Sample records for drug screening assay

  1. Assay to Screen Anti-metastatic Drugs

    Cancer.gov

    Scientists at the NCI's Mouse Cancer Genetics Program have developed a research tool, a murine cell line model (JygMC(A)) with a reporter construct, of spontaneous metastatic mammary carcinoma that resembles the human breast cancer metastatic process in a triple negative mammary tumor. The assay is useful for screening compounds that specifically inhibit pathways involved in mammary carcinoma and can improve clinical management of of triple negative breast cancer that are greatly refractory to conventional chemo and radiotherapy.

  2. New colorimetric cytotoxicity assay for anticancer-drug screening.

    PubMed

    Skehan, P; Storeng, R; Scudiero, D; Monks, A; McMahon, J; Vistica, D; Warren, J T; Bokesch, H; Kenney, S; Boyd, M R

    1990-07-01

    We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry. PMID:2359136

  3. Simple Fibroblast-Based Assay for Screening of New Antimicrobial Drugs against Mycobacterium tuberculosis

    Microsoft Academic Search

    Takemasa Takii; Yoshifumi Yamamoto; Taku Chiba; Chiyoji Abe; John T. Belisle; Patrick J. Brennan; Kikuo Onozaki

    2002-01-01

    In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimyco- bacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly

  4. 3D inverted colloidal crystals in realistic cell migration assays for drug screening applications.

    PubMed

    da Silva, Joakim; Lautenschläger, Franziska; Kuo, Cheng-Hwa R; Guck, Jochen; Sivaniah, Easan

    2011-12-01

    Screening drugs for their specific impact on cell mechanics, in addition to targeting adhesion and proteolysis, will be important for successfully moderating migration in infiltrative disorders including cancer metastasis. We present 3D inverted colloidal crystals made of hydrogel as a realistic cell migration assay, where the geometry and stiffness can be set independently to mimic the tissue requirements in question. We show the utility of this 3D assay for drug screening purposes, specifically in contrast to conventional 2D migration studies, by surveying the effects of commonly used cytoskeletal toxins that impact cell mechanics. This assay allows studying large cell numbers for good statistics but at single-cell resolution. PMID:22038190

  5. Radioligand binding assays in the drug discovery process: potential pitfalls of high throughput screenings.

    PubMed

    Noël, F; Mendonça-Silva, D L; Quintas, L E

    2001-02-01

    Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results. PMID:11258048

  6. Development of a yeast bio-assay to screen anti-mitochondrial drugs.

    PubMed

    Kock, Johan L F; Swart, Chantel W; Ncango, Desmond M; Kock, Johan L F; Munnik, Ingrid A; Maartens, Marleen M J; Pohl, Carolina H; van Wyk, Pieter W J

    2009-09-01

    Previous studies show that acetylsalicylic acid (aspirin) at low concentrations affects yeast sexual structure development in a similar fashion than oxygen depletion. This is ascribed to its anti-mitochondrial action. In this study, we report the same for other anti-inflammatory (i.e. ibuprofen, indomethacin, salicylic acid, benzoic acid) as well as anticancer (Lonidamine) drugs, also known for inhibiting mitochondrial activity in mammalian cells. This is shown by a unique yeast bio-assay, with the mitochondrion-dependent sexual structure, riboflavin production, and hyphal morphology of the yeast Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique "anoxic" morphological pattern for this yeast. Only drugs associated with anti-mitochondrial activity presented such a pattern. This bio-assay may find application in the screening for novel drugs from various sources with anti-mitochondrial actions. PMID:19496752

  7. A Generally Applicable, High-Throughput Screening–Compatible Assay to Identify, Evaluate, and Optimize Antimicrobial Agents for Drug Therapy

    Microsoft Academic Search

    Gerald Kleymann; Hans-Otto Werling

    2004-01-01

    Efficacy and tolerability are the key criteria for a successful medication in the clinic. Therefore, a new test method to obtain selective and active lead molecules has been developed. Recently, this novel screening strategy enabled a breakthrough in drug discovery in the field of herpes viruses. Here the authors report that this assay is a generally applicable screening test, which

  8. Cheburator Software for Automatically Calculating Drug Inhibitory Concentrations from In Vitro Screening Assays

    PubMed Central

    Nevozhay, Dmitry

    2014-01-01

    In the process of new cancer drug development, as the first step of their assessment, their activities are usually studied in vitro against a panel of cancer cell lines. The results of these in vitro drug screening assays are commonly expressed as inhibitory concentration 50% (IC50): the concentration of the tested agent that inhibits the proliferation of the cancer cell population to 50% of the theoretically possible effect (absolute IC50) or maximum effect practically achieved by the drug (relative IC50). The currently available software for calculating IC50 values requires manual data entry, is time consuming, and is prone to calculation errors. Thus, we have developed open source, free, easy-to-use software for performing standardized data evaluations and automatically calculating the IC50. This software eliminates the laborious and error-prone manual entry of data, substantially reduces the amount of time spent for data analysis. It has been extensively used in our department as the main tool for in vitro data processing during the past several years and can be useful for other research groups working in the area of anticancer drug discovery, either alone or combined with other software packages. The current version of our program, Cheburator, together with sample data, source code, and documentation, is freely available at the following URL: http://www.cheburator.nevozhay.com (it is free for academic use, but a license is required for commercial use). PMID:25184280

  9. Development and Validation of a Luminescence-based, Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

    PubMed Central

    Lalli, Cristiana; Guidi, Alessandra; Gennari, Nadia; Altamura, Sergio; Bresciani, Alberto; Ruberti, Giovina

    2015-01-01

    Background Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. Methodology/Principal Findings The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. Conclusions The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies. PMID:25635836

  10. Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

    PubMed Central

    Varela, Rubén Eduardo M; Muñoz, Diana Lorena; Robledo, Sara M.; Kolli, Bala K.; Dutta, Sujoy; Chang, Kwang Poo; Muskus, Carlos

    2013-01-01

    Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known anti-leishmanial drugs, i. e. amphotericin B and glucantime®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus. PMID:19303871

  11. Model for high-throughput screening of multitarget drugs in chemical neurosciences: synthesis, assay, and theoretic study of rasagiline carbamates.

