsp.

    Directory of Open Access Journals (Sweden)

    Nisan Bhattacharyya

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

  18. One-step seeding of neural stem cells with vitronectin-supplemented medium for high throughput screening assays

    Science.gov (United States)

    Dai, Sheng; Li, Rong; Long, Yan; Titus, Steve; Zhao, Jinghua; Huang, Ruili; Xia, Menghang; Zheng, Wei

    2017-01-01

    Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research. PMID:27647668

  19. Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan.

    Science.gov (United States)

    Mizukami, Keijiro; Chang, Hye-Sook; Yabuki, Akira; Kawamichi, Takuji; Kawahara, Natsuko; Hayashi, Daisuke; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Yamato, Osamu

    2011-11-01

    Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR primer-induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.

  20. Cytochrome P450 2C24: expression, tissue distribution, high-throughput assay, and pharmacological inhibition

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2012-04-01

    Full Text Available Cytochrome P450 (CYP-mediated epoxidation of arachidonic acid (AA contributes to important biological functions, including the pain-relieving responses produced by analgesic drugs. However, the relevant epoxygenase(s remain unidentified. Presently, we describe the tissue distribution, high-throughput assay, and pharmacological characteristics of the rat epoxygenase CYP2C24. Following cloning from male rat liver, recombinant baculovirus containing the C-terminal His-tagged cDNA was constructed and used to express the protein in Spodoptera frugiperda (Sf9 cells. Enzymatic activity was detected with membranes, NADPH regenerating system and CYP reductase, and optimized for high throughput screening by use of the Vivid Blue© BOMCC fluorescence substrate. Quantitative real-time PCR identified CYP2C24 m-RNA in liver, kidney, heart, lung, gonad and brain. Screening of CYP2C24 activity against a panel of inhibitors showed a very strong correlation with activity against the human homologue CYP2C19. In agreement with recent findings on CYP2C19, the epoxygenase blockers PPOH and MS-PPOH inhibited CYP2C24 only weakly, confirming that these drugs are not universal epoxygenase inhibitors. Finally, comparisons of the CYP2C24 inhibitor profile with anti-analgesic activity suggests that this isoform does not contribute to brain analgesic drug action. The present methods and pharmacological data will aid in study of the biological significance of this CYP isoform.

  1. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    Science.gov (United States)

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D

    2007-04-20

    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  2. Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

    NARCIS (Netherlands)

    Wang, S.; Aarts, J.M.M.J.G.; Evers, N.M.; Peijnenburg, A.A.C.M.; Rietjens, I.; Bovee, T.F.H.

    2012-01-01

    Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or

  3. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    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.

  4. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.

    2012-01-01

    present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell...... populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important...

  5. Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms

    Directory of Open Access Journals (Sweden)

    Pettit George R

    2009-10-01

    Full Text Available Abstract Background Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms. Methods Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt®, exposed to the oxidation reduction indicator Alamar blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC was defined as ≤ 50% Alamar blue reduction and a purple/blue well 60 min after the addition of Alamar blue. Because all of the approved antibiotics had MBICs >128 μg/ml (most >2048 μg/ml, lysostaphin and Conflikt®, with relatively low MBICs, were used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide (XTT reduction and viable counts (CFU/ml for S. aureus ATCC 29213 and three clinical isolates. Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. Results Overall, Alamar blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or Conflikt®, Alamar blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99 and with CFU/ml (r = 0.92-0.98. For one of the clinical isolates, the results were moderately correlated for Conflikt® (r = 0.76, Alamar blue vs. XTT; r = 0.81, Alamar blue vs. CFU/ml and had excellent correlation for lysostaphin (r = 0.95, Alamar blue vs. XTT; r = 0.97, Alamar blue vs. CFU/ml. Conclusion A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to S. aureus biofilms. The described method

  6. A high throughout assay for human papillomavirus genotypes with fluorescence polarization

    Institute of Scientific and Technical Information of China (English)

    张菊; 闫小君; 孙建中; 陈中灿; 高艳娥; 白玉杰; 刘智广

    2003-01-01

    Objective To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA. Methods Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence. Results Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B,11 and 16 (4), HPV11,16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11,16 and 18 (1)] and one HPV quadruple infection (HPV6B,11,16 and 18) were detected in cervical cancer scrapes.Conclusions The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV

  7. A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

    Science.gov (United States)

    Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

    2016-07-03

    Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

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

    Science.gov (United States)

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

    2015-07-01

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

  9. Photo- and biophysical studies of lectin-conjugated fluorescent nanoparticles: reduced sensitivity in high density assays.

    Science.gov (United States)

    Wang, Yaqi; Gildersleeve, Jeffrey C; Basu, Amit; Zimmt, Matthew B

    2010-11-18

    Lectin-conjugated, fluorescent silica nanoparticles (fNP) have been developed for carbohydrate-based histopathology evaluations of epithelial tissue biopsies. The fNP platform was selected for its enhanced emissive brightness compared to direct dye labeling. Carbohydrate microarray studies were performed to compare the carbohydrate selectivity of the mannose-recognizing lectin Concanavalin A (ConA) before and after conjugation to fluorescent silica nanoparticles (ConA-fNP). These studies revealed surprisingly low emission intensities upon staining with ConA-fNP compared to those with biotin-ConA/Cy3-streptavidin staining. A series of photophysical and biophysical characterizations of the fNP and ConA-fNP conjugates were performed to probe the low sensitivity from fNP in the microarray assays. Up to 1200 fluorescein (FL) and 80 tetramethylrhodamine (TR) dye molecules were incorporated into 46 nm diameter fNP, yielding emissive brightness values 400 and 35 times larger than the individual dye molecules, respectively. ConA lectin conjugated to carboxylic acid surface-modified nanoparticles covers 15-30% of the fNP surface. The CD spectra and mannose substrate selectivity of ConA conjugated to the fNP differed slightly compared to that of soluble ConA. Although, the high emissive brightness of fNP enhances detection sensitivity for samples with low analyte densities, large fNP diameters limit fNP recruitment and binding to samples with high analyte densities. The high analyte density and nearly two-dimensional target format of carbohydrate microarrays make probe size a critical parameter. In this application, fNP labels afford minimal sensitivity advantage compared to direct dye labeling.

  10. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  11. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2008-01-01

    Aim:To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs.Methods:The donor DNA duplex,with a sequence identical to the U5 end of HIV-1 long terminal repeats,is labeled at its 5' end with biotin (BIO).The target DNA duplex is labeled at its 3' end with digoxin (DIG).IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product.Streptavidin-coated magnetic beads were used to capture the product,and the amount of DIG was measured as the ST reaction product.The assay was optimized in 96-well microplate format for high-throughput screening purpose.Moreover,the assay was applied in a ST reaction character study,and the efficiency of the assay in the identification of antiviral compounds was tested.Results:The end-point values,measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings.The ST reaction char-acter and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays.The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9.Conclusion:The assay presented here has been proven to be rapid,sensitive,and specific for the detection of IN ST activity,the reaction character study,as well as for the identification of antiviral drugs targeting IN.

  12. A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

    Directory of Open Access Journals (Sweden)

    Mindy I Davis

    Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

  13. HUITWU:An Efficient Algorithm for High-Utility Itemset Mining in Transaction Databases

    Institute of Scientific and Technical Information of China (English)

    Shi-Ming Guo; Hong Gao

    2016-01-01

    Mining high-utility itemsets (HUIs) from a transaction database refers to the discovery of itemsets with high utilities like profits. Most of existing studies discover HUIs from a transaction database in two phases. In phase 1, different overestimation methods are applied to calculate the upper bounds of the utilities of itemsets. Since the overestimated utilities of itemsets are adopted, the itemsets whose overestimated utilities are no less than a user-specified threshold are selected as candidate HUIs, and they are verified by scanning the database one more time in phase 2. However, a large number of candidate HUIs incur two problems: 1) it requires excessive memory to store these candidates;2) it needs a large amount of running time to calculate their exact utilities. Vertical data format has been applied to mine HUIs recently. However this kind of method cannot deal with transactions with the same items effectively so that the size of database cannot be reduced sufficiently. The overall performance of algorithms is degraded consequently. Thus an algorithm HUITWU is proposed in this paper for mining HUIs. A novel data structure HUITWU-Tree is adopted to efficiently calculate the utilities of itemsets in a database. Extensive studies with both sparse and dense datasets have demonstrated that our proposed algorithm is more than an order of magnitude faster and consumes less memory than the state-of-the-art algorithms.

  14. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay.

    Science.gov (United States)

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung

    2014-09-01

    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm(2). A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times.

  15. Research Activity in Computational Physics utilizing High Performance Computing: Co-authorship Network Analysis

    Science.gov (United States)

    Ahn, Sul-Ah; Jung, Youngim

    2016-10-01

    The research activities of the computational physicists utilizing high performance computing are analyzed by bibliometirc approaches. This study aims at providing the computational physicists utilizing high-performance computing and policy planners with useful bibliometric results for an assessment of research activities. In order to achieve this purpose, we carried out a co-authorship network analysis of journal articles to assess the research activities of researchers for high-performance computational physics as a case study. For this study, we used journal articles of the Scopus database from Elsevier covering the time period of 2004-2013. We extracted the author rank in the physics field utilizing high-performance computing by the number of papers published during ten years from 2004. Finally, we drew the co-authorship network for 45 top-authors and their coauthors, and described some features of the co-authorship network in relation to the author rank. Suggestions for further studies are discussed.

  16. Commutability of the CRM 470 C-reactive protein value in the Dade Behring N High Sensitivity CRP assay.

    Science.gov (United States)

    Johnson, A Myron; Ledue, Thomas B; Collins, Marilyn F

    2003-02-01

    Certified Reference Material 470 (CRM 470) demonstrates commutability with both the manufacturer's calibrator and with dilutions of serum pools in the Dade Behring N High Sensitivity assay for C-reactive protein (CRP). Both regression and back calibration show similar nonlinearity for all materials, largely due to the method of calibration curve fitting used in this assay. Significant differences in values among the currently available commercial assays can be largely overcome by using appropriate calibration curve fitting and the recommended value transfer protocol, which includes a minimum of two assay runs on each of at least 3 separate days, with weight correction of all reconstitutions and dilutions. An initial weight-corrected dilution should be made each day because of the relatively high level of CRP in CRM 470. In our opinion, the degree of nonlinearity, imprecision, and differences in values in currently available assays renders the use of fixed clinical decision cut-points questionable for high-sensitivity CRP. An alternative approach is suggested.

  17. Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: "The Worminator".

    Science.gov (United States)

    Storey, Bob; Marcellino, Chris; Miller, Melissa; Maclean, Mary; Mostafa, Eman; Howell, Sue; Sakanari, Judy; Wolstenholme, Adrian; Kaplan, Ray

    2014-12-01

    A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the "Worminator" system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application "WormAssay", developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic) parasites (e.g. Brugia malayi). We adapted this assay for use with microscopic parasites by modifying the software to support a full frame analysis mode that applies the motion algorithm to the entire video frame. Thus, the motility of all parasites in a given well are recorded and measured simultaneously. Assays performed on third-stage larvae (L3) of the bovine intestinal nematode Cooperia spp., as well as microfilariae (mf) of the filarioid nematodes B. malayi and Dirofilaria immitis, yielded reproducible dose responses using the macrocyclic lactones ivermectin, doramectin, and moxidectin, as well as the nicotinic agonists, pyrantel, oxantel, morantel, and tribendimidine. This new computer based-assay is simple to use, requires minimal new investment in equipment, is robust across nematode genera and developmental stage, and does not require subjective scoring of motility by an observer. Thus, the "Worminator" provides a relatively low-cost platform for developing genera- and stage-specific assays with high efficiency and reproducibility, low labor input

  18. Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format

    Science.gov (United States)

    Davis, Mindy I.; Sasaki, Atsuo T.; Simeonov, Anton

    2015-01-01

    Summary ipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we describe a method to prepare a DMSO-based lipid mixture that enables the 1536-well screening of the lipid kinase phosphatidylinositol-5-phosphate 4-kinase α (PI5P4Kα) utilizing the D-myo-di16-PtIns(5)P substrate in quantitative high-throughput screening (qHTS) format using the ADP-Glo™ technology to couple the production of ADP to a bioluminescent readout. PMID:26552670

  19. INFLUENCE OF STUDENT ENGLISH UTILITY AND TEACHER EFFICACY ON ENGLISH PROFICIENCY OF HIGH SCHOOL STUDENTS

    Directory of Open Access Journals (Sweden)

    Ruth A. ORTEGA-DELA CRUZ

    2016-12-01

    Full Text Available Learning second language considers a number of factors that influence the manner in which the language is taught. Understanding of the learners’ goals and motivation for learning is one. Using descriptive-correlational research design, this study determined the influence of student English utility and teacher efficacy on the students’ English proficiency. A total of 101 students from first year to fourth year level served as the respondents of the study. The study quantified the students’ perception towards English utility and their evaluation of English teacher efficacy which employed a researcher-made survey questionnaire. Results revealed high positive perceptions of students towards English utility. Analysis of variance revealed significant differences in the perceptions of high school students on the efficacy of their English teachers. Correlation coefficients indicated a positive linear relationship among the given variables. The p-value revealed significant relationship of teacher efficacy (r = .691, p-value = .000 and English utility (r = .467, p-value = .000 to students’ English proficiency. Results of regression statistics revealed that English utility has no significant influence on the student English proficiency. Therefore, the main factor that must still be considered then should be the teacher. Finally, there is an explicit indication that high level of teachers’ efficacy performing in teaching has much powerful influence on the English proficiency of high school students. Thus improving the methods of teaching English provides a better way of motivating students to achieve higher levels of proficiency in the future.

  20. Design and analysis of experiments with high throughput biological assay data.

    Science.gov (United States)

    Rocke, David M

    2004-12-01

    The design and analysis of experiments using gene expression microarrays is a topic of considerable current research, and work is beginning to appear on the analysis of proteomics and metabolomics data by mass spectrometry and NMR spectroscopy. The literature in this area is evolving rapidly, and commercial software for analysis of array or proteomics data is rarely up to date, and is essentially nonexistent for metabolomics data. In this paper, I review some of the issues that should concern any biologists planning to use such high-throughput biological assay data in an experimental investigation. Technical details are kept to a minimum, and may be found in the referenced literature, as well as in the many excellent papers which space limitations prevent my describing. There are usually a number of viable options for design and analysis of such experiments, but unfortunately, there are even more non-viable ones that have been used even in the published literature. This is an area in which up-to-date knowledge of the literature is indispensable for efficient and effective design and analysis of these experiments. In general, we concentrate on relatively simple analyses, often focusing on identifying differentially expressed genes and the comparable issues in mass spectrometry and NMR spectroscopy (consistent differences in peak heights or areas for example). Complex multivariate and pattern recognition methods also need much attention, but the issues we describe in this paper must be dealt with first. The literature on analysis of proteomics and metabolomics data is as yet sparse, so the main focus of this paper will be on methods devised for analysis of gene expression data that generalize to proteomics and metabolomics, with some specific comments near the end on analysis of metabolomics data by mass spectrometry and NMR spectroscopy.

  1. Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.

    Science.gov (United States)

    de Cózar, Cristina; Caballero, Iván; Colmenarejo, Gonzalo; Sanz, Laura M; Álvarez-Ruiz, Emilio; Gamo, Francisco-Javier; Cid, Concepción

    2016-10-01

    The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.

  2. High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic vivid substrates.

    Science.gov (United States)

    Marks, Bryan D; Goossens, Tony A; Braun, Heidi A; Ozers, Mary S; Smith, Ronald W; Lebakken, Connie; Trubetskoy, Olga V

    2003-01-01

    CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

  3. A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays

    Directory of Open Access Journals (Sweden)

    Linda G. Lee

    2013-10-01

    Full Text Available We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting.

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

    Science.gov (United States)

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

  5. A highly sensitive quantitative real-time PCR assay for determination of mutant JAK2 exon 12 allele burden.

    Directory of Open Access Journals (Sweden)

    Lasse Kjær

    Full Text Available Mutations in the Janus kinase 2 (JAK2 gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.

  6. Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence.

    Science.gov (United States)

    Lorenz, Natalie; Loh, Jacelyn M S; Moreland, Nicole J; Proft, Thomas

    2017-03-01

    The lack of standardised protocols for the assessment of functional antibodies has hindered Streptococcus pyogenes research and the development of vaccines. A robust, high throughput opsonophagocytic bactericidal assay to determine protective antibodies in human and rabbit serum has been developed that utilises bioluminescence as a rapid read out.

  7. Mobile natural gas leak surveys indicate that two utilities have high false negative rates

    Science.gov (United States)

    von Fischer, J. C.; Brewer, P. E.; Chamberlain, S.; Gaylord, A.; von Fischer, J.

    2016-12-01

    In the distribution systems that carry natural gas to consumers, leaks need to be discovered for safety reasons and to reduce greenhouse gas emissions. However, few utilities have adopted newer laser-based technologies that have greater sensitivity and precision, and instead rely on "industry standard" equipment that is far less sensitive. In partnership with the Environmental Defense Fund and Google, we mapped natural gas leaks in the domains of two anonymous utilities (Utility "A" and "B") using high sensitivity Picarro methane analyzers in Google Street View Cars. Surprisingly, when we shared these results with utilities, their survey crews were unable to find most of the leaks that our survey indicated (84% in A and 80% in B). To investigate this phenomenon, our team visited a subset of the leaks in each utility domain (n=32 in A and n=30 in B), and worked alongside utility surveyors to search the leak indication area, using a Los Gatos Research ultraportable methane analyzer to pinpoint leak locations. We found evidence of natural gas leaks at 69% and 68% of the locations in Utilities A and B respectively where survey crews had found nothing. We describe this as a "false negative" rate for the utility because the utility survey falsely indicated that there was no leak at these locations. Of these false negatives, 7% (n=2 of 32 in A, n=2 of 30 in B) were determined to be Grade 1 leaks requiring immediate repair due to high safety risk. Instrument sensitivity appears to explain some of the false negative rates. In particular, use of some industry standard equipment appears to have created a false sense of confidence among utility surveyors that leaks were not present. However, there was also evidence of communication failures and that surveyors did not use optimal approaches in their search. Based on these findings, we suggest that: 1) mobile deployment of high-precision methane analyzers can help find more natural gas leaks, and 2) use of some hand-held survey

  8. Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-01-01

    Full Text Available Abstract A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM in tablets. The formation of a colored charge-transfer (CT complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml-1. The limits of detection and quantitation were 0.3 and 1 μg ml-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

  9. Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens.