    PubMed

    Alonso, Nerea; Caamaño, Olga; Romero-Duran, Francisco J; Luan, Feng; D S Cordeiro, M Natália; Yañez, Matilde; González-Díaz, Humberto; García-Mera, Xerardo

    2013-10-16

    The disappointing results obtained in recent clinical trials renew the interest in experimental/computational techniques for the discovery of neuroprotective drugs. In this context, multitarget or multiplexing QSAR models (mt-QSAR/mx-QSAR) may help to predict neurotoxicity/neuroprotective effects of drugs in multiple assays, on drug targets, and in model organisms. In this work, we study a data set downloaded from CHEMBL; each data point (>8000) contains the values of one out of 37 possible measures of activity, 493 assays, 169 molecular or cellular targets, and 11 different organisms (including human) for a given compound. In this work, we introduce the first mx-QSAR model for neurotoxicity/neuroprotective effects of drugs based on the MARCH-INSIDE (MI) method. First, we used MI to calculate the stochastic spectral moments (structural descriptors) of all compounds. Next, we found a model that classified correctly 2955 out of 3548 total cases in the training and validation series with Accuracy, Sensitivity, and Specificity values>80%. The model also showed excellent results in Computational-Chemistry simulations of High-Throughput Screening (CCHTS) experiments, with accuracy=90.6% for 4671 positive cases. Next, we reported the synthesis, characterization, and experimental assays of new rasagiline derivatives. We carried out three different experimental tests: assay (1) in the absence of neurotoxic agents, assay (2) in the presence of glutamate, and assay (3) in the presence of H2O2. Compounds 11 with 27.4%, 8 with 11.6%, and 9 with 15.4% showed the highest neuroprotective effects in assays (1), (2), and (3), respectively. After that, we used the mx-QSAR model to carry out a CCHTS of the new compounds in >400 unique pharmacological tests not carried out experimentally. Consequently, this model may become a promising auxiliary tool for the discovery of new drugs for the treatment of neurodegenerative diseases. PMID:23855599

  12. Model for High-Throughput Screening of Multitarget Drugs in Chemical Neurosciences: Synthesis, Assay, and Theoretic Study of Rasagiline Carbamates

    PubMed Central

    2013-01-01

    The disappointing results obtained in recent clinical trials renew the interest in experimental/computational techniques for the discovery of neuroprotective drugs. In this context, multitarget or multiplexing QSAR models (mt-QSAR/mx-QSAR) may help to predict neurotoxicity/neuroprotective effects of drugs in multiple assays, on drug targets, and in model organisms. In this work, we study a data set downloaded from CHEMBL; each data point (>8000) contains the values of one out of 37 possible measures of activity, 493 assays, 169 molecular or cellular targets, and 11 different organisms (including human) for a given compound. In this work, we introduce the first mx-QSAR model for neurotoxicity/neuroprotective effects of drugs based on the MARCH-INSIDE (MI) method. First, we used MI to calculate the stochastic spectral moments (structural descriptors) of all compounds. Next, we found a model that classified correctly 2955 out of 3548 total cases in the training and validation series with Accuracy, Sensitivity, and Specificity values > 80%. The model also showed excellent results in Computational-Chemistry simulations of High-Throughput Screening (CCHTS) experiments, with accuracy = 90.6% for 4671 positive cases. Next, we reported the synthesis, characterization, and experimental assays of new rasagiline derivatives. We carried out three different experimental tests: assay (1) in the absence of neurotoxic agents, assay (2) in the presence of glutamate, and assay (3) in the presence of H2O2. Compounds 11 with 27.4%, 8 with 11.6%, and 9 with 15.4% showed the highest neuroprotective effects in assays (1), (2), and (3), respectively. After that, we used the mx-QSAR model to carry out a CCHTS of the new compounds in >400 unique pharmacological tests not carried out experimentally. Consequently, this model may become a promising auxiliary tool for the discovery of new drugs for the treatment of neurodegenerative diseases. PMID:23855599

  13. Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity Assay for hERG Channels

    PubMed Central

    Su, Xiaojing; Young, Edmond W.K.; Underkofler, Heather A. S.; Kamp, Timothy J.; January, Craig T.; Beebe, David J.

    2011-01-01

    Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval, and hence risk for life threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. We validate our microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP) and poly(dimethylsiloxane) (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG), and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Our results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. We demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. We further showed that experimental procedures can be streamlined by using direct in-channel immunofluorescent staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with five different drugs suggested that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules. PMID:21131594

  14. A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility

    PubMed Central

    Smout, Michael J.; Kotze, Andrew C.; McCarthy, James S.; Loukas, Alex

    2010-01-01

    Background Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. PMID:21103363

  15. DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

    PubMed

    Miyagi, Toru; Shiotani, Bunsyo; Miyoshi, Ryuya; Yamamoto, Takuya; Oka, Takanori; Umezawa, Kazuo; Ochiya, Takahiro; Takano, Mikihisa; Tahara, Hidetoshi

    2014-07-01

    Nuclear factor-?B (NF-?B) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-?B, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-?B (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-?B binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-?B binding site of the competitor. Evaluation of two NF-?B inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-?B), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins. PMID:24724610