    Science.gov (United States)

    Saponari, Maria; Loconsole, Giuliana; Liao, Hui-Hong; Jiang, Bo; Savino, Vito; Yokomi, Raymond K

    2013-11-01

    A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for 'Candidatus Liberibacter asiaticus' (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series

  10. The clinical utility of tuberculin skin test and interferon-γ release assay in the diagnosis of active tuberculosis among young adults: a prospective observational study

    Directory of Open Access Journals (Sweden)

    Lee Sei Won

    2011-04-01

    Full Text Available Abstract Background The roles of the tuberculin skin test (TST and QuantiFERON®-TB Gold In-Tube assay (QFT-IT in the diagnosis of active tuberculosis (TB are not clear in young adults. We evaluated the diagnostic accuracy of the TST and QFT-IT in smear-negative TB among young adults with no underlying disease. Methods We prospectively enrolled 166 young participants 20-29 years of age with suspected active TB in a military hospital of South Korea. The TST and QFT-IT were performed for all participants. Results Of the 143 patients included in the analysis, active TB was diagnosed in 100 (69.9%. There were 141 male patients, none of whom had immunosuppressive disease. The sensitivity, specificity, positive predictive value (PPV, and negative predictive value (NPV of TST were 94% (95% CI, 87-98%, 88% (95% CI, 74-96%, 95% (95% CI, 88-98%, and 86% (95% CI, 72-94%, respectively. The sensitivity, specificity, PPV, and NPV of the QFT-IT were 93% (95% CI, 86-97%, 95% (95% CI, 81-99%, 98% (95% CI, 92-99%, and 84% (95% CI, 69-93%, respectively. No significant differences were found between the TST and QFT-IT in any statistic. Conclusions Both the TST and QFT-IT showed high sensitivity and specificity in differentiating active TB from other diseases. The diagnostic accuracy of these two tests did not differ significantly when applied to this clinical population of young, immunocompetent adults in whom neonatal BCG vaccination was common, there was no history of previous TB and in whom suspicion of TB was high. Trial registration ClinicalTrials.gov: NCT00982969

  11. Utility value of a T-cell interferon-γ release assay based on recombinant Mycobacterium tuberculosis 11kD protein in the diagnosis of tuberculosis

    Institute of Scientific and Technical Information of China (English)

    张丽帆

    2014-01-01

    Objective To evaluate the diagnostic efficiency of a T-cell interferon-γrelease assay based on recombinant Mycobacterium tuberculosis(MTB)11kD protein for diagnosing tuberculosis.Methods This prospective study enrolled inpatients with suspected tuberculosis at PUMCH to examine the diagnostic sensitivity,specificity,predictive value(PV)and likelihood ratio(LR)of T-cell interferon-γrelease assays based on recombinant MTB-11kD

  12. Utility of a single nasal polymerase chain reaction assay in predicting absence of skin and environmental contamination in hospitalized patients with past methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Guerrero, Dubert M; Wagner, Matthew; Carson, Grace; Hanish, Christine; Thompson, Jody; Orr, Megan; Roth, Felix; Carson, Paul J

    2016-06-01

    We evaluated hospitalized patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) for persistent colonization and need for contact precautions. Up to 3 daily cultures of nares, skin, and any present wounds were compared with a single nasal polymerase chain reaction (PCR) assay. Most patients (76.2%) were no longer colonized with MRSA. A single PCR assay was sufficient to exclude persistent colonization and environmental contamination and remove the contact precautions.

  13. Development of a high-throughput colorimetric Zika virus infection assay.

    Science.gov (United States)

    Müller, Janis A; Harms, Mirja; Schubert, Axel; Mayer, Benjamin; Jansen, Stephanie; Herbeuval, Jean-Philippe; Michel, Detlef; Mertens, Thomas; Vapalahti, Olli; Schmidt-Chanasit, Jonas; Münch, Jan

    2017-04-01

    Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.

  14. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two...

  15. Selection for high levamisole resistance in Haemonchus contortus monitored with an egg-hatch assay

    NARCIS (Netherlands)

    Hoekstra, R.; Borgsteede, F.H.M.; Boersema, J.H.; Roos, M.H.

    1997-01-01

    To investigate the characteristics of selection for levamisole resistance in Haemonchus contortus, the consecutive nematode generations of an in vivo selection were monitored with a newly developed egg-hatch assay. The in vivo selection was started with a population not previously exposed to any ant

  16. High-throughput micro plate vanillin assay for determination of tannin in sorghum grain

    Science.gov (United States)

    Sorghum tannins are phenolic compounds that offer health promoting antioxidant properties. The conventional HCl-vanillin assay for determining tannin content is a time-consuming method for screening large sample sets as seen in association mapping panels or breeder nursery samples. The objective of ...

  17. High-sensitive Troponin T assay for the diagnosis of acute myocardial infarction: An economic evaluation

    NARCIS (Netherlands)

    A. Vaidya (Anil); J.L. Severens (Hans); B.W.C. Bongaerts (Brenda); C.B.J.M. Cleutjens (Kitty); P. Nelemans (Patricia); L. Hofstra (Leo); M.P. van Dieijen-Visser (Marja); E.A.L. Biessen (Erik)

    2014-01-01

    markdownabstract__Abstract__ Background: Delayed diagnosis and treatment of Acute Myocardial Infarction (AMI) has a major adverse impact on prognosis in terms of both morbidity and mortality. Since conventional cardiac Troponin assays have a low sensitivity for diagnosing AMI in the first hours

  18. A highly sensitive cell assay for validation of purification regimes of alginates.

    Science.gov (United States)

    Leinfelder, U; Brunnenmeier, F; Cramer, H; Schiller, J; Arnold, K; Vásquez, J A; Zimmermann, U

    2003-10-01

    Among the hydrogels used for microencapsulation of cells and tissues, alginate has been and will continue to be one of the most important biomaterials. A mandatory requirement for clinical immunoisolated transplantations is the reproducible production of biocompatible alginate. As shown here for alginates extracted from freshly collected algal stipes, the current assays used for validation of the quality of the alginate are not sufficient to screen for impurities arising from spores of gram-positive bacteria (and related contaminants). To assess the quality of alginate, we have developed a cell assay based on the induction of apoptosis in Jurkat cells. This assay allows in combination with the "modified mixed lymphocyte" assay a rapid and sensitive screening for any fibrosis-inducing impurities in alginate samples (even during the purification regime) as demonstrated by transplantation experiments performed in parallel with BB rats (exhibiting an elevated macrophage activity). The results clearly demonstrate that the quality of the input algal material is of key relevance for the production of transplantation-grade alginate.

  19. High-sensitive Troponin T assay for the diagnosis of acute myocardial infarction: An economic evaluation

    NARCIS (Netherlands)

    A. Vaidya (Anil); J.L. Severens (Hans); B.W.C. Bongaerts (Brenda); C.B.J.M. Cleutjens (Kitty); P. Nelemans (Patricia); L. Hofstra (Leo); M.P. van Dieijen-Visser (Marja); E.A.L. Biessen (Erik)

    2014-01-01

    markdownabstract__Abstract__ Background: Delayed diagnosis and treatment of Acute Myocardial Infarction (AMI) has a major adverse impact on prognosis in terms of both morbidity and mortality. Since conventional cardiac Troponin assays have a low sensitivity for diagnosing AMI in the first hours aft

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through...

  1. 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.

  2. Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists

    Institute of Scientific and Technical Information of China (English)

    Rui ZHANG; Pang-ke YAN; Cai-hong ZHOU; Jia-yu LIAO; Ming-wei WANG

    2007-01-01

    Aim: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts, cAMP measurements were camed out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 recep-tor agonists following HTS of 16 000 samples. Results: EC50 values of the positive control compounds (β-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z' factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA1 receptor showing ≥20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screen- ing confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conclusion: A series of validation studies demonstrated that the homogeneous calcium mobili-zation assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation.

  3. A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    Full Text Available BACKGROUND: Pre-existing immunity to Vaccinia Tian Tan virus (VTT resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. METHODOLOGY/PRINCIPAL FINDINGS: A new anti-Vaccinia virus (VACV neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT. Using this assay, we found a low prevalence of NAb to VTT (7.6% in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. CONCLUSION: A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in

  4. A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

    Science.gov (United States)

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui

  5. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.

    Directory of Open Access Journals (Sweden)

    Windy A Boyd

    Full Text Available BACKGROUND: Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. METHODOLOGY/PRINCIPAL FINDINGS: Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants, and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50 of 2 microM. CONCLUSIONS/SIGNIFICANCE: The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or

  6. A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

    Science.gov (United States)

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

  7. Development of a Fluorescent Multiplex Assay for Detection of MSI-High Tumors

    Directory of Open Access Journals (Sweden)

    Jeffery W. Bacher

    2004-01-01

    of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.

  8. Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: “The Worminator”

    Directory of Open Access Journals (Sweden)

    Bob Storey

    2014-12-01

    Full Text Available A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the “Worminator” system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application “WormAssay”, developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic parasites (e.g. Brugia malayi. We adapted this assay for use with microscopic parasites by modifying the software to support a full frame analysis mode that applies the motion algorithm to the entire video frame. Thus, the motility of all parasites in a given well are recorded and measured simultaneously. Assays performed on third-stage larvae (L3 of the bovine intestinal nematode Cooperia spp., as well as microfilariae (mf of the filarioid nematodes B. malayi and Dirofilaria immitis, yielded reproducible dose responses using the macrocyclic lactones ivermectin, doramectin, and moxidectin, as well as the nicotinic agonists, pyrantel, oxantel, morantel, and tribendimidine. This new computer based-assay is simple to use, requires minimal new investment in equipment, is robust across nematode genera and developmental stage, and does not require subjective scoring of motility by an observer. Thus, the “Worminator” provides a relatively low-cost platform for developing genera- and stage-specific assays with high efficiency and

  9. High-Voltage DC-DC Converter Topology for PV Energy Utilization - Investigation and Implementation

    DEFF Research Database (Denmark)

    Sanjeevikumar, Padmanaban; Blaabjerg, Frede; Wheeler, Patrick

    2017-01-01

    This paper exploited the utilization of photovoltaic (PV) energy system with high-voltage (HV) output DC-DC converter. Classical boost converters are used for both renewable energy integration and HV applications, but limited by reducing output/efficiency in performance. Moreover, as parasitic...... elements suppress the power transfer ratio, converter needs to maximize the PV energy utilization. This investigation study focused to include additional parasitic elements (voltage-lift technique) to a standard DC-DC buck converter and to overcome all the above drawbacks to maximize the PV power...

  10. Navy and the HARV: High angle of attack tactical utility issues

    Science.gov (United States)

    Sternberg, Charles A.; Traven, Ricardo; Lackey, James B.

    1994-01-01

    This presentation will highlight results from the latest Navy evaluation of the HARV (March 1994) and focus primarily on the impressions from a piloting standpoint of the tactical utility of thrust vectoring. Issue to be addressed will be mission suitability of high AOA flight, visual and motion feedback cues associated with operating at high AOA, and the adaptability of a pilot to effectively use the increased control power provided by the thrust vectoring system.

  11. Utilization of a Buffered Dielectric to Achieve High Field-Effect Carrier Mobility in Graphene Transistors

    OpenAIRE

    Farmer, Damon B.; Chiu, Hsin-Ying; Lin, Yu-Ming; Jenkins, Keith A.; Xia, Fengnian; Avouris, Phaedon

    2009-01-01

    We utilize an organic polymer buffer layer between graphene and conventional gate dielectrics in top-gated graphene transistors. Unlike other insulators, this dielectric stack does not significantly degrade carrier mobility, allowing for high field-effect mobilities to be retained in top-gate operation. This is demonstrated in both two-point and four-point analysis, and in the high-frequency operation of a graphene transistor. Temperature dependence of the carrier mobility suggests that phono...

  12. The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

    OpenAIRE

    Akinosho, Hannah; Yee, Kelsey; Close, Dan; Ragauskas, Arthur

    2014-01-01

    First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing (CBP) applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes toward the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermoc...

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

    Science.gov (United States)

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-02-19

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

  14. High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

    Science.gov (United States)

    Zhai, Dayong; Godoi, Paulo; Sergienko, Eduard; Dahl, Russell; Chan, Xochella; Brown, Brock; Rascon, Justin; Hurder, Andrew; Su, Ying; Chung, Thomas D Y; Jin, Chaofang; Diaz, Paul; Reed, John C

    2012-03-01

    Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

  15. The Application of a Highly Purified Rat Leydig Cell Assay as a Complement to the H295R Steroidogenesis Assay for the Evaluation of Toxicant Induced Alterations in Testosterone Production

    Data.gov (United States)

    U.S. Environmental Protection Agency — The greater dynamic range of testosterone production in a highly purified rat Leydig cell assay permitted the detection of chemical induced inhibition that was not...

  16. 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.

  17. 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.

  18. High-throughput spectrophotometric assay of reactive oxygen species in serum.

    Science.gov (United States)

    Hayashi, Ikue; Morishita, Yukari; Imai, Kazue; Nakamura, Masakazu; Nakachi, Kei; Hayashi, Tomonori

    2007-07-10

    The derivatives of reactive oxygen metabolites (D-ROM) test has been developed to determine the amount of oxygen-centered free radicals in a blood sample as a marker of oxidative stress. This study aims to improve the D-ROM test and develop an automated assay system by use of a clinical chemistry analyzer. Five microliters of serum was added to 1 well of a 96-well microtiter plate for a total 240microl of reaction solution containing alkylamine and metals. This was followed by automatic mixing, incubation and measurement of reactive oxygen species (ROS) levels as a color development at 505nm using a spectrophotometer with catalytic capability for transition metals. This assay system was used to measure serum levels of ROS in cigarette smokers and never-smokers, by way of example. The levels of serum ROS determined by this system correlate with the amounts of free radicals and peroxides, which reacted with various molecules in the body and formed stable metabolites. This test can use frozen sera as well as fresh ones. The inter- and intra-deviation of this system was within 5% and showed consistent linearity in the range between 4 and 500mg/l of hydrogen peroxides. Serum ROS levels among smokers increased with the number of cigarettes smoked per day (36.5% increment per pack per day; P<0.0001). This assay system will be a simple, inexpensive, and reliable tool for assessing oxidative stress in human populations. Our preliminary results on cigarette smoking imply that this assay system has potential for application in various epidemiological and clinical settings.

  19. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    Energy Technology Data Exchange (ETDEWEB)

    Esposito, Anthony M. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Cheung, Pamela [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Swartz, Talia H.; Li, Hongru [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Tsibane, Tshidi [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Durham, Natasha D. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Basler, Christopher F. [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Felsenfeld, Dan P. [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Chen, Benjamin K., E-mail: benjamin.chen@mssm.edu [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States)

    2016-03-15

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  20. A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

    Science.gov (United States)

    Lucas, Andrew T; O'Neal, Sara K; Santos, Charlene M; White, Taylor F; Zamboni, William C

    2016-02-01

    Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

  1. Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.

    Science.gov (United States)

    Hattori, Mitsu; Torii, Chiharu; Yagihashi, Tatsuhiko; Izumi, Kosuke; Suda, Naoto; Ohyama, Kimie; Takahashi, Takao; Moriyama, Keiji; Kosaki, Kenjiro

    2009-10-01

    Russell-Silver syndrome (RSS) is characterized by prenatal and postnatal growth retardation, triangular facies, and fifth-finger clinodactyly. Half of all patients with RSS have hypomethylation of the differentially methylated region of the H19 gene on chromosome 11p15.5. Hence, a quantitative methylation analysis of this region can be useful for the molecular diagnosis of RSS. However, conventional assays based on bisulfite clone sequencing are rather time and labor consuming and are not suitable for clinical use. In the present study, we investigated a possible method of quantitatively determining H19 hypomethylation in RSS patients using a combined bisulfite restriction analysis (COBRA)-denaturing high-performance liquid chromatography (DHPLC) assay; in this combined assay, polymerase chain reaction products amplified from the H19 differentially methylated region of bisulfite-treated genomic DNA were analyzed using a COBRA assay, which detects methylation-dependent sequence differences in the bisulfite-treated genomic DNA using a restriction enzyme analysis. We designed the assay so that a restriction enzyme (HinfI) would cut the methylated, but not the unmethylated, template. The molar ratio between the cut and uncut fragments was measured using DHPLC, and the construction of a calibration curve enabled the methylation index for the original genomic DNA to be estimated. An analysis of seven RSS patients using the COBRA-DHPLC assay demonstrated that three of the seven RSS patients had a low methylation index of around 10%. A comparison of the methylation indices obtained using COBRA-DHPLC and conventional bisulfite clone sequencing revealed an excellent intermethod agreement. In summary, we have developed a robust, rapid, and cost-effective COBRA-DHPLC-based screening system for RSS.

  2. High throughput microwell spectrophotometric assay for olmesartan medoxomil in tablets based on its charge-transfer reaction with DDQ

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A.

    2014-03-01

    Full Text Available The study describes the development and validation of a new microwell-based spectrophotometric assay for determination of olmesartan medoxomil (OLM in tablets. The formation of a colored charge-transfer (CT complex between OLM as an n-electron donor and 2,3-dichloro- -5,6-dicyano-1,4-benzoquinone (DDQ as a p-electron acceptor was investigated, and employed as the basis for the development of the new assay. The proposed assay was conducted in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm with a microplate reader. Optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, a linear relationship with a good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 2-200 μg per well. The limits of detection and quantitation were 0.53 and 1.61 μg per well, respectively. No interference was observed from the excipients present in OLM tablets or from hydrochlorothiazide and amlodipine besylate that were co-formulated with OLM in some of its formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has a great practical value in the routine analysis of OLM in quality control laboratories, since it has a high throughput property and consumes low volumes of organic solvent. It thus offers a reduction in the exposure of analysts to the toxic effects of organic solvents, as well as a reduction in the cost of analysis.