  16. Homogeneous Cell and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

    Microsoft Academic Search

    Sheri Miraglia; Elana E. Swartzman; Julia Mellentin-Michelotti; Lolita Evangelista; Christopher Smith; Iwan Gunawan; Kenton Lohman; Edward M. Goldberg; Bala Manian; Pau-Miau Yuan

    1999-01-01

    High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay

  17. Current status of targets and assays for anti-HIV drug screening

    Microsoft Academic Search

    Ren-rong Tian; Qing-jiao Liao; Xu-lin Chen

    2007-01-01

    HIV\\/AIDS is one of the most serious public health challenges globally. Despite the great efforts that are being devoted to\\u000a prevent, treat and to better understand the disease, it is one of the main causes of morbidity and mortality worldwide. Currently,\\u000a there are 30 drugs or combinations of drugs approved by FDA. Because of the side-effects, price and drug resistance,

  18. Development of High-throughput and Robust Microfluidic Live Cell Assay Platforms for Combination Drug and Toxin Screening

    E-print Network

    Wang, Han

    2012-02-14

    ................................................................................................... ix CHAPTER I INTRODUCTION ................................................................................ 1 1.1. Combination Chemotherapy ........................................................ 1 1.2. Microfluidic Live Cell Assay... ....................................................... 2 1.3. Microfluidic High-throughput Screening Systems ...................... 5 1.4. Microfluidic Single Cell Assay Platforms ................................... 7 1.5. Organization of the Thesis...

  19. High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

    PubMed

    Fonsi, Massimiliano; Orsale, Maria V; Monteagudo, Edith

    2008-10-01

    In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. PMID:18812573

  20. A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors: Implications for Antiviral Drug Development

    PubMed Central

    GEISS, BRIAN J.; STAHLA-BEEK, HILLARY J.; HANNAH, AMANDA M.; GARI, HAMID H.; HENDERSON, BRITTNEY R.; SAEEDI, BEJAN J.; KEENAN, SUSAN M.

    2012-01-01

    There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5? end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development. PMID:21788392

  1. Rapid identification and drug susceptibility screening of ESAT-6 secreting Mycobacteria by a NanoELIwell assay.

    PubMed

    Nguyen, Yen H; Ma, Xin; Qin, Lidong

    2012-01-01

    To meet the global needs of tuberculosis (TB) control, a nanoELIwell device was developed as a multifunctional assay for TB diagnosis and drug susceptibility testing. The device integrates on-chip culturing of mycobacteria, immunoassay, and high-resolution fluorescent imaging. Mycobacterium smegmatis and Mycobacterium kansasii were used as models of Mycobacterium tuberculosis to evaluate device integrity by using antigens, Ag85 and ESAT-6, as biomarkers. As a result, the nanoELIwell device detected antigens released from a single bacterium within 24-48-hour culture. Antimycobacterial drug-treated M. smegmatis showed significant decreased in Ag85 antigen production when treated with ethambutol and no change in antigen production when treated with rifampin, demonstrating drug susceptibility and resistance, respectively. The nanoELIwell assay also distinguished the ESAT-6-secreting M. kansasii from the non-ESAT-6-secreting M. simiae. The combination of microwell technology and ELISA assay holds potential to the development of a rapid, sensitive, and specific diagnostics and susceptibility testing of TB. PMID:22957139

  2. A 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase

    Microsoft Academic Search

    Mesens Natalie; Steemans Margino; Hansen Erik; Peters Annelieke; Verheyen Geert; Vanparys Philippe

    2009-01-01

    Drug-induced phospholipidosis is caused by lysosomal accumulation of the drug, resulting in the disturbance of phospholipid degradation and a consequent excessive phospholipid accumulation. Depending on the type and number of tissues affected, phospholipidosis occurrence in test animals can raise safety issues, which may be critical for the risk assessment. Safety profiling of potential phospholipidosis-inducing drugs in the drug discovery phase

  3. Development of a phenotypic high-content assay to identify pharmacoperone drugs for the treatment of primary hyperoxaluria type 1 by high-throughput screening.

    PubMed

    Madoux, Franck; Janovick, Jo Ann; Smithson, David; Fargue, Sonia; Danpure, Christopher J; Scampavia, Louis; Chen, Yih-Tai; Spicer, Timothy P; Conn, P Michael

    2015-01-01

    Primary hyperoxaluria is a severe disease for which the best current therapy is dialysis or organ transplantation. These are risky, inconvenient, and costly procedures. In some patients, pyridoxine treatment can delay the need for these surgical procedures. The underlying cause of particular forms of this disease is the misrouting of a specific enzyme, alanine:glyoxylate aminotransferase (AGT), to the mitochondria instead of the peroxisomes. Pharmacoperones are small molecules that can rescue misfolded proteins and redirect them to their correct location, thereby restoring their function and potentially curing disease. In the present study, we miniaturized a cell-based assay to identify pharmacoperone drugs present in large chemical libraries to selectively correct AGT misrouting. This assay employs AGT-170, a mutant form of AGT that predominantly resides in the mitochondria, which we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. Over the course of a pilot screen of 1,280 test compounds, we achieved an average Z'-factor of 0.72±0.02, demonstrating the suitability of this assay for HTS. PMID:25710543

  4. Fluorescence Polarization Assays in Small Molecule Screening

    PubMed Central

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  5. Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.

    PubMed

    Gong, Edwin Yunhao; Bonfanti, Jean-François; Ivens, Tania; Van der Auwera, Marijke; Van Kerckhove, Barbara; Kraus, Guenter

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Réunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described. PMID:23821286

  6. Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay1

    Microsoft Academic Search

    Michael C. Alley; Dominic A. Scudiere; Anne Monks; Miriam L. Hursey; Maciej J. Czerwinski; Donald L. Fine; Betty J. Abbott; Joseph G. Mayo; Robert H. Shoemaker; Michael R. Boyd

    1988-01-01

    For the past 30 years strategies for the prcclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro\\/in vivo screening for selective

  7. Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

    PubMed

    Miraglia; Swartzman; Mellentin-Michelotti; Evangelista; Smith; Gunawan; Lohman; Goldberg; Manian; Yuan

    1999-01-01

    High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software. PMID:10838439

  8. TOXICITY SCREENING WITH ZEBRAFISH ASSAY

    EPA Science Inventory

    The proposed toxicity screening will help EPA to prioritize chemicals for further testing, and it may also alert chemical manufacturers that some of their commercial products may be toxic. The proposed toxicity pathway studies will improve the research community’s abi...

  9. A Novel Leishmania major Amastigote Assay in 96-Well Format for Rapid Drug Screening and Its Use for Discovery and Evaluation of a New Class of Leishmanicidal Quinolinium Salts

    PubMed Central

    Thomale, Katja; Bischof, Sebastian; Schneider, Christoph; Schultheis, Martina; Schwarz, Tobias; Moll, Heidrun

    2013-01-01

    In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC50s against intracellular amastigotes (IC50 < 1 ?g/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 ?g/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity. PMID:23587955

  10. Image-Based Screening of Signal Transduction Assays

    NSDL National Science Digital Library

    Peter Ramm (Brock University; Imaging Research REV)

    2003-04-08

    Imaging techniques have played a vital role in signal transduction research over several decades. Recently, industrialized macro- and micro-imaging systems have found application in drug discovery laboratories, where they increase the throughput and efficiency of drug screening. Macro-imagers are used for primary screening, where they favor compound conservation (through assay miniaturization), and achieve unprecedented rates of throughput. Micro-imaging systems achieve relatively high throughput, at the same time providing sub-cellular resolution with fixed or living cells. These micro-imaging analyses were previously conducted at very low throughput and, typically, were the sole domain of the academic researcher. Although both macro and micro forms of image-based screening remain technologies in development, they have already made substantial contributions to screening programs and will continue to do so.

  11. Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries

    E-print Network

    Hammock, Bruce D.

    Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted librariesPhos as the substrate. This assay was used to screen a wide variety of chemical entities, includ- ing a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides

  12. High-content screening for biofilm assays.

    PubMed

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

    2010-08-01

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

  13. A new, sensitive ecto-5'-nucleotidase assay for compound screening.

    PubMed

    Freundlieb, Marianne; Zimmermann, Herbert; Müller, Christa E

    2014-02-01

    Ecto-5'-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5'-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [³H]adenosine-5'-monophosphate (AMP) as a substrate. The reaction product [³H]adenosine was separated from [³H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [³H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z' factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay. PMID:24144488

  14. Nonradioactive trans-sialidase screening assay.

    PubMed

    Schrader, Silke; Schauer, Roland

    2006-01-01

    Trans-sialidase (TS; E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3-linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor, TS acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in TS has increased rapidly in recent years owing to its great relevance to the pathogenicity of trypanosomes and its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates. Recently, the authors described a newly developed nonradioactive screening test for monitoring TS activity (1). In this highly sensitive and specific assay, 4-methylumbelliferyl-beta-D-galactoside is used as acceptor substrate and sialyllactose as donor to fluorimetrically detect enzyme activity in the low mU range (approximately 0.1-1 mU/mL possible). The test can be applied to screen a large number of samples quickly and reliably during enzyme purification, for testing inhibitors, and for monitoring TS activity during the production of monoclonal antibodies (2). This chapter focuses on the main steps of this assay and gives detailed instructions for performing a nonradioactive TS 96-well-plate fluorescence test. In addition, it describes the controls necessary when starting to monitor an unknown TS and facts to be considered when testing new substrates and inhibitors. PMID:17072006

  15. A high-throughput three-dimensional cell migration assay for toxicity screening

    E-print Network

    ­5 . In vitro assays have been used as early screens and cheaper alternatives to animal models cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated screens for drug toxicity use animal models that are similar in composition and structure to the human

  16. Complement C5a receptor assay for high throughput screening

    SciTech Connect

    Harris, S.R.; Garlick, R.K.; Miller, J.J. Jr.; Harney, H.N.; Monroe, P.J. (E.I.Du Pont de Nemours, Biotechnology Systems Division, Billerica, MA (United States))

    1991-01-01

    The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a dilute, ready to use concentration. (125I)-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10-40 pM and there are 50,000-100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.

  17. Complement C5a receptor assay for high throughput screening.

    PubMed

    Harris, S R; Garlick, R K; Miller, J J; Harney, H N; Monroe, P J

    1991-01-01

    The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a dilute, ready to use concentration. [125I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10-40 pM and there are 50,000-100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor. PMID:1886076

  18. A RAINBOW TROUT TISSUE SLICE ASSAY FOR SCREENING ENVIRONMENTAL ESTROGENS

    EPA Science Inventory

    An in vitro fish liver tissue assay has been developed to test for estrogenic and anti-estrogenic effects of xenobiotics. The assay is part of a broader effort to provide multi-level screening of chemicals to enhance......