  3. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

    Science.gov (United States)

    Hung, Paul J; Lee, Philip J; Sabounchi, Poorya; Lin, Robert; Lee, Luke P

    2005-01-05

    We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. (c) 2004 Wiley Periodicals, Inc.

  4. High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

    Energy Technology Data Exchange (ETDEWEB)

    Wills, Lauren P. [MitoHealth Inc., Charleston, SC 29403 (United States); Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Beeson, Craig C. [MitoHealth Inc., Charleston, SC 29403 (United States); Peterson, Yuri K. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

    2013-10-15

    Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants.

  5. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry.

    Science.gov (United States)

    Westby, Mike; Smith-Burchnell, Caroline; Mori, Julie; Lewis, Marilyn; Mosley, Michael; Stockdale, Mark; Dorr, Patrick; Ciaramella, Giuseppe; Perros, Manos

    2007-03-01

    Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Delta32/Delta32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

  6. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity.

    Science.gov (United States)

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A; Schreiber, Stuart L; Ioset, Jean-Robert; Gray, David W

    2015-09-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

  7. A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

    Science.gov (United States)

    Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

    2013-06-21

    Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

  8. The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing.

    Science.gov (United States)

    Haglund, Caroline; Aleskog, Anna; Håkansson, Lena Douhan; Höglund, Martin; Jacobsson, Stefan; Larsson, Rolf; Lindhagen, Elin

    2010-05-04

    One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34(+) progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC(50)) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC(50) from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.

  9. CTL ELISPOT assay.

    Science.gov (United States)

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  10. Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

    Energy Technology Data Exchange (ETDEWEB)

    Algar, W. Russ; Tavares, Anthony J. [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6 (Canada); Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6 (Canada)

    2010-07-12

    A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.

  11. Rapid detection and classification of Salmonella enterica shedding in feedlot cattle utilizing Roka Bioscience Atlas Salmonella detection assay for the analysis of rectoanal mucosal swabs

    Science.gov (United States)

    With an increasing focus on preharvest food safety, rapid methods are required for the detection and quantification of foodborne pathogens such as Salmonella enterica in beef cattle. We validated the Atlas Salmonella Detection Assay (SEN), a nucleic acid amplification technology that targets Salmone...

  12. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

    NARCIS (Netherlands)

    Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

    2001-01-01

    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 and

  13. RABiT-II: Implementation of a High-Throughput Micronucleus Biodosimetry Assay on Commercial Biotech Robotic Systems.

    Science.gov (United States)

    Repin, Mikhail; Pampou, Sergey; Karan, Charles; Brenner, David J; Garty, Guy

    2017-02-23

    We demonstrate the use of high-throughput biodosimetry platforms based on commercial high-throughput/high-content screening robotic systems. The cytokinesis-block micronucleus (CBMN) assay, using only 20 μl whole blood from a fingerstick, was implemented on a PerkinElmer cell::explorer and General Electric IN Cell Analyzer 2000. On average 500 binucleated cells per sample were detected by our FluorQuantMN software. A calibration curve was generated in the radiation dose range up to 5.0 Gy using the data from 8 donors and 48,083 binucleated cells in total. The study described here demonstrates that high-throughput radiation biodosimetry is practical using current commercial high-throughput/high-content screening robotic systems, which can be readily programmed to perform and analyze robotics-optimized cytogenetic assays. Application to other commercial high-throughput/high-content screening systems beyond the ones used in this study is clearly practical. This approach will allow much wider access to high-throughput biodosimetric screening for large-scale radiological incidents than is currently available.

  14. Evaluation of hemoglobin A1c measurement from filter paper using high-performance liquid chromatography and immunoturbidimetric assay.

    Science.gov (United States)

    Wu, Yonghua; Yang, Xu; Wang, Haining; Li, Zhenrong; Wang, Tiancheng

    2017-04-01

    Glycated hemoglobin (HbA1c) measurement from whole blood (WB) samples is inconvenient for epidemic surveillance and self-monitoring of glycemic level. We evaluated HbA1c measurement from WB blotted on filter paper (FP), which can be easily transported to central laboratories, with high-performance liquid chromatography (HPLC) and immunoturbidimetric assay (ITA). WB was applied to Whatman filter paper. By using HPLC and WB samples as reference methods, these FP samples were evaluated on HPLC and ITA. Inter- and intra-assay variation, WB vs. FP agreement and sample stability at 20-25 °C and -70 °C were assessed by statistical analysis. Results showed that the coefficient of variation (CV, %) of FP samples for HPLC and ITA were 0.44-1.02% and 1.47-2.72%, respectively (intra-assay); 2.13-3.56% and 3.21-4.82%, respectively (inter-assay). The correlation of WB HPLC with FP analyzed using HPLC and ITA are both significant (p < 0.001). Sample stability showed that FP method up to 5 days at 20-25 °C and 5 weeks at -70 °C is accurate and reproducible. In conclusion, FP samples analyzed by HPLC and ITA can both provide an alternative to WB for HbA1c measurement, supporting the use of FP method in epidemic surveillance and healthcare units.

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

    Science.gov (United States)

    Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

    2016-11-25

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

  16. Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries

    Directory of Open Access Journals (Sweden)

    Hiroshi Shinozuka

    2016-08-01

    Full Text Available Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX ConnectTM Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R2 > 0.77, and that observed from MiSeq sequencing (R2 = 0.82. Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

  17. Validation of a uv spectrophotometric and a reverse high performance methods for the assay of codeine phosphate in tablets

    OpenAIRE

    Romero, Diego; Gil, Andrés; Rojas,Jaime

    2009-01-01

    In this paper we present the results obtained during the validation of a UV spectrometric meted for the assay of codeine phosphate in tablets and in uniformity of content test. Also we report the results obtained in the validation of a reverse high performance method developed for the determination of codeine phosphate in the dissolution test and in the quality control with release purpose. As stationary bounded phase we used octadecylsilane and a mobile phase composed of acetonitrile-water-t...

  18. A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions.

    Science.gov (United States)

    Dehdashti, Seameen J; Abbott, Jennifer; Nguyen, Dac-Trung; McKew, John C; Williamson, Peter R; Zheng, Wei

    2013-08-01

    Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1% and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3'-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.

  19. Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes.

    Science.gov (United States)

    Zhang, Kailin; He, Xiulan; Liu, Yang; Yu, Ping; Fei, Junjie; Mao, Lanqun

    2017-06-20

    Development of new principles and methods for cerebral ATP assay is highly imperative not only for determining ATP dynamics in brain but also for understanding physiological and pathological processes related to ATP. Herein, we for the first time demonstrate that micrometer scale ion current rectification (MICR) at a polyimidazolium brush-modified micropipette can be used as the signal transduction output for the cerebral ATP assay with a high selectivity. The rationale for ATP assay is essentially based on the competitive binding ability between positively charged polyimidazolium and ATP toward negatively charged ATP aptamer. The method is well responsive to ATP with a good linearity within a concentration range from 5 nM to 100 nM, and high selectivity toward ATP. These properties essentially enable the method to determine the cerebral ATP by combining in vivo microdialysis. The basal dialysate level of ATP in rat brain cortex is determined to be 11.32 ± 2.36 nM (n = 3). This study demonstrates that the MICR-based sensors could be potentially used for monitoring neurochemicals in cerebral systems.

  20. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

    Directory of Open Access Journals (Sweden)

    Steven L Walker

    Full Text Available Reporter-based assays underlie many high-throughput screening (HTS platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv. ARQiv differs from current "high-content" (e.g., confocal imaging-based whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1 Rapid; achieving true HTS capacities (i.e., >50,000 units per day, 2 Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5, and 3 Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1 Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors, 2 Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3 Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

  1. RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

    Science.gov (United States)

    Stephen J. Amish,; Paul A. Hohenlohe,; Sally Painter,; Robb F. Leary,; Muhlfeld, Clint C.; Fred W. Allendorf,; Luikart, Gordon

    2012-01-01

    Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

  2. A GA-Based Approach to Hide Sensitive High Utility Itemsets

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    Chun-Wei Lin

    2014-01-01

    Full Text Available A GA-based privacy preserving utility mining method is proposed to find appropriate transactions to be inserted into the database for hiding sensitive high utility itemsets. It maintains the low information loss while providing information to the data demanders and protects the high-risk information in the database. A flexible evaluation function with three factors is designed in the proposed approach to evaluate whether the processed transactions are required to be inserted. Three different weights are, respectively, assigned to the three factors according to users. Moreover, the downward closure property and the prelarge concept are adopted in the proposed approach to reduce the cost of rescanning database, thus speeding up the evaluation process of chromosomes.

  3. Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

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    Andrea Fuso

    Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

  4. Identification of low level gamma-irradiation of meats by high sensitivity comet assay

    Science.gov (United States)

    Miyahara, Makoto; Saito, Akiko; Ito, Hitoshi; Toyoda, Masatake

    2002-03-01

    The detection of low levels of irradiation in meats (pork, beef, and chicken) using the new comet assay was investigated in order to assess the capability of the procedure. The new assay includes a process that improves its sensitivity to irradiation and a novel evaluation system for each slide (influence score and comet-type distribution). Samples used were purchased at retailers and were irradiated at 0.5 and 2kGy at 0°C. The samples were processed to obtain comets. Slides were evaluated by typing comets, calculating the influence score and analyzing the comet-type distribution chart of shown on the slide. Influence scores of beef, pork, and chicken at 0kGy were 287(SD=8.0), 305 (SD=12.9), and 320 (SD=21.0), respectively. Those at 500Gy, were 305 (SD=5.3), 347 (SD=10.6), and 364 (12.6), respectively. Irradiation levels in food were successfully determined. Sensitivity to irradiation differed among samples (chicken>pork>beef).

  5. [Evaluation of high-sensitivity HBsAg quantitative assay for HBV genotype].

    Science.gov (United States)

    Takagi, Kazumi; Tanaka, Yasuhito; Hiramatu, Kumiko; Kani, Satomi; Tatematsu, Kanako; Naganuma, Hatsue; Ueno, Tetsuo; Gotou, Takaaki; Wakimoto, Yukio; Mizokami, Masashi

    2009-07-01

    The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations has been reported in HBV-infected patients during anti-viral treatment. HBV genotypes A and D are ubiquitous and scattered worldwide, especially northern America as well as Europe, whereas genotypes B and C are common in Asia. The aim of this study was to evaluate a new version of the Sysmex HBsAg quantitative kit based on Chemiluminescence Enzyme Immunoassay. Sera collected from 172 patients infected with any of the four major genotypes A to D (HBV/A, n = 18; B, n = 25; C, n = 84; D, n = 45), including the genotype D cases with weak reaction in the previous version of the kit. The new version of the kit having additional monoclonal antibody, showed improved sensitivity compared to the previous version as well as robust correlation with another quantitative HBsAg assay: the Abbot Architect. Observed during lamivudine therapy, increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for the four HBV genotypes common worldwide. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response or resistance to anti-viral therapy.

  6. A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

    Science.gov (United States)

    Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole; Essangui, Estelle; Diesburg, Steven; Mouliom, Abas; Melingui, Bernard; Manga, Jeanne; Donkeu, Christiane; Epote, Annie; Texier, Gaëtan; LaBarre, Paul; Burton, Robert

    2016-01-01

    Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in

  7. Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay.

    Directory of Open Access Journals (Sweden)

    Aruna D Balgi

    Full Text Available The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.

  8. A novel high-throughput screening assay for HCN channel blocker using membrane potential-sensitive dye and FLIPR.

    Science.gov (United States)

    Vasilyev, Dmitry V; Shan, Qin J; Lee, Yan T; Soloveva, Veronica; Nawoschik, Stanley P; Kaftan, Edward J; Dunlop, John; Mayer, Scott C; Bowlby, Mark R

    2009-10-01

    Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

  9. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Xiao-yi ZHANG; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2007-01-01

    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  10. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    Energy Technology Data Exchange (ETDEWEB)

    Koivunen, Marja E. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Dettmer, Katja [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Vermeulen, Roel [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); Bakke, Berit [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); National Institute of Occupational Health, Oslo (Norway); Gee, Shirley J. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Hammock, Bruce D. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States)]. E-mail: bdhammock@ucdavis.edu

    2006-07-21

    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL{sup -1}), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL{sup -1}) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R {sup 2} values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R {sup 2} = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Xiaoming; Fu, Afu [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Luo, Kathy Qian, E-mail: kluo@ntu.edu.sg [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)

    2012-02-24

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

  12. The Utilization of High-Frequency Gravitational Waves for Global Communications

    Directory of Open Access Journals (Sweden)

    Robert M L Baker

    2012-10-01

    Full Text Available For over 1000 years electromagnetic radiation has been utilized for long-distance communication. Smoke signals, heliographs, telegraphs, telephones and radio have all served our previous communication needs. Nevertheless, electromagnetic radiation has one major difficulty: it is easily absorbed. In this paper we consider a totally different radiation, a radiation that is not easily absorbed: gravitational radiation. Such radiation, like gravity itself, is not absorbed by earth, water or any material substance. In particular we discuss herein means to generate and detect high-frequency gravitational waves or HFGWs, and how they can be utilized for communication. There are two barriers to their practical utilization: they are extremely difficult to generate (a large power required to generate very weak GWs and it is extremely difficult to detect weak GWs. We intend to demonstrate theoretically in this paper their phase-coherent generation utilizing an array of in-phase microelectro-mechanical systems or MEMS resonator elements in which the HFGW flux is proportional to the square of the number of elements. This process solves the transmitter difficulty. Three HFGW detectors have previously been built; but their sensitivity is insufficient for meaningful HFGW reception; greater sensitivity is necessary. A new Li-Baker HFGW detector, discussed herein, is based upon a different measurement technique than the other detectors and is predicted to achieve a sensitivity to satisfy HFGW communication needs.

  13. Utility of loop-mediated isothermal amplification assay, polymerase chain reaction, and elisa for diagnosis of leptospirosis in South Indian patients

    Directory of Open Access Journals (Sweden)

    Mallika Sengupta

    2017-01-01

    Full Text Available Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines' criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis. Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05. Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

  14. Utility of Loop-mediated Isothermal Amplification Assay, Polymerase Chain Reaction, and ELISA for Diagnosis of Leptospirosis in South Indian Patients

    Science.gov (United States)

    Sengupta, Mallika; Prabhakar, Abhilash Kundavaram Paul; Satyendra, Sowmya; Thambu, David; Abraham, Ooriapadickal Cherian; Balaji, Veeraraghavan; Chen, Hua-Wei; Chao, Chien-Chung; Ching, Wei-Mei; Prakash, John Antony Jude

    2017-01-01

    Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days) were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP) assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines’ criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis). Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05). Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

  15. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

    Directory of Open Access Journals (Sweden)

    Jianjun Guo

    Full Text Available BACKGROUND: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. METHODOLOGY/PRINCIPAL FINDINGS: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. CONCLUSIONS/SIGNIFICANCE: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  16. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [ORNL; Morrell-Falvey, Jennifer L [ORNL; Labbe, Jessy L [ORNL; Muchero, Wellington [ORNL; Kalluri, Udaya C [ORNL; Tuskan, Gerald A [ORNL; Chen, Jay [ORNL

    2012-01-01

    Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  17. Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

    Science.gov (United States)

    Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.

    2015-01-01

    We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

  18. Testing the genotoxicity of some perfumes with high diethylphthalate (DEP levels using comet assay

    Directory of Open Access Journals (Sweden)

    Iman Al-Saleh

    2015-05-01

    Full Text Available The presence of phthalates in perfumes has gained recently some attention since these chemicals are added sometimes intentionally as a fixative. Our previous study tested 47 branded perfumes sold in Saudi market and found 68% of tested samples had diethylphthalate (DEP, above reported threshold limit of 1 ppm. Of these phthalates, DEP was found to have the highest mean value (1621.63 ppm. These results enticed us to test the potential genotoxicity of 7 brands in which their DEP contents were in the range of 1.06 ppm to 23649.3 ppm in human TK-6 cells using single cell gel electrophoresis assay (comet assay. Cells were exposed to 400 µl perfume for 2 hrs, at room temperature. Tail moment was (TM used as a metric measure for DNA damage and 25 cells per sample were determined by image analysis software. Four perfumes with DEP above 40 ppm produced significant DNA damage in TK-6 cells with TM of 54.27 ± 1.04, n=500 compare to the other 3 perfumes with DEP < 2 ppm (21.94 ± 1.09, n=300, untreated cells (2.99 ± 0.17, n=250 and cells induced with ethanol (33.76 ± 1.4, n=75 or methanol (23.82 ± 1.84, n=100 which are the vehicles used in perfume's preparations. The results suggest that DEP in perfumes might be one of the ingredients that provoked DNA damage; however, further investigation is required confirming our observation.

  19. High-level neutron-coincidence-counter (HLNCC) implementation: assay of the plutonium content of mixed-oxide fuel assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Foley, J E; Bosler, G E

    1982-04-01

    The portable High-Level Neutron Coincidence Counter is used to assay the /sup 240/Pu-effective loading of a reference mixed-oxide fuel assembly by neutron coincidence counting. We have investigated the effects on the coincidence count rate of the total fuel loading (UO/sub 2/ + PuO/sub 2/), the fissile loading, the fuel rod diameter, and the fuel rod pattern. The coincidence count rate per gram of /sup 240/Pu-effective per centimeter is primarily dependent on the total fuel loading of the assembly; the higher the loading, the higher the coincidence count rate. Detailed procedures for the assay of mixed-oxide fuel assemblies are developed.

  20. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    Science.gov (United States)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  1. A novel high throughput screening assay for binding affinities of perfluoroalkyl iodide for estrogen receptor alpha and beta isoforms.

    Science.gov (United States)

    Song, Wenting; Zhao, Lixia; Sun, Zhendong; Yang, Xiaoxi; Zhou, Qunfang; Jiang, Guibin

    2017-12-01

    Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9h, and 40nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E2) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks

  2. Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals

    Science.gov (United States)

    Wong, Chin Lin; Lam, Ai-Leen; Smith, Maree T.; Ghassabian, Sussan

    2016-01-01

    The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight

  3. 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

  4. High-performance tonpilz transducers utilizing d32-cut piezoelectric single crystals

    Science.gov (United States)

    Rehrig, Paul W.; Hackenberger, Wesley S.; Jiang, Xiaoning; Meyer, Richard J.; Geng, Xuecang

    2003-08-01

    High frequency sonar is becoming ever more important to the Navy through expanded use of unmanned underwater vehicles (UUV). Proposed missions for many UUV's involve shallow water operation where broad bandwidth is required making these applications ideal candidates to use single crystal piezoelectrics. In addition, many UUV sonar systems have commercial uses including oceanographic research, oil and mineral prospecting, salvage, and undersea equipment inspection and maintenance. The properties of single crystal piezoelectrics were exploited for broad bandwidth, high frequency sonar. Crystal sonar investigations based on Tonpilz transducers utilizing the '33' resonance mode have shown limitations on bandwidth due to less than ideal resonator aspect ratio. This is a result of the crystals' low elastic stiffness, which leads to short resonators with large lateral dimensions. To address this issue an alternative design was proposed utilizing the '32' resonance mode with the resonating length oriented along a special crystallographic cut. 'Crystals with this orientation are known to have high properties; d32 values as high as 1600 pC/N have been observed. Since prestress for such a design is applied perpendicular to the poling direction, '32' mode Tonpilz elements exhibit lower loss and higher reliability than '33' mode designs. The feasibility of such '32' mode Tonpilz resonators will be presented as determined through property measurements and finite element analysis. The targeted application for this work is broadband (>100%), high frequency (45 kHz) synthetic aperture arrays for unmanned underwater vehicles.

  5. The diagnostic utility of High-Sensitivity Cardiac Troponin T in acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    Manal Mohsen

    2016-03-01

    Conclusion: Our study demonstrated highly significant absolute and relative kinetic changes in hs-TnT levels in patients with AMI. The hs-TnT can be used for early rule in patients with MI as it could detect 97% of cases with MI whose Troponin I was negative and could detect 100% of cases of MI after 6–8 h compared to Troponin I which could detect only 21.2% of cases. This can be of great importance in the future introduction and use of the new high sensitive assay as a non-invasive reliable diagnostic tool to replace the currently used invasive diagnostic techniques for patients with ACS.

  6. A facile and green strategy for preparing newly-designed 3D graphene/gold film and its application in highly efficient electrochemical mercury assay.

    Science.gov (United States)

    Shi, Lei; Wang, Yan; Ding, Shiming; Chu, Zhenyu; Yin, Yu; Jiang, Danfeng; Luo, Jingyi; Jin, Wanqin

    2017-03-15

    In this work, we report a facile and green strategy for in situ and one step preparation of a novel 3D graphene/gold (G/Au) film. Triggering with unique driving force from hydrothermal growth, a 3D interlaced graphene framework with hierarchically porous structures was directly attached on a gold substrate pretreated with a self-assembled monolayer. Simultaneously, highly dispersive Au nanoparticles with tunable morphologies were anchored on the framework utilizing generated graphene as an endogenous reductant. Newly-designed 3D G/Au film possessed excellent properties of significantly large specific area, good electrical conductivity, high structure stability and substrate binding strength, etc. As a paradigm, an electrochemical Hg(2+) biosensor was constructed on 3D G/Au film, in which an exonuclease III-assisted target recycling was introduced. Impressively, an ultralow detection limit of 50 aM (S/N=3), a wide linear range from 0.1 fM to 0.1μM, a high selectivity and a good reliability for Hg(2+) assay in real water and serum samples were realized using prepared biosensor. It is highly envisioned that this work opens the door towards simply fabricating varying types of 3D graphene based hybrid films, and such G/Au film will have widespread applications in electroanalysis and electrocatalysis.

  7. High educational attainment moderates the association between dental health-care supply and utilization in Europe.

    Science.gov (United States)

    Schulz, Maike; Kunst, Anton E; Brockmann, Hilke

    2016-02-01

    In line with the theory of supplier-induced demand, an increased physician density often goes along with a higher utilization of medical services, including dental services. This study aimed to assess whether dentist density and self-employment are related to dental care use, and whether these relationships are moderated by patients' educational attainment. We used data from the Survey of Health, Ageing, and Retirement in Europe (SHARE) from over 20,000 respondents, 50 + yr of age, in 13 countries. We conducted multilevel logistic regressions with probability and type of dental treatment on individual education, country-specific dentist density, and dentist remuneration, and their cross-level interaction. Patients with a high educational level were more likely to report a dentist visit (OR = 2.1, 95% CI: 1.912-2.305) and to receive preventive care (OR = 1.9, 95% CI: 1.697-2.043) than those with a low educational level (reference category). Cross-level interaction effects indicated that high dentist density decreases dental care utilization differences between patients with high (OR = 0.996, 95% CI: 0.993-0.999), medium (OR = 0.995, 95% CI: 0.991-0.999), and low education levels. This was also true for prevention use (OR = 0.996, 95% CI: 0.992-0.999 for patients with a high education level, and OR = 0.996, 95% CI: 0.993-1.000 for patients with a medium education level). The findings suggest that although dentist density is positively associated with dental care utilization, patients have differing susceptibilities to dental care supply.

  8. A cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding.

    Science.gov (United States)

    Capul, Althea A; de la Torre, Juan Carlos

    2008-12-05

    Several arenaviruses cause hemorrhagic fever (HF) disease in humans for which there are no licensed vaccines, and current therapy is limited to the use of ribavirin (Rib) that is only partially effective and associated with significant side effects. In addition, compelling evidence indicates that the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Therefore, it is important to develop novel and effective anti-arenaviral drugs. The arenavirus Z protein is the driving force of arenavirus budding, and PPPY and PTAP late (L) domain motifs within Z are critical for Z-mediated budding, which involves the interaction of Z with a variety of host cellular factors. Compounds capable of inhibiting these virus-host cell interactions represent candidate anti-arenaviral drugs. The identification of these candidate compounds would be facilitated by the availability of a Z budding assay amenable to high-throughput screens (HTS). To this end, we have developed a novel assay that allows for rapid and quantitative assessment of Z-mediated budding. We provide evidence that this novel assay is amenable to HTS to identify small molecule inhibitors of Z-mediated budding, as well as to uncover cellular genes contributing to arenavirus budding.

  9. Evaluation of high sensitivity C-reactive protein assay in cerebrospinal fluid on the Dimension RxL analyzer

    Directory of Open Access Journals (Sweden)

    Jozo Ćorić

    2012-04-01

    Full Text Available Introduction: Low sensitivity and specificity in traditional laboratory tests became insufficient for accurate diagnostics and initiation of proper treatment of patients infected with bacterial meningitis. High sensitivity C reactive protein (hsCRP may be an appropriate supplement for rapid diagnosis of bacterial meningitis. The subject of our investigation was the determination of C- reactive protein in cerebrospinal fluid (CSF duringacute bacterial meningitis.Methods: HsCRP was analysed by a sensitive immunoturbidimetric assay using the Dimension RxL analyser (Siemens. Cerebrospinal fluid concentrations of C-reactive protein have been measured in 20 patients(age range,1 to 50 years presenting with acute bacterial meningitis and also in a non-infected, non-inflamed control group (n=25.Results: The accuracy and precision of the method proved to be satisfactory. Repeatability of serial sampling for hsCRP described by coefficient of variation were CV=2.1-4.5%. This assay hsCRP in cerebrospinal fluid demonstrates adequate performance characteristics for routine clinical use. Elevated levels of CRP were found in 95% patients with bacterial meningitis. The mean CRP value in 25 uninfected control group was 0.25 mg/L (range 0.10-0.55. The mean CRP for patients with bacterial meningitis was 21.4 mg/L (range 0.40-100.Conclusions: A sensitive assay for CRP in CSF would be an useful adjunct to conventional investigation of acute infective meningitis.

  10. A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents.

    Science.gov (United States)

    Bergsdorf, Christian; Kropp-Goerkis, Carmen; Kaehler, Irene; Ketscher, Lars; Boemer, Ulf; Parczyk, Karsten; Bader, Benjamin

    2008-02-01

    Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio

  11. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    Science.gov (United States)

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.

  12. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

    Directory of Open Access Journals (Sweden)

    Lötzerich Mark

    2010-10-01

    Full Text Available Abstract Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2 detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens

  13. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

    Science.gov (United States)

    2010-01-01

    Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes

  14. High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Posaconazole and Vincristine in Rat Plasma

    Directory of Open Access Journals (Sweden)

    Hadeel A. Khalil

    2015-01-01

    Full Text Available Purpose. Developing a validated HPLC-DAD method for simultaneous determination of posaconazole (PSZ and vincristine (VCR in rat plasma. Methods. PSZ, VCR, and itraconazole (ITZ were extracted from 200 μL plasma using diethyl ether in the presence of 0.1 M sodium hydroxide solution. The organic layer was evaporated in vacuo and dried residue was reconstituted and injected through HC-C18 (4.6 × 250 mm, 5 μm column. In the mobile phase, acetonitrile and 0.015 M potassium dihydrogen orthophosphate (30 : 70 to 80 : 20, linear gradient over 7 minutes pumped at 1.5 mL/min. VCR and PSZ were measured at 220 and 262 nm, respectively. Two Sprague Dawley rats were orally dosed PSZ followed by iv dosing of VCR and serial blood sampling was performed. Results. VCR, PSZ, and ITZ were successfully separated within 11 min. Calibration curves were linear over the range of 50–5000 ng/mL for both drugs. The CV% and % error of the mean were ≤18% and limit of quantitation was 50 ng/mL for both drugs. Rat plasma concentrations of PSZ and VCR were simultaneously measured up to 72 h and their calculated pharmacokinetics parameters were comparable to the literature. Conclusion. The assay was validated as per ICH guidelines and is appropriate for pharmacokinetics drug-drug interaction studies.

  15. New high-throughput screening protease assay based upon supramolecular self-assembly.

    Energy Technology Data Exchange (ETDEWEB)

    Whitten, David G.; Tang, Yanli; Zhou, Zhijun; Achyuthan, Komandoor E.

    2008-11-01

    We previously demonstrated that the supramolecular self-assembly of cyanines could be useful for developing fluorescent enzymatic assays. We took that concept a step further by synthesizing a covalent adduct of the tetrapeptide Asp-Glu-Val-Asp (DEVD) and a cyanine (DEVD-cyanine). The DEVD-cyanine due to its canonical sequence was recognized and hydrolyzed by the proteases, Caspase-3 and -7 in 96- or 384-microwell plate reactions. The catalytically liberated cyanine self-assembled upon scaffolds of carboxymethylamylose (CMA), carboxymethylcellulose (CMC), or a mixture of CMA and CMC resulting in a J aggregate exhibiting bright fluorescence at a 470 nm emission wavelength (optimum signal/background using excitation wavelengths of 415-440 nm). The fluorescence intensity increased with enzyme and substrate concentrations or reaction time and exhibited classical saturation profiles of a rectangular hyperbola. Saturation of the reaction was at 30 U/mL (1 {micro}g/mL) Caspase-3 and 250 {micro}M DEVD-cyanine. The reaction kinetics was linear between 1 and 20 min and saturated at 60 min. The affinity constant (Km) for DEVD-cyanine was 23 {micro}M, similar to those of previously reported values for other DEVD substrates of Caspase-3. Maximal fluorescence emission was observed by using a mixture of CMA and CMC scaffolds at 65 and 35 {micro}M, respectively. The reaction kinetics of Caspase-7 executed in a 384-well plate was similar to the reaction kinetics of Caspase-3 conducted in a 96-well plate. We believe that this is the first demonstration of a cyanine liberated from a covalent adduct due to protease action, leading to supramolecular self-assembly and the detection of protease activity.

  16. Clinical Use of Ultrasensitive Cardiac Troponin I Assay in Intermediate- and High-Risk Surgery Patients

    Directory of Open Access Journals (Sweden)

    Flávia Kessler Borges

    2013-01-01

    Full Text Available Background. Cardiac troponin levels have been reported to add value in the detection of cardiovascular complications in noncardiac surgery. A sensitive cardiac troponin I (cTnI assay could provide more accurate prognostic information. Methods. This study prospectively enrolled 142 patients with at least one Revised Cardiac Risk Index risk factor who underwent noncardiac surgery. cTnI levels were measured postoperatively. Short-term cardiac outcome predictors were evaluated. Results. cTnI elevation was observed in 47 patients, among whom 14 were diagnosed as having myocardial infarction (MI. After 30 days, 16 patients had major adverse cardiac events (MACE. Excluding patients with a final diagnosis of MI, predictors of cTnI elevation included dialysis, history of heart failure, transoperative major bleeding, and elevated levels of pre- and postoperative N-terminal pro-B-type natriuretic peptide (NT-proBNP. Maximal cTnI values showed the highest sensitivity (94%, specificity (75%, and overall accuracy (AUC 0.89; 95% CI 0.80–0.98 for postoperative MACE. Postoperative cTnI peak level (OR 9.4; 95% CI 2.3–39.2 and a preoperative NT-proBNP level ≥917 pg/mL (OR 3.47; 95% CI 1.05–11.6 were independent risk factors for MACE. Conclusions. cTnI was shown to be an independent prognostic factor for cardiac outcomes and should be considered as a component of perioperative risk assessment.

  17. Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials.

    Science.gov (United States)

    Qiu, Tian A; Nguyen, Thu Ha Thi; Hudson-Smith, Natalie V; Clement, Peter L; Forester, Dona-Carla; Frew, Hilena; Hang, Mimi N; Murphy, Catherine J; Hamers, Robert J; Feng, Z Vivian; Haynes, Christy L

    2017-02-07

    Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.

  18. Utilizing High Pressure Processing to Induce Structural Changes in Dairy and Meat Products

    DEFF Research Database (Denmark)

    Orlien, Vibeke

    2017-01-01

    . In this article the HP modification of milk and meat proteins is evaluated in relation to the changed molecular functionality and product texture. The underlying mechanisms of the pressure-induced molecular changes are surveyed and related to practical applications in the view of HP-produced milk and meat......High pressure (HP) is capable of modifying the functional properties of milk and meat proteins by pressure-induced changes of the molecular structure. Therefore, HP treatment of milk and meat has been extensively investigated to understand, clarify, and utilize HP processing in the food industry...... products...

  19. Method and apparatus for measuring gravitational acceleration utilizing a high temperature superconducting bearing

    Energy Technology Data Exchange (ETDEWEB)

    Hull, John R. (Downers Grove, IL)

    2000-01-01

    Gravitational acceleration is measured in all spatial dimensions with improved sensitivity by utilizing a high temperature superconducting (HTS) gravimeter. The HTS gravimeter is comprised of a permanent magnet suspended in a spaced relationship from a high temperature superconductor, and a cantilever having a mass at its free end is connected to the permanent magnet at its fixed end. The permanent magnet and superconductor combine to form a bearing platform with extremely low frictional losses, and the rotational displacement of the mass is measured to determine gravitational acceleration. Employing a high temperature superconductor component has the significant advantage of having an operating temperature at or below 77K, whereby cooling may be accomplished with liquid nitrogen.

  20. Method and Apparatus for measuring Gravitational Acceleration Utilizing a high Temperature Superconducting Bearing

    Energy Technology Data Exchange (ETDEWEB)

    Hull, John R.

    1998-11-06

    Gravitational acceleration is measured in all spatial dimensions with improved sensitivity by utilizing a high temperature superconducting (HTS) gravimeter. The HTS gravimeter is comprised of a permanent magnet suspended in a spaced relationship from a high temperature superconductor, and a cantilever having a mass at its free end is connected to the permanent magnet at its fixed end. The permanent magnet and superconductor combine to form a bearing platform with extremely low frictional losses, and the rotational displacement of the mass is measured to determine gravitational acceleration. Employing a high temperature superconductor component has the significant advantage of having an operative temperature at or below 77K, whereby cooling maybe accomplished with liquid nitrogen.

  1. Simultaneous High Performance Liquid Chromatography Assay of Pentoxifylline, Mupirocin, Itraconazole, and Fluticasone Propionate in Humco™ Lavare Wound Base

    Directory of Open Access Journals (Sweden)

    Troy Purvis

    2015-11-01

    Full Text Available This article details the elements used in the method verification for the simultaneous high performance liquid chromatography (HPLC assay of Pentoxifylline, Mupirocin, Itraconazole, and Fluticasone Propionate in Humco™ Lavare Wound base. The method was proven to be linear over 50%–150% of the nominal concentration of the standards. The method was proven to be accurate over 50%–150%, with 98%–102% recovery of the actives from spiked placeboes over that range. The method was shown to be specific to the analytes listed and precise, yielding acceptable results for system reproducibility and method repeatability. The method, as written, is considered to have been verified.

  2. Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior

    DEFF Research Database (Denmark)

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Arvidson, Sandra Marie Neumann

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim...... was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses...... in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans....

  3. A universal indicator dye pH assay for crystallization solutions and other high-throughput applications.

    Science.gov (United States)

    Newman, Janet; Sayle, Roger A; Fazio, Vincent J

    2012-08-01

    In protein crystallization, as well as in many other fields, it is known that the pH at which experiments are performed is often the key factor in the success or failure of the trials. With the trend towards plate-based high-throughput experimental techniques, measuring the pH values of solutions one by one becomes prohibitively time- and reagent-expensive. As part of an HT crystallization facility, a colour-based pH assay that is rapid, uses very little reagent and is suitable for 96-well or higher density plates has been developed.

  4. A modified parallel artificial membrane permeability assay for evaluating the bioconcentration of highly hydrophobic chemicals in fish.