  19. Comparison of alamar blue and MTT assays for high through-put screening.

    PubMed

    Hamid, R; Rotshteyn, Y; Rabadi, L; Parikh, R; Bullock, P

    2004-10-01

    The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol. PMID:15251189

  20. A novel assay for high-throughput screening of anti-Alzheimer’s disease drugs to determine their efficacy by real-time monitoring of changes in PC12 cell proliferation

    PubMed Central

    HOU, XUE-QIN; YAN, RONG; YANG, CONG; ZHANG, LEI; SU, RU-YU; LIU, SI-JUN; ZHANG, SHI-JIE; HE, WEN-QING; FANG, SHU-HUAN; CHENG, SHU-YI; SU, ZI-REN; CHEN, YUN-BO; WANG, QI

    2014-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by the accumulation of senile plaque and neurofibrilary tangle formation in the brain, including the cerebral cortex and hippocampus. Nowadays, the first-line treatment for AD is the application of acetylcholinesterase inhibitors. However, acetylcholinesterase inhibitors are basically anti-symptomatic for a limited aspect of AD pathology and are associated with serious side-effects. With the advantage of multiple targets, pathways and systems, Chinese herbal compounds hold promising potential for the development of drugs for the treatment of AD. Over the past few years, with the development of Chinese herbal compounds and in vitro pharmacological studies, cell-based disease models are one of the main methods used to screen Chinese herbal compounds for potential efficacy. Testing the efficacy of possible anti-Alzheimer’s disease drugs and the development of new drugs are hindered by the lack of objective high-throughput screening methods. Currently, the assessment of the effects of drugs is usually made by MTT assays, involving laborious, subjective, low-throughput methods. Herein, we suggest a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess the effective composition of Chinese herbal compounds by assessing amyloid-? peptide A?1-42-induced apoptosis in PC12 cells. We detected the proliferation and motility of the cells using a fully automated high-throughput and real-time system. We quantitatively assessed cell motility and determined the real-time IC50 values of various anti-AD drugs that intervene in several developmental stages of A?1-42-induced apoptosis in PC12 cells, Then, we identified the optimal time phase by curative efficacy. Our data indicate that this technique may aid in the discovery and development of novel anti-Alzheimer’s disease drugs. It is possible to utilize a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance of 9–12. The impedance is displayed as a dimensionless parameter termed the cell index, which is directly proportional to the total area of tissue culture well that is covered by the cells. Hence, the cell index can be used to monitor cell adhesion, spreading, morphological variation and cell density. PMID:24378397

  1. Two High Throughput Screen Assays for Measurement of TNF-? in THP-1 Cells

    PubMed Central

    Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

    2011-01-01

    Tumor Necrosis Factor-? (TNF-?), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-?, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-? production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-? assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-? assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-? assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-? inhibitors. We also identified several novel inhibitors of TNF-?, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-? assays are robust and suitable for high throughput screening. PMID:21643507

  2. Combined Analysis of Phenotypic and Target-Based Screening in Assay Networks.

    PubMed

    Swamidass, S Joshua; Schillebeeckx, Constantino N; Matlock, Matthew; Hurle, Mark R; Agarwal, Pankaj

    2014-02-21

    Small-molecule screens are an integral part of drug discovery. Public domain data in PubChem alone represent more than 158 million measurements, 1.2 million molecules, and 4300 assays. We conducted a global analysis of these data, building a network of assays and connecting the assays if they shared nonpromiscuous active molecules. This network spans both phenotypic and target-based screens, recapitulates known biology, and identifies new polypharmacology. Phenotypic screens are extremely important for drug discovery, contributing to the discovery of a large proportion of new drugs. Connections between phenotypic and biochemical, target-based screens can suggest strategies for repurposing both small-molecule and biologic drugs. For example, a screen for molecules that prevent cell death from a mutated version of superoxide-dismutase is linked with ALOX15. This connection suggests a therapeutic role for ALOX15 inhibitors in amyotrophic lateral sclerosis. An interactive version of the network is available online (http://swami.wustl.edu/flow/assay_network.html). PMID:24563424

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

    PubMed Central

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

    2009-01-01

    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. PMID:19492073

  4. Phenotypic screening in cancer drug discovery - past, present and future.

    PubMed

    Moffat, John G; Rudolph, Joachim; Bailey, David

    2014-08-01

    There has been a resurgence of interest in the use of phenotypic screens in drug discovery as an alternative to target-focused approaches. Given that oncology is currently the most active therapeutic area, and also one in which target-focused approaches have been particularly prominent in the past two decades, we investigated the contribution of phenotypic assays to oncology drug discovery by analysing the origins of all new small-molecule cancer drugs approved by the US Food and Drug Administration (FDA) over the past 15 years and those currently in clinical development. Although the majority of these drugs originated from target-based discovery, we identified a significant number whose discovery depended on phenotypic screening approaches. We postulate that the contribution of phenotypic screening to cancer drug discovery has been hampered by a reliance on 'classical' nonspecific drug effects such as cytotoxicity and mitotic arrest, exacerbated by a paucity of mechanistically defined cellular models for therapeutically translatable cancer phenotypes. However, technical and biological advances that enable such mechanistically informed phenotypic models have the potential to empower phenotypic drug discovery in oncology. PMID:25033736

  5. An axenic amastigote system for drug screening.

    PubMed Central

    Callahan, H L; Portal, A C; Devereaux, R; Grogl, M

    1997-01-01

    Currently available primary screens for selection of candidate antileishmanial compounds are not ideal. The choices include screens that are designed to closely reflect the situation in vivo but are labor-intensive and expensive (intracellular amastigotes and animal models) and screens that are designed to facilitate rapid testing of a large number of drugs but do not use the clinically relevant parasite stage (promastigote model). The advent of successful in vitro culture of axenic amastigotes permits the development of a primary screen which is quick and easy like the promastigote screen but still representative of the situation in vivo, since it uses the relevant parasite stage. We have established an axenic amastigote drug screening system using a Leishmania mexicana strain (strain M379). A comparison of the 50% inhibitory concentration (IC50) drug sensitivity profiles of M379 promastigotes, intracellular amastigotes, and axenic amastigotes for six clinically relevant antileishmanial drugs (sodium stibogluconate, meglumine antimoniate, pentamidine, paromomycin, amphotericin B, WR6026) showed that M379 axenic amastigotes are a good model for a primary drug screen. Promastigote and intracellular amastigote IC50s differed for four of the six drugs tested by threefold or more; axenic amastigote and intracellular amastigote IC50s differed by twofold for only one drug. This shows that the axenic amastigote susceptibility to clinically used reference drugs is comparable to the susceptibility of amastigotes in macrophages. These data also suggest that for the compounds tested, susceptibility is intrinsic to the parasite stage. This contradicts previous hypotheses that suggested that the activities of antimonial agents against intracellular amastigotes were solely a function of the macrophage. PMID:9087496