    Science.gov (United States)

    Kwon, Jung-Hwan; Escher, Beate I

    2008-03-01

    Low cost in vitro tools are needed at the screening stage of assessment of bioaccumulation potential of new and existing chemicals because the number of chemical substances that needs to be tested highly exceeds the capacity of in vivo bioconcentration tests. Thus, the parallel artificial membrane permeability assay (PAMPA) system was modified to predict passive uptake/ elimination rate in fish. To overcome the difficulties associated with low aqueous solubility and high membrane affinity of highly hydrophobic chemicals, we measured the rate of permeation from the donor poly(dimethylsiloxane)(PDMS) disk to the acceptor PDMS disk through aqueous and PDMS membrane boundary layers and term the modified PAMPA system "PDMS-PAMPA". Twenty chemicals were selected for validation of PDMS-PAMPA. The measured permeability is proportional to the passive elimination rate constant in fish and was used to predict the "minimum" in vivo elimination rate constant. The in vivo data were very close to predicted values except for a few polar chemicals and metabolically active chemicals, such as pyrene and benzo[a]pyrene. Thus, PDMS-PAMPA can be an appropriate in vitro system for nonmetabolizable chemicals. Combination with metabolic clearance rates using a battery of metabolic degradation assays would enhance the applicability for metabolizable chemicals.

  5. Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

    Science.gov (United States)

    Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

    2015-03-01

    The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

  6. The design of a high-efficiency neutron counter for waste drums to provide optimized sensitivity for plutonium assay

    Energy Technology Data Exchange (ETDEWEB)

    Menlove, H.O.; Beddingfield, D.H.; Pickrell, M.M. [Los Alamos National Lab., NM (United States)] [and others

    1997-11-01

    An advanced passive neutron counter has been designed to improve the accuracy and sensitivity for the nondestructive assay of plutonium in scrap and waste containers. The High-Efficiency Neutron Counter (HENC) was developed under a Cooperative Research Development Agreement between the Los Alamos National Laboratory and Canberra Industries. The primary goal of the development was to produce a passive assay system for 200-L drums that has detectability limits and multiplicity counting features that are superior to previous systems. A detectability limit figure of merit (FOM) was defined that included the detector efficiency, the neutron die-away time, and the detector`s active volume and density that determine the cosmic-ray background. Monte Carlo neutron calculations were performed to determine the parameters to provide an optimum FOM. The system includes the {sup 252}Cf {open_quotes}add-a-source{close_quotes} feature to improve the accuracy as well as statistical filters to reduce the cosmic-ray spallation neutron background. The final decision gave an efficiency of 32% for plutonium with a detector {sup 3}He tube volume that is significantly smaller than for previous high-efficiency systems for 200-L drums. Because of the high efficiency of the HENC, we have incorporated neutron multiplicity counting for matrix corrections for those cases where the plutonium is localized in nonuniform hydrogenous materials. The paper describes the design and performance testing of the advanced system. 5 refs., 8 figs., 3 tabs.

  7. Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology.

    Directory of Open Access Journals (Sweden)

    Antonio M Ramos

    Full Text Available BACKGROUND: The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs. This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay. METHODOLOGY/PRINCIPAL FINDINGS: A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI were sequenced using Illumina's Genome Analyzer (GA. The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%. Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF for all scorable SNPs was 0.274. CONCLUSIONS/SIGNIFICANCE: Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.

  8. Introduction and Utilization of High Priced HCV Medicines across Europe; Implications for the Future

    Science.gov (United States)

    de Bruijn, Winnie; Ibáñez, Cristina; Frisk, Pia; Bak Pedersen, Hanne; Alkan, Ali; Vella Bonanno, Patricia; Brkičić, Ljiljana S.; Bucsics, Anna; Dedet, Guillaume; Eriksen, Jaran; Fadare, Joseph O.; Fürst, Jurij; Gallego, Gisselle; Godói, Isabella P.; Guerra Júnior, Augusto A.; Gürsöz, Hakkı; Jan, Saira; Jones, Jan; Joppi, Roberta; Kerman, Saim; Laius, Ott; Madzikwa, Newman; Magnússon, Einar; Maticic, Mojca; Markovic-Pekovic, Vanda; Massele, Amos; Ogunleye, Olayinka; O'Leary, Aisling; Piessnegger, Jutta; Sermet, Catherine; Simoens, Steven; Tiroyakgosi, Celda; Truter, Ilse; Thyberg, Magnus; Tomekova, Kristina; Wladysiuk, Magdalena; Vandoros, Sotiris; Vural, Elif H.; Zara, Corinne; Godman, Brian

    2016-01-01

    Background: Infection with the Hepatitis C Virus (HCV) is a widespread transmittable disease with a diagnosed prevalence of 2.0%. Fortunately, it is now curable in most patients. Sales of medicines to treat HCV infection grew 2.7% per year between 2004 and 2011, enhanced by the launch of the protease inhibitors (PIs) boceprevir (BCV) and telaprevir (TVR) in addition to ribavirin and pegylated interferon (pegIFN). Costs will continue to rise with new treatments including sofosbuvir, which now include interferon free regimens. Objective: Assess the uptake of BCV and TVR across Europe from a health authority perspective to offer future guidance on dealing with new high cost medicines. Methods: Cross-sectional descriptive study of medicines to treat HCV (pegIFN, ribavirin, BCV and TVR) among European countries from 2008 to 2013. Utilization measured in defined daily doses (DDDs)/1000 patients/quarter (DIQs) and expenditure in Euros/DDD. Health authority activities to influence treatments categorized using the 4E methodology (Education, Engineering, Economics and Enforcement). Results: Similar uptake of BCV and TVR among European countries and regions, ranging from 0.5 DIQ in Denmark, Netherlands and Slovenia to 1.5 DIQ in Tayside and Catalonia in 2013. However, different utilization of the new PIs vs. ribavirin indicates differences in dual vs. triple therapy, which is down to factors including physician preference and genotypes. Reimbursed prices for BCV and TVR were comparable across countries. Conclusion: There was reasonable consistency in the utilization of BCV and TVR among European countries in comparison with other high priced medicines. This may reflect the social demand to limit the transmission of HCV. However, the situation is changing with new curative medicines for HCV genotype 1 (GT1) with potentially an appreciable budget impact. These concerns have resulted in different prices across countries, with their impact on budgets and patient outcomes

  9. Optical Frequency Optimization of a High Intensity Laser Power Beaming System Utilizing VMJ Photovoltaic Cells

    Science.gov (United States)

    Raible, Daniel E.; Dinca, Dragos; Nayfeh, Taysir H.

    2012-01-01

    An effective form of wireless power transmission (WPT) has been developed to enable extended mission durations, increased coverage and added capabilities for both space and terrestrial applications that may benefit from optically delivered electrical energy. The high intensity laser power beaming (HILPB) system enables long range optical 'refueling" of electric platforms such as micro unmanned aerial vehicles (MUAV), airships, robotic exploration missions and spacecraft platforms. To further advance the HILPB technology, the focus of this investigation is to determine the optimal laser wavelength to be used with the HILPB receiver, which utilizes vertical multi-junction (VMJ) photovoltaic cells. Frequency optimization of the laser system is necessary in order to maximize the conversion efficiency at continuous high intensities, and thus increase the delivered power density of the HILPB system. Initial spectral characterizations of the device performed at the NASA Glenn Research Center (GRC) indicate the approximate range of peak optical-to-electrical conversion efficiencies, but these data sets represent transient conditions under lower levels of illumination. Extending these results to high levels of steady state illumination, with attention given to the compatibility of available commercial off-the-shelf semiconductor laser sources and atmospheric transmission constraints is the primary focus of this paper. Experimental hardware results utilizing high power continuous wave (CW) semiconductor lasers at four different operational frequencies near the indicated band gap of the photovoltaic VMJ cells are presented and discussed. In addition, the highest receiver power density achieved to date is demonstrated using a single photovoltaic VMJ cell, which provided an exceptionally high electrical output of 13.6 W/sq cm at an optical-to-electrical conversion efficiency of 24 percent. These results are very promising and scalable, as a potential 1.0 sq m HILPB receiver of

  10. High Performance Liquid Chromatography Tandem Mass Spectrometry Assay for the Determination of Cobinamide in Pig Plasma

    Science.gov (United States)

    McCracken, Brent A.; Brittain, Matthew K.

    2015-01-01

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely utilized for the analysis of compounds in biological matrices due to its selectivity and sensitivity. This study describes the application of an LC-MS/MS-based approach toward the analysis of cobinamide in Yorkshire pig plasma. The selectivity, accuracy, precision, recovery, linearity, range, carryover, sensitivity, matrix effect, interference, stability, reproducibility, and ruggedness of the method were investigated in pig plasma. The accuracy and precision of the method was determined to be within 10% over three different days over a range of concentrations (25–10,000 ng/mL) that spanned more than two orders of magnitude. The lower limit of quantitation (LLOQ) for dicyanocobinamide was determined to be 25 ng/mL in pig plasma. Carryover was acceptable, as the area response of the carryover blanks were ≤15% of the area response of the nearest LLOQ standard for the analyte, while it was nonexistent for the internal standard. Specificity was ensured using six different lots of pig plasma. While the matrix effects of dicyanocobinamide in plasma were enhanced, ginsenoside Rb1 experienced signal suppression under the described conditions. The absolute recovery results for both compounds were consistent, precise, and reproducibly lower than expected at ~60% for dicyanocobinamide and ~22% for ginsenoside Rb1, confirming that a matrix standard curve was required for accurate quantitation. Cobinamide was shown to be very stable in matrix at various storage conditions including room temperature, refrigerated, and frozen at time intervals of 20 hours, 4 days, and 60 days respectively. This method was demonstrated to be sensitive, reproducible, stable, and rugged, and it should be applicable to the analysis of cobinamide in other biological matrices and species. PMID:26540437

  11. A whole-cell assay for the high throughput screening of calmodulin antagonists.

    Science.gov (United States)

    Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

    2008-04-01

    Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca(2+), CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.

  12. Utilizing wideband AMC structures for high-gain inkjet-printed antennas on lossy paper substrate

    KAUST Repository

    Cook, Benjamin Stassen

    2013-01-01

    Significant gain and bandwidth improvement of inkjet-printed antennas with integrated artificial magnetic conductor (AMC) is achieved by utilizing wideband ground-backed frequency selective surfaces (FSSs) to overcome the high losses of organic substrates such as paper. A microstrip-fed monopole mounted on an artificial magnetic conductor is demonstrated to improve the gain by 5 dB over previous works and exhibit much wider impedance bandwidth while maintaining a thin antenna profile and a 20% electrical size reduction. The effect of AMC bandwidth on substrate losses and the gain reduction caused by finite AMC array effects are investigated in an effort to produce high-gain, miniaturized, low-cost wearable and structure mount antennas. © 2013 IEEE.

  13. Use of high-stability composts in recreational areas: assays on cold season turf grasses

    Energy Technology Data Exchange (ETDEWEB)

    Gomez de Barreda-Ferraz, D.; Albiach, M. R.; Pomares, F.; Ingelmo, F.; Canet, R.

    2009-07-01

    Recreational and sport areas, steadily increasing on number and occupied surface, show great interest as consumers of large amounts of organic products. High-quality composts could be used to improve soil properties, increasing its water-hold capacity and reducing the amounts of synthetic fertilizers needed to support the vegetal cover. (Author)

  14. Device and method for enhanced collection and assay of chemicals with high surface area ceramic

    Energy Technology Data Exchange (ETDEWEB)

    Addleman, Raymond S.; Li, Xiaohong Shari; Chouyyok, Wilaiwan; Cinson, Anthony D.; Bays, John T.; Wallace, Krys

    2016-02-16

    A method and device for enhanced capture of target analytes is disclosed. This invention relates to collection of chemicals for separations and analysis. More specifically, this invention relates to a solid phase microextraction (SPME) device having better capability for chemical collection and analysis. This includes better physical stability, capacity for chemical collection, flexible surface chemistry and high affinity for target analyte.

  15. An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants

    NARCIS (Netherlands)

    Bountagkidou, O.; Klift, van der E.J.C.; Tsimidou, M.Z.; Ordoudi, S.A.; Beek, van T.A.

    2012-01-01

    An on-line HPLC (high performance liquid chromatography) method for the rapid screening of individual antioxidants in mixtures was developed using crocin as a substrate (i.e. oxidation probe) and 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH)) in phosphate buffer (pH 7.5) as a radical generator

  16. A highly sensitive gold nanoparticle-based colorimetric probe for pyrophosphate using a competition assay approach.

    Science.gov (United States)

    Kim, Sudeok; Eom, Min Sik; Kim, Seung Kyung; Seo, Seong Hyeok; Han, Min Su

    2013-01-01

    In this study, a mixture of [Zn(2)(1,3-bis[bis(2-pyridylmethyl)aminomethyl]benzene)](4+) ([Zn(2)(BBPAB)](4+)) and 11-mercaptoundecylphosphoric acid functionalized gold nanoparticles (Phos-AuNPs) is shown to be a highly sensitive colorimetric probe that can easily detect pyrophosphate (PPi) at less than 200 nM with the naked eye.

  17. Comparison of cardiac TnI outliers using a contemporary and a high-sensitivity assay on the Abbott Architect platform.

    Science.gov (United States)

    Ryan, J B; Southby, S J; Stuart, L A; Mackay, R; Florkowski, C M; George, P M

    2014-07-01

    Assays for cardiac troponin (cTn) have undergone improvements in sensitivity and precision in recent years. Increased rates of outliers, however, have been reported on various cTn platforms, typically giving irreproducible, falsely higher results. We aimed to evaluate the outlier rate occurring in patients with elevated cTnI using a contemporary and high-sensitivity assay. All patients with elevated cTnI (up to 300 ng/L) performed over a 21-month period were assayed in duplicate. A contemporary assay (Abbott STAT Troponin-I) was used for the first part of the study and subsequently a high-sensitivity assay (Abbott STAT High-Sensitive Troponin-I) was used. Outliers exceeded a calculated critical difference (CD) (CD = z × √2 × SDAnalytical) where z = 3.5 (for probability of 0.0005) and critical outliers also were on a different side of the decision level. The respective outlier and critical outlier rates were 0.22% and 0.10% for the contemporary assay (n = 4009) and 0.18% and 0.13% for the high-sensitivity assay (n = 3878). There was no significant reduction in outlier rate between the two assays (χ(2) = 0.034, P = 0.854). Fifty-six percent of outliers occurred in samples where cTn was an 'add-on' test (and was stored and refrigerated prior to assay). Despite recent improvements in cTn methods, outliers (including critical outliers) still occur at a low rate in both a contemporary and high-sensitivity cTnI assay. Laboratory and clinical staff should be aware of this potential analytical error, particularly in samples with suboptimal sample handling such as add-on tests. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  18. Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals

    Directory of Open Access Journals (Sweden)

    Chin Lin eWong

    2016-03-01

    Full Text Available The direct peptide reactivity assay (DPRA is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium and high concentrations and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme and non-sensitizers with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF, cysteine- (Ac-RFAACAA and lysine- (Ac-RFAAKAA containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7% and glass (47.3% vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2,4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further

  19. Four-year post-exposure assay of vegetation surrounding project pinstripe: demonstration of the utility of delayed damage appraisals. [Larrea divaricata, Ephedra funerea, Atriplex confertifolia

    Energy Technology Data Exchange (ETDEWEB)

    Ragsdale, H.L. (Emory Univ., Atlanta); Rhoads, W.A.

    1974-01-01

    This report illustrates the feasibility of using temporally-delayed vegetation assays to determine radiation damage, by documenting the radiation damage resulting from the accidental venting of radioactive materials during Project Pinstripe, Frenchman's Flat, Nevada Test Site, in April, 1966. Evidence of desert shrub radiation damage was first observed and photographed, in April, 1968. Systematic study of the vegetation was initiated in October, 1970, and evidence of radiation damage documented over 72.9 hectares adjacent to the vent. Beta doses were estimated at 15--21 krads based on gamma exposure dose measurements. The minimum beta dose estimate was substantially greater than the theoretical lethal dose for the shrub, Larrea divaricata. Radiation damage to the shrubs, Larrea divaricata, Ephedra funerea, and Atriplex confertifolia was expressed as differential bud mortality, partial death of shrub crowns with and without crown regrowth, and total shrub crown death without crown regrowth. Each of the shrub populations was statistically different from its control population with respect to the distribution of individuals among damage classes. Generally, damage patterns were similar to those observed at two previously-studied Plowshare events.

  20. HIGH-DOSE BIOTIN TREATMENT FOR SECONDARY PROGRESSIVE MULTIPLE SCLEROSIS MAY INTERFERE WITH THYROID ASSAYS.

    Science.gov (United States)

    Minkovsky, Alissa; Lee, Mark N; Dowlatshahi, Mitra; Angell, Trevor E; Mahrokhian, Lilian S; Petrides, Athena K; Melanson, Stacy E F; Marqusee, Ellen; Woodmansee, Whitney W

    2016-01-01

    To review cases and increase awareness in clinicians treating patients who may be taking biotin. We describe the presentation and workup of a woman with secondary progressive multiple sclerosis on high dose biotin with laboratory studies suggestive of thyrotoxicosis. Plasma samples showed laboratory evidence of elevated thyroid hormone levels with elevated free thyroxine >7.8 ng/dl (reference interval (RI) 0.9-1.7 ng/dl) and decreased thyroid stimulating hormone <0.02 uIU/ml (RI 0.50-5.70 uIU/ml). Laboratory values normalized when biotin was withheld prior to repeat testing. Our case report demonstrates that ingestion of high dose biotin in multiple sclerosis patients can cause interference with laboratory assessment of thyroid function. This interference causes laboratory values suggestive of thyrotoxicosis and can lead to unnecessary evaluation. Clinicians should be aware of the risk of laboratory interference in this patient demographic.

  1. Post-exercise substrate utilization after a high glucose vs. high fructose meal during negative energy balance in the obese.

    Science.gov (United States)

    Tittelbach, T J; Mattes, R D; Gretebeck, R J

    2000-10-01

    To assess the effects of negative energy balance on the metabolic response of a meal containing either glucose or fructose as the primary source of carbohydrate after exercise in obese individuals in energy balance, or negative energy balance. Fourteen adults with mean body mass index (BMI) 30.3 +/- 1 kg/m2, age 26 +/- 2 years, and weight 93.5 +/- 5.4 kg, adhered to an energy-balanced (EB) or a negative energy-balanced (NEB) diet for 6 days. On Day 7, subjects exercised at 70% VO2peak for 40 minutes then consumed either high glucose (50 g of glucose, HG) or high fructose (50 g of fructose, HF) liquid meal. Substrate utilization was measured by indirect calorimetry for 3 hours. Blood samples were collected before exercise and 0, 30, 60, 120, and 180 minutes after consuming the meal. The HG produced 15.9% greater glycemic (p fructose in supplements/meals may provide no additional benefit in terms of substrate utilization during a weight loss program involving diet and exercise.

  2. Highly selective azadipeptide nitrile inhibitors for cathepsin K: design, synthesis and activity assays.

    Science.gov (United States)

    Ren, Xing-Feng; Li, Hong-Wei; Fang, Xuexun; Wu, Yuqing; Wang, Lincong; Zou, Shuxue

    2013-02-21

    We have developed a series of azadipeptide nitriles with different P3 groups. A triaryl meta-phenyl derivative, compound 13, was not only a potent inhibitor for cathepsin K (K(i) = 0.0031 nM), but also highly selective over both cathepsins B and S (~1000-fold). A protein-ligand docking study performed on the series provided a possible explanation why compound 13 could be significantly more potent than the others, especially compound 12 in the same series.

  3. [Comparison of refractometric and ultraviolet detection methods in lactulose assay by high performance liquid chromatography].

    Science.gov (United States)

    Vaccher, C; Berthelot, P; Flouquet, N; Debaert, M

    1990-01-01

    Dosage of lactulose and determination of related sugars (lactose, fructose, galactose, épilactose) were undertaken by high performance liquid chromatography (HPLC). The present note described two modes of detection used simultaneously and compared: refractive index detector (RI) and multiwavelength ultraviolet detector (UV). HPLC is performed with an aminopropylsylil phase as stationary phase and an isocratic mobile phase containing acetonitrile and aqueous sodium phosphate. The results are outlined and discussed.

  4. Design of a Binocular Pupil and Gaze Point Detection System Utilizing High Definition Images

    Directory of Open Access Journals (Sweden)

    Yilmaz Durna

    2017-05-01

    Full Text Available This study proposes a novel binocular pupil and gaze detection system utilizing a remote full high definition (full HD camera and employing LabVIEW. LabVIEW is inherently parallel and has fewer time-consuming algorithms. Many eye tracker applications are monocular and use low resolution cameras due to real-time image processing difficulties. We utilized the computer’s direct access memory channel for rapid data transmission and processed full HD images with LabVIEW. Full HD images make easier determinations of center coordinates/sizes of pupil and corneal reflection. We modified the camera so that the camera sensor passed only infrared (IR images. Glints were taken as reference points for region of interest (ROI area selection of the eye region in the face image. A morphologic filter was applied for erosion of noise, and a weighted average technique was used for center detection. To test system accuracy with 11 participants, we produced a visual stimulus set up to analyze each eye’s movement. Nonlinear mapping function was utilized for gaze estimation. Pupil size, pupil position, glint position and gaze point coordinates were obtained with free natural head movements in our system. This system also works at 2046 × 1086 resolution at 40 frames per second. It is assumed that 280 frames per second for 640 × 480 pixel images is the case. Experimental results show that the average gaze detection error for 11 participants was 0.76° for the left eye, 0.89° for right eye and 0.83° for the mean of two eyes.

  5. Assay of tolnaftate in human skin samples after in vitro penetration studies using high performance liquid chromatography.

    Science.gov (United States)

    Kezutyte, Toma; Kornysova, Olga; Maruska, Audrius; Briedis, Vitalis

    2010-01-01

    Abstract: Tolnaftate, an antifungal of thiocarbamate class, is used topically in 1% formulations. Its penetration into skin layers is a prerequisite for tolnaftate action against dermatophytes. The aim of this work was to optimize and validate a simple, rapid, accurate and reproducible procedure for tolnaftate assay in human skin samples and to apply this procedure for in vitro tolnaftate penetration studies. High performance liquid chromatography (HPLC) method with UV detection was used to validate tolnaftate assay for linearity, specificity, accuracy, precision, limit of quantitation, limit of detection, drug extraction recovery and stability in skin extracts. In vitro tolnaftate penetration studies were carried out using flow-through diffusion cells, mounted with human skin. Epidermis and dermis, separated by heat-separation method, were extracted using ultrasonication in methanol. Linear range of the analytical procedure was within 0.6-100 pg/mL. The assay was specific, accurate (within-day and between-day recovery values were 98.2-104.2% and 98.7-101.4%, respectively) and precise (within-day and between-day imprecision was = 3.8%). Mean extraction recoveries of tolnaftate from epidermis and dermis were satisfactory and reaching 90%. In vitro skin penetration studies revealed that after application of 1% (w/w) tolnaftate solution in polyethylene glycol 400 for 24 hours, the mean amount of tolnaftate penetrating into the epidermis and dermis was 2.60 +/- 0.28 microg/cm2 and 0.92 +/- 0.12 microg/cm2, respectively. A validated reliable HPLC method could be recommended for biopharmaceutical evaluation of tolnaftate preparations and studies of pharmacokinetics in human skin after in vitro penetration studies.

  6. High-density SNP assay development for genetic analysis in maritime pine (Pinus pinaster).

    Science.gov (United States)

    Plomion, C; Bartholomé, J; Lesur, I; Boury, C; Rodríguez-Quilón, I; Lagraulet, H; Ehrenmann, F; Bouffier, L; Gion, J M; Grivet, D; de Miguel, M; de María, N; Cervera, M T; Bagnoli, F; Isik, F; Vendramin, G G; González-Martínez, S C

    2016-03-01

    Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.

  7. Utility of specimens positive for Neisseria gonorrhoeae by the Aptima Combo 2 assay for assessment of strain diversity and antibiotic resistance.

    Science.gov (United States)

    Pabbaraju, Kanti; Wong, Sallene; Song, Joanna J; Singh, Ameeta E; Read, Ron; Drews, Steven J

    2013-12-01

    In our jurisdiction, the Aptima Combo 2 assay (Gen-Probe, Inc.) is used to detect Neisseria gonorrhoeae from specimens collected at clinics for sexually transmitted infections (STI) and from select community patients. In addition, swabs are also collected for N. gonorrhoeae culture, susceptibility testing, and sequence typing (ST). Since only a small proportion of samples from provincial cases undergo culture, the available trends in antimicrobial susceptibility and predominant strain types may not be representative of all N. gonorrhoeae infections. Due to the limitations facing the use of N. gonorrhoeae culture to understand these trends in the general community, we performed a molecular analysis for markers of cephalosporin resistance and ST determination by using nucleic acid extracts of specimens sent for Aptima testing. Thirty-four samples submitted for both Aptima testing and N. gonorrhoeae culture from the same anatomic location (within 24 h) were included in the study. Sequence type was determined based on the sequence of the por and tbpB genes, and amino acid changes in the PBP 2 protein, encoded by the penA gene, were considered representative for the assessment of antimicrobial susceptibility. Sequence identity of 100% was observed between the sequences obtained from Aptima-analyzed samples and culture samples. Sequencing results showed an association between decreased susceptibility to extended-spectrum cephalosporins (ESC(ds)), tbp allele 110, ST 1407, and amino acid changes (G545S, I312M, and V316T) in the PBP 2 protein. Our data, generated based on a few representative genes, suggest that gonococcal samples positive by Aptima testing can be used to determine single nucleotide polymorphisms associated with ESC(ds) and the sequence type based on molecular strain typing. Confirmation of these findings may obviate the need for gonorrhea culture in the future.

  8. 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%.

  9. A multilocus assay reveals high nucleotide diversity and limited differentiation among Scandinavian willow grouse (Lagopus lagopus

    Directory of Open Access Journals (Sweden)

    Quintela Maria

    2008-12-01

    Full Text Available Abstract Background There is so far very little data on autosomal nucleotide diversity in birds, except for data from the domesticated chicken and some passerines species. Estimates of nucleotide diversity reported so far in birds have been high (~10-3 and a likely explanation for this is the generally higher effective population sizes compared to mammals. In this study, the level of nucleotide diversity has been examined in the willow grouse, a non-domesticated bird species from the order Galliformes, which also holds the chicken. The willow grouse (Lagopus lagopus has an almost circumpolar distribution but is absent from Greenland and the north Atlantic islands. It primarily inhabits tundra, forest edge habitats and sub-alpine vegetation. Willow grouse are hunted throughout its range, and regionally it is a game bird of great cultural and economical importance. Results We sequenced 18 autosomal protein coding loci from approximately 15–18 individuals per population. We found a total of 127 SNP's, which corresponds to 1 SNP every 51 bp. 26 SNP's were amino acid replacement substitutions. Total nucleotide diversity (πt was between 1.30 × 10-4 and 7.66 × 10-3 (average πt = 2.72 × 10-3 ± 2.06 × 10-3 and silent nucleotide diversity varied between 4.20 × 10-4and 2.76 × 10-2 (average πS = 9.22 × 10-3 ± 7.43 × 10-4. The synonymous diversity is approximately 20 times higher than in humans and two times higher than in chicken. Non-synonymous diversity was on average 18 times lower than the synonymous diversity and varied between 0 and 4.90 × 10-3 (average πa = 5.08 × 10-4 ± 7.43 × 103, which suggest that purifying selection is strong in these genes. FST values based on synonymous SNP's varied between -5.60 × 10-4 and 0.20 among loci and revealed low levels of differentiation among the four localities, with an overall value of FST = 0.03 (95% CI: 0.006 – 0.057 over 60 unlinked loci. Non-synonymous SNP's gave similar results. Low

  10. NITROGEN UTILIZATION BY DAIRY GOATS OFFERED DIFFERENT NITROGEN SOURCES AS SUPPLEMENTS IN HIGH ISOCALORIC ENERGY CONCENTRATES

    Directory of Open Access Journals (Sweden)

    A.R.S. Asih

    2014-10-01

    Full Text Available Twelve growing female goats (Anglo-Nubian were assigned to a multiple latin square designexperiment to evaluate the effectiveness of additions of nitrogen (N supplements to a high isocaloricenergy ration on N utilization. In this experiment, microbial synthesis and N balance were assessed. Thedaily rations were either unsupplemented barley meal (BM, or BM supplemented with one of threenitrogen sources. All rations were isocaloric (3.0 Mcal ME/kg DM and the N supplements weresoybean meal (BSBM, cottonseed meal (BCSM or urea (BU to provide 2.9% N in the concentratecomponent. The unsupplemented BM contained 1.7% N. The addition of N supplements to the rationenhanced N utilization in dairy goats. The organic matter (OM intake, N intake, N balance, andmicrobial N synthesis for BM, BSBM, BCSM and BU were 660.5 g, 721.9 g, 728.1g and 703.5 g; 13.5g, 21.5 g, 20.9 g and 20.7 g; 2.7 g; 7.1 g, 5.4 g, and 5.7 g; and 14.1 g 19.1 g, 19.1 g, and 20.0 g,respectively. It can be concluded that when sufficient dietary energy was available for ruminal microbialactivities, the source of N did not affect N balance, and microbial N synthesis.

  11. Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining.

    Science.gov (United States)

    Zhao, Dong S; Roy, Binayak; McCormick, Matthew T; Kuhr, Werner G; Brazill, Sara A

    2003-05-01

    In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).

  12. Application of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 high volume clinical chemistry analyzer

    NARCIS (Netherlands)

    K. Boer; T. Deufel; D. Schmidt; S. Streck; M. Kiehntopf

    2006-01-01

    Objectives: Evaluation of the performance of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 analyzer. Design and Methods: Imprecision studies were performed and patient samples were assayed by EMIT assay and by LC-MS/MS. Results: Limit of quantification was established at 2.8 mu g/L. A

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  15. A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

    Science.gov (United States)

    Garver, W S; Kemp, J D; Kuehn, G D

    1992-12-01

    Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

  16. A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening.

    Science.gov (United States)

    Hamel, Laura D; Deschenes, Robert J; Mitchell, David A

    2014-09-01

    Palmitoylation, the posttranslational thioester-linked modification of a 16-carbon saturated fatty acid onto the cysteine residue of a protein, has garnered considerable attention due to its implication in a multitude of disease states. The signature DHHC motif (Asp-His-His-Cys) identifies a family of protein acyltransferases (PATs) that catalyze the S-palmitoylation of target proteins via a two-step mechanism. In the first step, autopalmitoylation, palmitate is transferred from palmitoyl-CoA to the PAT, creating a palmitoyl:PAT intermediate and releasing reduced CoA. The palmitoyl moiety is then transferred to a protein substrate in the second step of the reaction. We have developed an in vitro, single-well, fluorescence-based enzyme assay that monitors the first step of the PAT reaction by coupling the production of reduced CoA to the reduction of NAD(+) using the α-ketoglutarate dehydrogenase complex. This assay is suitable for determining PAT kinetic parameters, elucidating lipid donor specificity and measuring PAT inhibition by 2-bromopalmitate. Finally, it can be used for high-throughput screening (HTS) campaigns for modulators of protein palmitoylation. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Oates, Matthew R.; Clarke, William; Zimlich, Alden; Hage, David S

    2002-10-11

    Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was L-thyroxine (also known as T{sub 4}). In this technique, a sample containing thyroxine was first combined with an excess of anti-T{sub 4} antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T{sub 4}. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T{sub 4} in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.

  18. Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis.

    Science.gov (United States)

    Fabbri, Rita; Montagna, Michele; Balbi, Teresa; Raffo, Enrico; Palumbo, Franca; Canesi, Laura

    2014-08-01

    Emerging contaminants (such as Endocrine disrupting chemicals-EDCs, brominated and perfluorinated compounds-BFRs and PFCs, pharmaceuticals) are chemicals currently not included in regulatory monitoring programs, and whose fate and biological impacts are poorly understood. Assessment of ecosystem health with respect to these chemicals is of particular concern also in the marine environment: in this respect, data on the effects on early life stages are important to establish the sensitivity of marine species. In this work, the acute (48 h) bivalve embryo toxicity test was applied for screening the developmental effects of different emerging contaminants in the Mediterranean mussel Mytilus galloprovincialis. The assay was adapted to 96-microwell plates, and standardized in order to obtain to normal D-shaped larvae with acceptability of test results based on negative control and positive control (copper) comparable with those reported in literature for Mytilus spp. The effects of different model compounds representative of EDCs (Nonylphenol-NP and Bisphenol A-BPA), BFRs (Tetrabromobisphenol A-TBBPA), PFCs (perfluorooctanoid acid-PFOA and perfluorooctane sulphonate-PFOAS) and pharmaceuticals (Ibuprofen-IBU, Diclofenac-DCF, Bezafibrate-BEZA) in a wide concentration range (0.01-0.1-1-10-100-1000 μg/L) were evaluated. The assay proved as a sensitive tool for high throughput screening of emerging contaminants in a marine species, leading to production of significant amounts of data that may be useful for regulatory purposes.

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

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

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

  20. Tackling heterogeneity: a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco.

    Directory of Open Access Journals (Sweden)

    Natalia Piotrzkowski

    Full Text Available Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

  1. A highly efficient nonchemical method for isolating live nematodes (Caenorhabditis elegans) from soil during toxicity assays.

    Science.gov (United States)

    Kim, Shin Woong; Moon, Jongmin; An, Youn-Joo

    2015-01-01

    The success of soil toxicity tests using Caenorhabditis elegans may depend in large part on recovering the organisms from the soil. However, it can be difficult to learn the International Organization for Standardization/ASTM International recovery process that uses the colloidal silica flotation method. The present study determined that a soil-agar isolation method provides a highly efficient and less technically demanding alternative to the colloidal silica flotation method. Test soil containing C. elegans was arranged on an agar plate in a donut shape, a linear shape, or a C curve; and microbial food was placed outside the soil to encourage the nematodes to leave the soil. The effects of ventilation and the presence of food on nematode recovery were tested to determine the optimal conditions for recovery. A linear arrangement of soil on an agar plate that was sprinkled with microbial food produced nearly 83% and 90% recovery of live nematodes over a 3-h and a 24-h period, respectively, without subjecting the nematodes to chemical stress. The method was tested using copper (II) chloride dihydrate, and the resulting recovery rate was comparable to that obtained using colloidal silica flotation. The soil-agar isolation method portrayed in the present study enables live nematodes to be isolated with minimal additional physicochemical stress, making it a valuable option for use in subsequent sublethal tests where live nematodes are required. © 2014 SETAC.

  2. A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

    Science.gov (United States)

    Lohman, Gregory J S; Bauer, Robert J; Nichols, Nicole M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Evans, Thomas C

    2016-01-29

    DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

  3. A high-throughput assay for quantification of starch hydrolase inhibition based on turbidity measurement.

    Science.gov (United States)

    Liu, Tingting; Song, Lixia; Wang, Hongyu; Huang, Dejian

    2011-09-28

    A high-throughput method for rapid determination of starch hydrolase inhibition was developed using a 96-well microplate UV-vis reader to monitor the turbidity decrease over time. The area under the curve of turbidity measured over time was used to quantify the inhibitory effect of polyphenolic compounds on porcine pancreatic amylase, rat intestine α-glucosidase, and fungal amyloglucosidase. Acarbose equivalence (AE) was introduced for the first time and defined as IC50 of acarbose divided by the IC50 of the sample measured under the same 96-well plate. This way, the run-to-run variations are canceled out. Among the plant extracts tested, grape seed extracts (1,440 μmolAE/g) and cinnamon bark extracts (1600 μmolAE/g) are the most active in inhibiting rat intestine α-glucosidase. For porcine α-amylase inhibition, grape seed extracts (5710 μmol AE/g) are close to four times more active (equal weight basis) than acarbose (1550 μmolAE/g).