  6. Synthetic tumor networks for screening drug delivery systems.

    PubMed

    Prabhakarpandian, Balabhaskar; Shen, Ming-Che; Nichols, Joseph B; Garson, Charles J; Mills, Ivy R; Matar, Majed M; Fewell, Jason G; Pant, Kapil

    2015-03-10

    Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. A key factor is tumor microvasculature with complex effects including convective transport, high interstitial pressure and enhanced vascular permeability due to the presence of "leaky vessels". Current in vitro models of the tumor microenvironment for evaluating drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. In this study, we report on the development of a novel microfluidic platform that models the tumor microenvironment more accurately, with physiologically and morphologically realistic microvasculature including endothelial cell lined leaky capillary vessels along with 3D solid tumors. Endothelial cells and 3D spheroids of cervical tumor cells were co-cultured in the networks. Drug vehicle screening was demonstrated using GFP gene delivery by different formulations of nanopolymers. The synthetic tumor network was successful in predicting in vivo delivery efficiencies of the drug vehicles. The developed assay will have critical applications both in basic research, where it can be used to develop next generation delivery vehicles, and in drug discovery where it can be used to study drug transport and delivery efficacy in realistic tumor microenvironment, thereby enabling drug compound and/or delivery vehicle screening. PMID:25599856

  7. Lactate as a Novel Quantitative Measure of Viability in Schistosoma mansoni Drug Sensitivity Assays.

    PubMed

    Howe, Stephanie; Zöphel, Dorina; Subbaraman, Harini; Unger, Clemens; Held, Jana; Engleitner, Thomas; Hoffmann, Wolfgang H; Kreidenweiss, Andrea

    2015-02-01

    Whole-organism compound sensitivity assays are a valuable strategy in infectious diseases to identify active molecules. In schistosomiasis drug discovery, larval-stage Schistosoma allows the use of a certain degree of automation in the screening of compounds. Unfortunately, the throughput is limited, as drug activity is determined by manual assessment of Schistosoma viability by microscopy. To develop a simple and quantifiable surrogate marker for viability, we targeted glucose metabolism, which is central to Schistosoma survival. Lactate is the end product of glycolysis in human Schistosoma stages and can be detected in the supernatant. We assessed lactate as a surrogate marker for viability in Schistosoma drug screening assays. We thoroughly investigated parameters of lactate measurement and performed drug sensitivity assays by applying schistosomula and adult worms to establish a proof of concept. Lactate levels clearly reflected the viability of schistosomula and correlated with schistosomulum numbers. Compounds with reported potencies were tested, and activities were determined by lactate assay and by microscopy. We conclude that lactate is a sensitive and simple surrogate marker to be measured to determine Schistosoma viability in compound screening assays. Low numbers of schistosomula and the commercial availability of lactate assay reagents make the assay particularly attractive to throughput approaches. Furthermore, standardization of procedures and quantitative evaluation of compound activities facilitate interassay comparisons of potencies and, thus, concerted drug discovery approaches. PMID:25487803

  8. Reliability of the porphobilinogen screening assay.

    PubMed

    Buttery, J E; Carrera, A M; Pannall, P R

    1990-10-01

    A survey conducted by the Australian Association of Clinical Biochemists Porphyrin Working Party on urinary porphobilinogen screening showed good sensitivity (75-97.5%). This is contrary to reports in the literature and to our own observations. We therefore assessed a widely used screening method (Watson-Schwartz) and found poor sensitivity (40-69%), and even less sensitivity (28-53%) when the urine samples were normally coloured or concentrated. Thus the results obtained by the Working Party might mislead one to infer that the Watson-Schwartz method is reliable. PMID:2091000

  9. Sulfonylureas and Glinides as New PPAR? Agonists:. Virtual Screening and Biological Assays

    NASA Astrophysics Data System (ADS)

    Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph

    2007-12-01

    This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPAR? improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPAR? and exhibit PPAR? agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPAR?. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  10. Screening of antipsychotic drugs in animal models

    Microsoft Academic Search

    Ina Weiner; Inna Gaisler; Daniela Schiller; Amit Green; Lee Zuckerman; Daphna Joel

    2000-01-01

    Behavioral models of antipsychotic drug (APD) action in the rat are widely used for the screening and developing APDs. Valid models are not only required to be selective and specific for APDs, but also to be able to dissociate between typical and atypical APDs. In recent years, newer models have been developed that are claimed to model processes impaired in

  11. Rapid screening assay for calcium bioavailability studies

    SciTech Connect

    Luhrsen, K.R.; Hudepohl, G.R.; Smith, K.T.