  4. A Highly Sensitive Spectrophotometric Assay of Bleomycins Based on the Fading Reaction of Some Halofluorescein Dyes

    Institute of Scientific and Technical Information of China (English)

    LIU,Jiang-Tao; LIU,Zhong-Fang; HU,Xiao-Li; Kong,Ling; LIU,Shao-Pu

    2007-01-01

    In weak acidic medium, anticancer antibiotics bleomycin A5 (BLMA5) and bleomycin A2 (BLMA2) can react with halofluorescein dyes such as erythrosin (Ery), eosin Y (EY), eosin B (EB) and rose bengal (RB) by virtue of electrostatic attraction and hydrophobic force to form the ion-association complexes, which can result in the fading reactions of four halofluorescein dyes. The maximum fading wavelengths of these four dyes were located at 527 nm for Ery, 515 nm for EY, 517 nm for EB and 546 nm for RB, respectively. The decrements of absorbance (△A) were directly proportional to the concentrations of bleomycin in a certain range. A new method for the determination of bleomycins anticancer drugs based on fading reactions of halofluorescein dyes has been developed. The method was not only highly sensitive but also simple and rapid. The molar absorptivities (ε) ranged from 1.5 × 105 to 7.5 ×105 L·mol-1·cm-1. It was applied to determination of the bleomycins in human serum, urine and rabbit serum samples. In this work, the spectral properties and the optimum reaction conditions were investigated. The structure of ion-association complexes and the reaction mechanism were discussed.

  5. Detection of high-risk human papillomavirus infection in tonsillar specimens using 2 commercially available assays.

    Science.gov (United States)

    Cockerill, Cara C; Orvidas, Laura J; Moore, Eric J; Binnicker, Matthew J; Duresko, Brian J; Espy, Mark J; Cockerill, Franklin R; Tombers, Nicole M; Pritt, Bobbi S

    2016-12-01

    THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Performance evaluation of high-temperature superconducting current leads for electric utility SMES systems

    Science.gov (United States)

    Niemann, R. C.; Cha, Y. S.; Hull, J. R.; Rey, C. M.; Dixon, K. D.

    As part of the U.S. Department of Energy's Superconductivity Technology Program, Argonne National Laboratory and Babcock & Wilcox are developing high-temperature super-conductor (HTS) current leads for application to electric utility superconducting magnetic energy storage systems. A 16,000-A HTS lead has been designed and is being constructed. An evaluation program for component performance was conducted to confirm performance predictions and/or to qualify the design features for construction. Performance of the current lead assemblies will be evaluated in a test program that includes assembly procedures, tooling, and quality assurance; thermal and electrical performance; and flow and mechanical characteristics. Results of the evaluations to date are presented.

  7. The Open Cloud Testbed: A Wide Area Testbed for Cloud Computing Utilizing High Performance Network Services

    CERN Document Server

    Grossman, Robert; Sabala, Michal; Bennet, Collin; Seidman, Jonathan; Mambratti, Joe

    2009-01-01

    Recently, a number of cloud platforms and services have been developed for data intensive computing, including Hadoop, Sector, CloudStore (formerly KFS), HBase, and Thrift. In order to benchmark the performance of these systems, to investigate their interoperability, and to experiment with new services based on flexible compute node and network provisioning capabilities, we have designed and implemented a large scale testbed called the Open Cloud Testbed (OCT). Currently the OCT has 120 nodes in four data centers: Baltimore, Chicago (two locations), and San Diego. In contrast to other cloud testbeds, which are in small geographic areas and which are based on commodity Internet services, the OCT is a wide area testbed and the four data centers are connected with a high performance 10Gb/s network, based on a foundation of dedicated lightpaths. This testbed can address the requirements of extremely large data streams that challenge other types of distributed infrastructure. We have also developed several utiliti...

  8. A high throughput in vivo assay for taste quality and palatability.

    Directory of Open Access Journals (Sweden)

    R Kyle Palmer

    Full Text Available Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli. Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity. Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints. We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm. Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor. As a rat's tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser. The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever. Each disruption of the laser was counted as a lick. Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy. Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness. All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue. The technology is capable of generating multiple concentration-response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.

  9. 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.

  10. High-performance liquid chromatographic method for assay of otilonium bromide, diazepam, and related compounds in finished pharmaceutical forms.

    Science.gov (United States)

    Mannucci, C; Bertini, J; Cocchini, A; Perico, A; Salvagnini, F; Triolo, A

    1993-04-01

    A rapid, simple, stability-indicating assay procedure for otilonium bromide, a smooth muscle relaxant agent, and diazepam in composite tablet analysis was developed with high-performance liquid chromatography. The tablet matrix was dissolved with water, and drugs were extracted with acetonitrile containing an internal standard. An aliquot was centrifuged and chromatographed on a 5-microns, reversed-phase column with 0.5 M sodium acetate trihydrate buffer containing 5 mM 1-heptanesulfonic acid monohydrate sodium salt:methanol (30:70; v/v; adjusted to pH 6.0 with glacial acetic acid) as the mobile phase. The selectivity of the chromatographic system was demonstrated by resolving both compounds from various potential degradation products of each compound. The method is linear, quantitative, and reproducible.

  11. An Isocratic High-Performance Liquid Chromatography Assay for CYP7A1-Catalyzed Cholesterol 7α-Hydroxylation.

    Science.gov (United States)

    Waxman, David J; Chang, Thomas K H

    2006-01-01

    A normal-phase, isocratic high-performance liquid chromatography assay is described for cholesterol 7α-hydroxylation catalyzed by CYP7A1, which corresponds to the first and rate-limiting step in the conversion of cholesterol into bile acids. This method is based on the conversion of the primary cytochrome P450 metabolite, 7α-hydroxycholesterol, into 7α-hydroxy-4-cholesten-3-one in a reaction catalyzed by exogenous cholesterol oxidase, followed by chromatographic separation with monitoring at 254 nm. This technique is applicable to enzymatic studies for determination of cholesterol 7α- hydroxylation activity catalyzed by cDNA-expressed CYP7A1 and animal or human liver microsomes.

  12. Fully Integrated, Miniature, High-Frequency Flow Probe Utilizing MEMS Leadless SOI Technology

    Science.gov (United States)

    Ned, Alex; Kurtz, Anthony; Shang, Tonghuo; Goodman, Scott; Giemette. Gera (d)

    2013-01-01

    This work focused on developing, fabricating, and fully calibrating a flowangle probe for aeronautics research by utilizing the latest microelectromechanical systems (MEMS), leadless silicon on insulator (SOI) sensor technology. While the concept of angle probes is not new, traditional devices had been relatively large due to fabrication constraints; often too large to resolve flow structures necessary for modern aeropropulsion measurements such as inlet flow distortions and vortices, secondary flows, etc. Mea surements of this kind demanded a new approach to probe design to achieve sizes on the order of 0.1 in. (.3 mm) diameter or smaller, and capable of meeting demanding requirements for accuracy and ruggedness. This approach invoked the use of stateof- the-art processing techniques to install SOI sensor chips directly onto the probe body, thus eliminating redundancy in sensor packaging and probe installation that have historically forced larger probe size. This also facilitated a better thermal match between the chip and its mount, improving stability and accuracy. Further, the leadless sensor technology with which the SOI sensing element is fabricated allows direct mounting and electrical interconnecting of the sensor to the probe body. This leadless technology allowed a rugged wire-out approach that is performed at the sensor length scale, thus achieving substantial sensor size reductions. The technology is inherently capable of high-frequency and high-accuracy performance in high temperatures and harsh environments.

  13. Widespread utility of highly informative AFLP molecular markers across divergent shark species.

    Science.gov (United States)

    Zenger, Kyall R; Stow, Adam J; Peddemors, Victor; Briscoe, David A; Harcourt, Robert G

    2006-01-01

    Population numbers of many shark species are declining rapidly around the world. Despite the commercial and conservation significance, little is known on even the most fundamental aspects of their population biology. Data collection that relies on direct observation can be logistically challenging with sharks. Consequently, molecular methods are becoming increasingly important to obtain knowledge that is critical for conservation and management. Here we describe an amplified fragment length polymorphism method that can be applied universally to sharks to identify highly informative genome-wide polymorphisms from 12 primer pairs. We demonstrate the value of our method on 15 divergent shark species within the superorder Galeomorphii, including endangered species which are notorious for low levels of genetic diversity. Both the endangered sand tiger shark (Carcharodon taurus, N = 18) and the great white shark (Carcharodon carcharias, N = 7) displayed relatively high levels of allelic diversity. A total of 59 polymorphic loci (H(e) = 0.373) and 78 polymorphic loci (H(e) = 0.316) were resolved in C. taurus and C. carcharias, respectively. Results from other sharks (e.g., Orectolobus ornatus, Orectolobus sp., and Galeocerdo cuvier) produced remarkably high numbers of polymorphic loci (106, 94, and 86, respectively) from a limited sample size of only 2. A major constraint to obtaining much needed genetic data from sharks is the time-consuming process of developing molecular markers. Here we demonstrate the general utility of a technique that provides large numbers of informative loci in sharks.

  14. Basic concepts, status, opportunities, and challenges of electrical machines utilizing high-temperature superconducting (HTS) windings

    Energy Technology Data Exchange (ETDEWEB)

    Frauenhofer, J [Siemens AG, Automation and Drives, Large Drives (Germany); Grundmann, J; Klaus, G; Nick, W [Siemens AG, Corporate Technology, PO Box 3220, 91050 Erlangen (Germany)], E-mail: wolfgang.nick@siemens.com

    2008-02-15

    An overview of the different approaches towards achieving a marketable application of a superconducting electrical machine, either as synchronous motor or generator, will be given. This field ranges from relatively small industrial drives to utility generators with large power ratings, from the low speed and high torque of wind power generators and ship propulsion motors, to high speed generators attached to turbines. Essentially HTS machine technology offers several advantages such as compactness (weight and volume reduction), increased efficiency, and other operational benefits. The machine features have to be optimized with regard to the specific application, and different concepts were developed by internationally competing teams, with Siemens being one of them. The achieved status in these fields will be summarized, pointing to the specific technical challenges to overcome. For this purpose we have not only to consider the technology of manufacturing the HTS rotor winding itself, but also to check requirements and availability of supporting technologies. This ranges from new challenges posed to the non-superconducting ('conventional') components of such innovative HTS machines, manufacturing superconducting material in the coming transition from 1st to 2nd generation HTS tape, cryogenic technology including material behavior, to new and challenging tasks in simulating and predicting the performance of such machines by computational tools. The question of market opportunities for this technology obviously is a function of all these aspects; however, a strong tendency for the near future is seen in the area of high-torque ship propulsion.

  15. Basic concepts, status, opportunities, and challenges of electrical machines utilizing high-temperature superconducting (HTS) windings

    Science.gov (United States)

    Frauenhofer, J.; Grundmann, J.; Klaus, G.; Nick, W.

    2008-02-01

    An overview of the different approaches towards achieving a marketable application of a superconducting electrical machine, either as synchronous motor or generator, will be given. This field ranges from relatively small industrial drives to utility generators with large power ratings, from the low speed and high torque of wind power generators and ship propulsion motors, to high speed generators attached to turbines. Essentially HTS machine technology offers several advantages such as compactness (weight and volume reduction), increased efficiency, and other operational benefits. The machine features have to be optimized with regard to the specific application, and different concepts were developed by internationally competing teams, with Siemens being one of them. The achieved status in these fields will be summarized, pointing to the specific technical challenges to overcome. For this purpose we have not only to consider the technology of manufacturing the HTS rotor winding itself, but also to check requirements and availability of supporting technologies. This ranges from new challenges posed to the non-superconducting ("conventional") components of such innovative HTS machines, manufacturing superconducting material in the coming transition from 1st to 2nd generation HTS tape, cryogenic technology including material behavior, to new and challenging tasks in simulating and predicting the performance of such machines by computational tools. The question of market opportunities for this technology obviously is a function of all these aspects; however, a strong tendency for the near future is seen in the area of high-torque ship propulsion.

  16. High quality factor polymeric Fabry-Perot resonators utilizing a polymer waveguide.

    Science.gov (United States)

    Tadayon, Mohammad Amin; Baylor, Martha-Elizabeth; Ashkenazi, Shai

    2014-03-10

    Optical resonators are used in a variety of applications ranging from sensors to lasers and signal routing in high volume communication networks. Achieving a high quality (Q) factor is necessary for higher sensitivity in sensing applications and for narrow linewidth light emission in most lasing applications. In this work, we propose a new approach to achieve a very high Q-factor in polymeric Fabry-Perot resonators by conquering light diffraction inside the optical cavity. This can be achieved by inducing a refractive index feature inside the optical cavity that simply creates a waveguide between the two mirrors. This approach eliminates diffraction loss from the cavity and therefore the Q-factor is only limited by mirror loss and absorption. To demonstrate this claim, a device has been fabricated consisting of two dielectric Bragg reflectors with a 100 μm layer of photosensitive polymer between them. The refractive index of this polymer can be modified utilizing standard photo-lithography processes. The measured finesse of the fabricated device was 692 and the Q-factor was 55000.

  17. HPV Genotyping from the high risk mRNA Aptima assay- a direct approach using DNA from Aptima sample tubes.

    Science.gov (United States)

    Lillsunde Larsson, G; Kaliff, M; Bergengren, L; Karlsson, M G; Helenius, G

    2016-09-01

    The underlying cause of cervical cancer is an infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.

  18. New High-Performance Droplet Freezing Assay (HP-DFA) for the Analysis of Ice Nuclei with Complex Composition

    Science.gov (United States)

    Kunert, Anna Theresa; Scheel, Jan Frederik; Helleis, Frank; Klimach, Thomas; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2016-04-01

    Freezing of water above homogeneous freezing is catalyzed by ice nucleation active (INA) particles called ice nuclei (IN), which can be of various inorganic or biological origin. The freezing temperatures reach up to -1 °C for some biological samples and are dependent on the chemical composition of the IN. The standard method to analyze IN in solution is the droplet freezing assay (DFA) established by Gabor Vali in 1970. Several modifications and improvements were already made within the last decades, but they are still limited by either small droplet numbers, large droplet volumes or inadequate separation of the single droplets resulting in mutual interferences and therefore improper measurements. The probability that miscellaneous IN are concentrated together in one droplet increases with the volume of the droplet, which can be described by the Poisson distribution. At a given concentration, the partition of a droplet into several smaller droplets leads to finely dispersed IN resulting in better statistics and therefore in a better resolution of the nucleation spectrum. We designed a new customized high-performance droplet freezing assay (HP-DFA), which represents an upgrade of the previously existing DFAs in terms of temperature range and statistics. The necessity of observing freezing events at temperatures lower than homogeneous freezing due to freezing point depression, requires high-performance thermostats combined with an optimal insulation. Furthermore, we developed a cooling setup, which allows both huge and tiny temperature changes within a very short period of time. Besides that, the new DFA provides the analysis of more than 750 droplets per run with a small droplet volume of 5 μL. This enables a fast and more precise analysis of biological samples with complex IN composition as well as better statistics for every sample at the same time.

  19. High-throughput screening with a miniaturized radioligand competition assay identifies new modulators of human α2-adrenoceptors.

    Science.gov (United States)

    Fallarero, Adyary; Pohjanoksa, Katariina; Wissel, Gloria; Parkkisenniemi-Kinnunen, Ulla-Mari; Xhaard, Henri; Scheinin, Mika; Vuorela, Pia

    2012-12-18

    Human α(2)-adrenoceptors (α(2)-ARs) are rhodopsin-like G-protein coupled receptors, and potential drug targets. The three different human α(2)-AR subtypes α(2A), α(2B) and α(2C) are widely distributed in tissues, but so far only a few subtype-selective ligands have been identified. In this project, we set off to conduct a large chemical screen for activity on the human α(2B)-AR and studied the selectivity of the active compounds towards the human α(2A)- and α(2C)-AR subtypes. We employed a radioligand competition binding assay that was optimized and miniaturized into a robotic environment. Membrane fractions containing recombinant human receptor subtypes were prepared from stably transfected Chinese hamster ovary (CHO) cell lines. Initially identified hits were followed up and characterized, and chemoinformatics tools were applied to gain better understanding of the relevance of the results. After a primary screen against α(2B)-AR, 176 compounds of the 17,952 included in the library were declared as active at 10 μM, of which 89 compounds were further selected for potency and affinity determinations using the three human α(2)-AR subtypes. One of the identified positive hits was 2″,2″″-Bisepigallocatechin digallate, which was found to have high affinity at all three human α(2)-AR subtypes. This represents the first non-protonable molecule identified as able to interact with these receptors. Additionally, results obtained with a functional assay (agonist-induced stimulation of [(35)S]GTPγS binding) supported the identification of another positive hit, lysergol, as a partial agonist of the human α(2)-AR subtypes. The dataset of confirmed active chemical species represents a readily available, high quality source for follow-up studies. Altogether, these results provide novel research approaches for drug discovery of modulators of the α(2)-AR subtypes.