    1986-03-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium (/sup 47/Ca) in a whole body retention assay system. In this system, calcium sources are administered by oral gavage and subsequent counts are determined and corrected for isotopic decay. Unlike iron and zinc retention curves, which exhibit a 2-3 day equilibration period, calcium reaches equilibration after 24 hours. Autoradiographic analysis of the femurs indicate that the newly absorbed calcium is rapidly distributed to the skeletal system. Moreover, the isotope is distributed along the entire bone. Comparisons of calcium bioavailability were made using intrinsic/extrinsic labeled milk from two species i.e. rat and goat as well as CaCO/sub 3/. In addition, extrinsic labeled cow milk was examined. In the rat, the extrinsic labeled calcium from milk was better absorbed than the intrinsic calcium. This was not the case in goat milk or the calcium carbonate which exhibited no significant differences. Chromatographic analysis of the labeled milk indicates a difference in distribution of the /sup 47/Ca. From these data, the authors recommend the use of this assay system in calcium bioavailability studies. The labeling studies and comparisons indicate caution should be used, however, in labeling techniques and species milk comparison.

  12. Respirometric acute toxicity screening assay using Daphnia magna

    Microsoft Academic Search

    Alice Zitova; Maud Cross; Robert Hernan; John Davenport; Dmitri B. Papkovsky

    2009-01-01

    Oxygen consumption rate is a universal and sensitive biomarker of viability and metabolic status of aerobic organisms. We applied the optical oxygen sensing method to monitor the respiration of individual Daphnia magna (D. magna) and to develop a simple, automated screening assay for the assessment of acute toxicity of large numbers of chemical and environmental samples. D. magna were exposed

  13. Development of a Molecular Assay for Rapid Screening of

    E-print Network

    Wong, Pak Kin

    Development of a Molecular Assay for Rapid Screening of Chemopreventive Compounds Targeting Nrf2, AZ Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2 biosensor for rapid detection of antioxidant-responsive element (ARE)- bound Nrf2 protein. The development

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

    PubMed

    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. PMID:24871937

  15. Hepatic organoids for microfluidic drug screening.

    PubMed

    Au, Sam H; Chamberlain, M Dean; Mahesh, Shruthi; Sefton, Michael V; Wheeler, Aaron R

    2014-09-01

    We introduce the microfluidic organoids for drug screening (MODS) platform, a digital microfluidic system that is capable of generating arrays of individually addressable, free-floating, three-dimensional hydrogel-based microtissues (or 'organoids'). Here, we focused on liver organoids, driven by the need for early-stage screening methods for hepatotoxicity that enable a "fail early, fail cheaply" strategy in drug discovery. We demonstrate that arrays of hepatic organoids can be formed from co-cultures of HepG2 and NIH-3T3 cells embedded in hydrogel matrices. The organoids exhibit fibroblast-dependent contractile behaviour, and their albumin secretion profiles and cytochrome P450 3A4 activities are better mimics of in vivo liver tissue than comparable two-dimensional cell culture systems. As proof of principle for screening, MODS was used to generate and analyze the effects of a dilution series of acetaminophen on apoptosis and necrosis. With further development, we propose that the MODS platform may be a cost-effective tool in a "fail early, fail cheaply" paradigm of drug development. PMID:24984750

  16. XXIII. In vitro drug assays and statistical analysis

    Microsoft Academic Search

    Chiara Atzori; Elena Angeli; Fiorenza Agostoni; A. Mainini; M. Filippini; Antonietta Cargnel

    1998-01-01

    The development of in vitro drug tests to assess the efficacy of drugs against Pneumocystis carinii has been hindered by the lack of efficient methods for continous cultivation of the microorganism. However, different short-term culture systems have been proposed by many teams. In the present contribution an in vitro microplate drug assay and two statistical programs allowing the analysis of

  17. Measurement of multiple drugs in urine, water, and on surfaces using fluorescence covalent microbead immunosorbent assay.

    PubMed

    Smith, Jerome; Sammons, Deborah; Robertson, Shirley; Biagini, Raymond; Snawder, John

    2010-11-01

    There are a range of applications that require the measurement of multiple drugs such as urine analysis, drug determination in water, and screening for drug contamination on surfaces. Some of the procedures used such as enzyme-linked immunosorbent assay (ELISA) are simple but can only determine one drug at a time, and others such as GC-MS or LC-MS are complex, time-consuming, and expensive. In this study, fluorescence covalent microbead immunosorbent assay (FCMIA) was investigated as a simple method for the measurement of multiple drugs simultaneously in three matrices: diluted urine, water, and on surfaces. Five different drugs of abuse or their metabolites (methamphetamine, caffeine, benzoylecgonine (a metabolite of cocaine), tetrahydrocannabinol (THC), the active ingredient in marijuana, and oxycodone) were studied over the range 0-15?ng/ml. There was no measureable cross-reactivity among the drugs at the concentrations studied. Urine dilutions from 1/50 to 1/2.5 were studied and dilutions less than 1/20 had a significant effect on the methamphetamine assay but limited effects on the benzoylecgonine and oxycodone assays and almost no effect on the THC assay. For assays performed in 1/20 urine dilution, water, and diluted surface sampling buffer, least detectable doses (LDD) were 1?ng/ml or less for the drugs. Surfaces spiked with drugs were sampled with swabs wetted with surface sampling buffer and recoveries were linear over the range 0-100?ng/100?cm(2) surface loading for all drugs. FCMIA has potential to be used for the measurement of multiple drugs in the matrices studied. PMID:20942617

  18. High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

    Microsoft Academic Search

    Francesc Puig-Basagoiti; Tia S. Deas; Ping Ren; Mark Tilgner; David M. Ferguson; Pei-Yong Shi

    2005-01-01

    Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line

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

    PubMed Central

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

    2013-01-01

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

  20. Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

    PubMed Central

    Figuera-Losada, Mariana; Rojas, Camilo; Slusher, Barbara S.