  20. Utility of a multiplex reverse transcriptasepolymerase chain reaction assay (HemaVision in the evaluation of genetic abnormalities in Korean children with acute leukemia: a single institution study

    Directory of Open Access Journals (Sweden)

    Hye-Jin kim

    2013-06-01

    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; In children with acute leukemia, bone marrow genetic abnormalities (GA have prognostic significance, and may be the basis for minimal residual disease monitoring. Since April 2007, we have used a multiplex reverse transcriptase-polymerase chain reaction tool (HemaVision to detect of GA. &lt;b&gt;Methods:&lt;/b&gt; In this study, we reviewed the results of HemaVision screening in 270 children with acute leukemia, newly diagnosed at The Catholic University of Korea from April 2007 to December 2011, and compared the results with those of fluorescence in situ hybridization (FISH, and G-band karyotyping. &lt;b&gt;Results:&lt;/b&gt; Among the 270 children (153 males, 117 females, 187 acute lymphoblastic leukemia and 74 acute myeloid leukemia patients were identified. Overall, GA was detected in 230 patients (85.2%. HemaVision, FISH, and G-band karyotyping identified GA in 125 (46.3%, 126 (46.7%, and 215 patients (79.6%, respectively. TEL-AML1 (20.9%, 39/187 and AML1-ETO (27%, 20/74 were the most common GA in ALL and AML, respectively. Overall sensitivity of HemaVision was 98.4%, with false-negative results in 2 instances: 1 each for TEL-AML1 and MLL-AF4 . An aggregate of diseasesspecific FISH showed 100% sensitivity in detection of GA covered by HemaVision for actual probes utilized. G-band karyotype revealed GA other than those covered by HemaVison screening in 133 patients (49.3%. Except for hyperdiplody and hypodiploidy, recurrent GA as defined by the World Health Organizationthat were not screened by HemaVision, were absent in the karyotype. &lt;b&gt;Conclusion:&lt;/b&gt; HemaVision, supported by an aggregate of FISH tests for important translocations, may allow for accurate diagnosis of GA in Korean children with acute leukemia.

  1. Exogenous versus endogenous recovery of 25-hydroxyvitamins D2 and D3 in human samples using high-performance liquid chromatography and the DiaSorin LIAISON Total-D Assay.

    Science.gov (United States)

    Horst, Ronald L

    2010-07-01

    Demand for circulating 25-hydroxyvitamin D [25(OH)D] measurements has exploded due to its relationship with many serious health problems. The present study was designed to investigate the validity of samples "spiked" with 25-hydroxyvitamin D2 [25(OH)D2] or 25-hydroxyvitamin D3 [25(OH)D3] to determine their analytical recovery by the DiaSorin LIAISON 25 OH Vitamin D Total Assay (DiaSorin Assay) and high-performance liquid chromatography (HPLC). 25(OH)D was measured in nine volunteers taking large daily doses of vitamin D2 for 2 weeks. Samples were obtained pre-supplementation and 1 week following vitamin D2. Pre-supplementation samples were used for exogenous recovery studies by adding 25(OH)D2 or 25(OH)D3. Endogenous 25(OH)D [25(OH)D2 plus 25(OH)D3] concentrations reported by the DiaSorin Assay or detected by HPLC were in excellent agreement. However, exogenously added 25(OH)D2 and 25(OH)D3 were under-recovered by the DiaSorin Assay. NIST vitamin D standards containing serum from another species (horse) or exogenous 25(OH)D2 were similarly affected when using the DiaSorin Assay. Exogenous 25(OH)D2, 25(OH)D3 or serum from other species added to human samples is inappropriate in determining the analytical recovery of vitamin D compounds when using the DiaSorin Assay. Only endogenous 25(OH)D2 and/or 25(OH)D3 contained in human blood samples should be utilized for this purpose.

  2. Variation in neoadjuvant chemotherapy utilization for epithelial ovarian cancer at high volume hospitals in the United States and associated survival.

    Science.gov (United States)

    Barber, Emma L; Dusetzina, Stacie B; Stitzenberg, Karyn B; Rossi, Emma C; Gehrig, Paola A; Boggess, John F; Garrett, Joanne M

    2017-06-01

    To estimate variation in the use of neoadjuvant chemotherapy by high volume hospitals and to determine the association between hospital utilization of neoadjuvant chemotherapy and survival. We identified incident cases of stage IIIC or IV epithelial ovarian cancer in the National Cancer Database from 2006 to 2012. Inclusion criteria were treatment at a high volume hospital (>20 cases/year) and treatment with both chemotherapy and surgery. A logistic regression model was used to predict receipt of neoadjuvant chemotherapy based on case-mix predictors (age, comorbidities, stage etc). Hospitals were categorized by the observed-to-expected ratio for neoadjuvant chemotherapy use as low, average, or high utilization hospitals. Survival analysis was performed. We identified 11,574 patients treated at 55 high volume hospitals. Neoadjuvant chemotherapy was used for 21.6% (n=2494) of patients and use varied widely by hospital, from 5%-55%. High utilization hospitals (n=1910, 10 hospitals) had a median neoadjuvant chemotherapy rate of 39% (range 23-55%), while low utilization hospitals (n=2671, 14 hospitals) had a median rate of 10% (range 5-17%). For all ovarian cancer patients adjusting for clinical and socio-demographic factors, treatment at a hospital with average or high neoadjuvant chemotherapy utilization was associated with a decreased rate of death compared to treatment at a low utilization hospital (HR 0.90 95% CI 0.83-0.97 and HR 0.85 95% CI 0.75-0.95). Wide variation exists in the utilization of neoadjuvant chemotherapy to treat stage IIIC and IV epithelial ovarian cancer even among high volume hospitals. Patients treated at hospitals with low rates of neoadjuvant chemotherapy utilization experience decreased survival. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Statistics of assay validation in high throughput cell imaging of nuclear factor kappaB nuclear translocation.

    Science.gov (United States)

    Morelock, Maurice M; Hunter, Edward A; Moran, Timothy J; Heynen, Susanne; Laris, Casey; Thieleking, Michael; Akong, Michael; Mikic, Ivana; Callaway, Scott; DeLeon, Rodney P; Goodacre, Angela; Zacharias, David; Price, Jeffrey H

    2005-10-01

    This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF kappaB antagonist assay (inhibition of IL-1- induced NF kappaB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQtrade mark 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC method is described along with two-point (minimum-maximum) and multiple point titration statistical methods. As a ratio of compartment intensities that tend to change proportionally, FLIN was resistant to photobleaching errors. Two-point minimum-maximum statistical analyses yielded the following: a Z' of 0.174 with the data as n = 320 independent well samples; Z' by row data in a range of 0.393-0.933, with a mean of 0.766; by-plate Z' data of 0.310, 0.443, 0.545, and 0.794; and by-plate means of columns Z' data of 0.879, 0.927, 0.945, and 0.963. The mean 50% inhibitory concentration (IC50) for IL-1 receptor antagonist over all experiments was 213 ng/ml. The combined IC50 coefficients of variation (CVs) were 0.74%, 0.85%, 2.09%, and 2.52% for the four plates. Repeatability IC50 CVs were as follows: day to day 3.0%, row to row 8.0%, plate to plate 2.8%, and day to day 0.6%. The number of cells required for statistically resolvable

  4. Highly sensitive assay for the measurement of serotonin in microdialysates using capillary high-performance liquid chromatography with electrochemical detection.

    Science.gov (United States)

    Parrot, Sandrine; Lambás-Señas, Laura; Sentenac, Sabine; Denoroy, Luc; Renaud, Bernard

    2007-05-01

    A highly sensitive isocratic capillary high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) for the simultaneous measurement of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in microdialysates has been developed using a 0.5 mm i.d. capillary column and a 11-nL detection cell. This method, validated on both pharmacological and analytical bases, can be performed using injection volumes as low as 1 microL. The limits of detection were 5.6 x 10(-11)mol/L and 3.0 x 10(-9)mol/L for 5-HT and 5-HIAA. Several applications of the present method are given on microdialysates from rodent brain and human spinal cord.

  5. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2017-08-29

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  6. A high sensitivity field effect transistor biosensor for methylene blue detection utilize graphene oxide nanoribbon.

    Science.gov (United States)

    Lin, Ting-Chun; Li, Yan-Sheng; Chiang, Wei-Hung; Pei, Zingway

    2017-03-15

    In this work, we developed a field effect transistor (FET) biosensor utilizing solution-processed graphene oxide nanoribbon (GONR) for methylene blue (MB) sensing. MB is a unique material; one of its crucial applications is as a marker in the detection of biomaterials. Therefore, a highly sensitive biosensor with a low detection limit that can be fabricated simply in a noncomplex detection scheme is desirable. GONR is made by unzipping multiwall carbon nanotubes, which can be mass-produced at low temperature. The GONR-FET biosensor demonstrated a sensitivity of 12.5μA/mM (determined according to the drain current difference caused by the MB concentration change). The Raman spectra indicate that the materials quality of the GONR and the domain size for the C=C sp(2) bonding were both improved after MB detection. X-ray photoelectron spectroscopy revealed that the hydroxyl groups on the GONR were removed by the reductive MB. According to XPS and Raman, the positive charge is proposed to transfer from MB to GONR during sensing. This transfer causes charge in-neutrality in the GONR which is compensated by releasing •OH functional groups. With high sensitivity, a low detection limit, and a simple device structure, the GONR-FET sensor is suitable for sensing biomaterials.

  7. Highly intense monocycle terahertz vortex generation by utilizing a Tsurupica spiral phase plate

    Science.gov (United States)

    Miyamoto, Katsuhiko; Kang, Bong Joo; Kim, Won Tae; Sasaki, Yuta; Niinomi, Hiromasa; Suizu, Koji; Rotermund, Fabian; Omatsu, Takashige

    2016-12-01

    Optical vortex, possessing an annular intensity profile and an orbital angular momentum (characterized by an integer termed a topological charge) associated with a helical wavefront, has attracted great attention for diverse applications due to its unique properties. In particular for terahertz (THz) frequency range, several approaches for THz vortex generation, including molded phase plates consisting of metal slit antennas, achromatic polarization elements and binary-diffractive optical elements, have been recently proposed, however, they are typically designed for a specific frequency. Here, we demonstrate highly intense broadband monocycle vortex generation near 0.6 THz by utilizing a polymeric Tsurupica spiral phase plate in combination with tilted-pulse-front optical rectification in a prism-cut LiNbO3 crystal. A maximum peak power of 2.3 MW was obtained for THz vortex output with an expected topological charge of 1.15. Furthermore, we applied the highly intense THz vortex beam for studying unique nonlinear behaviors in bilayer graphene towards the development of nonlinear super-resolution THz microscopy and imaging system.

  8. The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

    Science.gov (United States)

    Ragauskas, Arthur; Akinosho, Hannah; Yee, Kelsey; Close, Dan

    2014-08-01

    First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes towards the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum’s role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum’s amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.

  9. High Hardware Utilization and Low Memory Block Requirement Decoding of QC-LDPC Codes

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ling; LIU Rongke; HOU Yi; ZHANG Xiaolin

    2012-01-01

    This paper presents a simple yet effective decoding for general quasi-cyclic low-density parity-check (QC-LDPC) codes,which not only achieves high hardware utility efficiency (HUE),but also brings about great memory block reduction without any performance degradation.The main idea is to split the check matrix into several row blocks,then to perform the improved message passing computations sequentially block by block.As the decoding algorithm improves,the sequential tie between the two-phase computations is broken,so that the two-phase computations can be overlapped which bring in high HUE.Two overlapping schemes are also presented,each of which suits a different situation.In addition,an efficient memory arrangement scheme is proposed to reduce the great memory block requirement of the LDPC decoder.As an example,for the 0.4 rate LDPC code selected from Chinese Digital TV Terrestrial Broadcasting (DTTB),our decoding saves over 80% memory blocks compared with the conventional decoding,and the decoder achieves 0.97 HUE.Finally,the 0.4 rate LDPC decoder is implemented on an FPGA device EP2S30 (speed grade-5).Using 8 row processing units,the decoder can achieve a maximum net throughput of 28.5 Mbps at 20 iterations.

  10. A noninvasive high frequency oscillation ventilator: Achieved by utilizing a blower and a valve.

    Science.gov (United States)

    Yuan, YueYang; Sun, JianGuo; Wang, Baicun; Feng, Pei; Yang, ChongChang

    2016-02-01

    After the High Frequency Oscillatory Ventilation (HFOV) has been applied in the invasive ventilator, the new technique of noninvasive High Frequency Oscillatory Ventilation (nHFOV) which does not require opening the patient's airway has attracted much attention from the field. This paper proposes the design of an experimental positive pressure-controlled nHFOV ventilator which utilizes a blower and a special valve and has three ventilation modes: spontaneous controlled ventilation combining HFOV, time-cycled ventilation combining HFOV (T-HFOV), and continuous positive airway pressure ventilation combining HFOV. Experiments on respiratory model are conducted and demonstrated the feasibility of using nHFOV through the control of fan and valve. The experimental ventilator is able to produce an air flow with small tidal volume (VT) and a large minute ventilation volume (MV) using regular breath tubes and nasal mask (e.g., under T-HFOV mode, with a maximum tidal volume of 100 ml, the minute ventilation volume reached 14,400 ml). In the process of transmission, there is only a minor loss of oscillation pressure. (Under experimental condition and with an oscillation frequency of 2-10 Hz, peak pressure loss was around 0%-50% when it reaches the mask.).

  11. The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

    Energy Technology Data Exchange (ETDEWEB)

    Akinsho, Hannah [Georgia Institute of Technology, Atlanta; Yee, Kelsey L [ORNL; Close, Daniel M [ORNL; Ragauskas, Arthur [University of Tennessee, Knoxville

    2014-01-01

    First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing (CBP) applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes toward the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum's role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum's amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.

  12. The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

    Directory of Open Access Journals (Sweden)

    Arthur eRagauskas

    2014-08-01

    Full Text Available First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes towards the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum’s role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum’s amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.

  13. Utilization of potato starch processing wastes to produce animal feed with high lysine content.

    Science.gov (United States)

    Li, Ying; Liu, Bingnan; Song, Jinzhu; Jiang, Cheng; Yang, Qian

    2015-02-01

    This work aims to utilize wastes from the potato starch industry to produce single-cell protein (SCP) with high lysine content as animal feed. In this work, S-(2-aminoethyl)-L-cysteine hydrochloride-resistant Bacillus pumilus E1 was used to produce SCP with high lysine content, whereas Aspergillus niger was used to degrade cellulose biomass and Candida utilis was used to improve the smell and palatability of the feed. An orthogonal design was used to optimize the process of fermentation for maximal lysine content. The optimum fermentation conditions were as follows: temperature of 40°C, substrate concentration of 3%, and natural pH of about 7.0. For unsterilized potato starch wastes, the microbial communities in the fermentation process were determined by terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes. Results showed that the dominant population was Bacillus sp. The protein quality as well as the amino acid profile of the final product was found to be significantly higher compared with the untreated waste product at day 0. Additionally, acute toxicity test showed that the SCP product was non-toxic, indicating that it can be used for commercial processing.

  14. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    Full Text Available BACKGROUND: High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. CONCLUSIONS: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  15. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.

    2007-01-01

    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery....... The 'functionality' assay displayed concentration dependent sensitivity to interference for ammonium sulphate and Tris(hydroxymethyl)-amino-methane, but was essentially unaffected by all other salts and buffer combinations tested. The immunoturbidimetric assays described here are generically applicable to polyclonal...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

  16. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  17. A high throughput colorimetric assay of β-1,3-D-glucans by Congo red dye.

    Science.gov (United States)

    Semedo, Magda C; Karmali, Amin; Fonseca, Luís

    2015-02-01

    Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (>20 nm) in UV-Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-D-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-D-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-D-glucans was investigated in several mushroom species. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. 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.

  19. Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis.

    Science.gov (United States)

    Saito, Ryoichi; Kasai, Ayako; Ogihara, Shinji; Yamada, Kageto; Tao, Kazuyuki

    2015-09-01

    We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

  20. Evaluation of a novel real-time fluorescent polymerase chain reaction assay for high-risk human papilloma virus DNA genotypes in cytological cervical screening

    OpenAIRE

    Cheng, Jiaoying; BIAN, MEILU; CONG, XIAO; SUN, AIPING; Li, Min; Ma, Li; Chen, Ying; Liu,Jun

    2012-01-01

    It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses T...

  1. "How I do it: utilization of high-pressure sealants in aortic reconstruction"

    Directory of Open Access Journals (Sweden)

    Elefteriades John A

    2009-06-01

    Full Text Available Abstract Background Suture-line hemostasis, reinforcement of friable tissue, and adhesion prevention are key concerns for patients undergoing cardiac surgery for aortic reconstruction. Failure to secure hemostasis at anastomotic junctures and reinforce fragile tissue may lead to increased blood loss, additional blood product requirements, increased operative time, and, in extreme cases, reoperation. Patients with aortic pathology may also be at higher risk for reoperation, and adhesion formation from prior surgery is an added risk at resternotomy. The advent of high-pressure sealants has been of benefit in helping to alleviate these perioperative challenges. Methods The author utilizes two high-pressure sealants for aortic reconstructive procedures. The first is made of two polymers of polyethylene glycol (PEG [Coseal®, Baxter Healthcare, Corporation], and is used to secure anastomotic suture-line hemostasis and for adhesion prevention. The second is a bovine serum albumin-glutaraldehyde (BSAG glue [BioGlue®, CryroLife, Inc.], used for the repair of dissected aortic tissue and in reinforcing ("tanning" fragile aortic tissues. The techniques for application in select aortic reconstruction procedures are described. Results To substantiate the hemostatic clinical benefit observed by the author, 60 consecutive major thoracic aortic operations in 57 patients in whom PEG sealant was used were retrospectively reviewed. Although comparisons with other agents were not performed for this descriptive report, bleeding results were very favorable for these types of operations. The strong clinical impression is that topical hemostatic application of PEG sealant to anastomotic suture lines is helpful in preventing bleeding. Conclusion In major aortic reconstructive procedures the need for anastomotic sealing performance, reinforcement of friable tissues, and adhesion prevention should not be underrated. High-pressure surgical sealants represent an important

  2. The utility of high b-value DWI in evaluation of ischemic stroke at 3 T

    Energy Technology Data Exchange (ETDEWEB)

    Cihangiroglu, Mutlu, E-mail: mcihangiroglu@yeditepe.edu.tr [Department of Radiology, Yeditepe University Hospital, Istanbul (Turkey); Citci, Beyza, E-mail: bcitci@yeditepe.edu.tr [Department of Neurology, Yeditepe University Hospital, Istanbul (Turkey); Kilickesmez, Ozgur, E-mail: okilickesmez@yeditepe.edu.tr [Department of Radiology, Yeditepe University Hospital, Istanbul (Turkey); Firat, Zeynep, E-mail: zfirat@yeditepe.edu.tr [Department of