    2014-01-01

    Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor ?B, and mitogen-activated protein kinases. PMID:23945875

  1. High Throughput Screening for Anti–Trypanosoma cruzi Drug Discovery

    PubMed Central

    Alonso-Padilla, Julio; Rodríguez, Ana

    2014-01-01

    The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti–T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti–T. cruzi drug entities in the near future, are reviewed here. PMID:25474364

  2. Structure-based drug screening and ligand-based drug screening with machine learning.

    PubMed

    Fukunishi, Yoshifumi

    2009-05-01

    The initial stage of drug development is the hit (active) compound search from a pool of millions of compounds; for this process, in silico (virtual) screening has been successfully applied. One of the problems of in silico screening, however, is the low hit ratio in relation to the high computational cost and the long CPU time. This problem becomes serious in structure-based in silico screening. The major reason is the low accuracy of the estimation of protein-compound binding free energy. The problem of ligand-based in silico screening is that the conventional quantitative structure-activity relationship (QSAR) approach is not effective at predicting new hit compounds with new scaffolds. Recently, machine-learning approaches have been applied to in silico drug screening to overcome the above problems. We review here machine-learning approaches for both structure-based and ligand-based drug screening. Machine learning is used to improve database enrichment in two ways, namely by improving the docking score calculated by the protein-compound docking program and by calculating the optimal distance between the feature vectors of active and inactive compounds. Both approaches require compounds that are known to be active with respect to the target protein. In structure-based screening, the former approach is mainly used with a protein-compound affinity matrix. In ligand-based screening, both the former and latter approaches are used, and the latter approach can be applied to various kinds of descriptors, such as 1D/2D descriptors/fingerprints and the affinity fingerprint given by the protein-compound affinity matrix. PMID:19442067

  3. Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

    PubMed

    Ordas, Anita; Raterink, Robert-Jan; Cunningham, Fraser; Jansen, Hans J; Wiweger, Malgorzata I; Jong-Raadsen, Susanne; Bos, Sabine; Bates, Robert H; Barros, David; Meijer, Annemarie H; Vreeken, Rob J; Ballell-Pages, Lluís; Dirks, Ron P; Hankemeier, Thomas; Spaink, Herman P

    2015-02-01

    The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans. PMID:25385118

  4. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification . The in vitro HIV drug resistance genotype assay...

  5. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification . The in vitro HIV drug resistance genotype assay...

  6. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification . The in vitro HIV drug resistance genotype assay...

  7. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay...

  8. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866...In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification . The in vitro HIV drug resistance genotype assay...

  9. UrbanSolutionsCenter Developing Detached Plant Assay for Screening Major Turfgrass Diseases

    E-print Network

    UrbanSolutionsCenter Developing Detached Plant Assay for Screening Major Turfgrass Diseases Background Breeding for disease resistance in St. Augustinegrass [Stenotapphrum secundatum (Walt.) Kuntz throughput turfgrass disease screening program, utilizing in vitro techniques allows for screening of large

  10. Human stem cell-derived cardiomyocytes in cellular impedance assays: bringing cardiotoxicity screening to the front line.

    PubMed

    Peters, Matthew F; Lamore, Sarah D; Guo, Liang; Scott, Clay W; Kolaja, Kyle L

    2015-04-01

    Cardiovascular (CV) toxicity is a leading cause of drug attrition and withdrawal. Introducing in vitro assays with higher throughput should permit earlier CV hazard identification and enable medicinal chemists to design-out liabilities. Heretofore, development of in vitro CV assays has been limited by the challenge of replicating integrated cardiovascular physiology while achieving the throughput and consistency required for screening. These challenges appear to be met with a combination of human stem cell-derived cardiomyocytes (CM) which beat spontaneously and monitoring the response with technology that can assess drug-induced changes in voltage dependent contraction such as cellular impedance which has been validated with excellent predictivity for drug-induced arrhythmia and contractility. Here, we review advances in cardiomyocyte impedance with emphasis on stem cell-derived cardiomyocyte models for toxicity screening. Key perspectives include: the electrical principles of impedance technology, impedance detection of cardiomyocyte beating, beat parameter selection/analysis, validation in toxicity and drug discovery, and future directions. As a conclusion, an in vitro screening cascade is proffered using the downstream, inclusive detection of CM impedance assays as a primary screen followed by complementary CM assays chosen to enable mechanism-appropriate follow-up. The combined approach will enhance testing for CV liabilities prior to traditional in vivo models. PMID:25134468

  11. A fluorescence-based rapid screening assay for cytotoxic compounds.

    PubMed

    Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E; Garza, Kristine; Aguilera, Renato J

    2004-12-24

    A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death. PMID:15555600

  12. Resources for Criminal Background Check, Fingerprinting, Drug Screen and Immunizations

    E-print Network

    Hart, Gus

    , Opiates (including Codeine, Morphine, and Heroin) and phencyclidine (PCP). A Five-panel drug screen tests Codeine, Morphine, and Heroin), and Phencyclidine (PCP). IMMUNIZATIONS Company Address Telephone Hours

  13. Clinical evaluation and use of urine screening for drug abuse.

    PubMed Central

    Saxon, A J; Calsyn, D A; Haver, V M; Delaney, C J

    1988-01-01

    Urine drug screening is indicated to evaluate patients who show mental status or behavioral changes and to monitor the abstinence of drug abusers. The appropriate timing for collecting urine specimens may vary depending on the suspected drug of abuse and on laboratory factors. Laboratories use a variety of techniques to do urine screens, and these must be understood by clinicians ordering the screens to interpret results correctly. In treating drug-abusing patients, clinicians must apply structured reinforcement in conjunction with urine screen results to aid patients in achieving abstinence. PMID:3176489

  14. A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics

    PubMed Central

    Iqbal, Junaid; Kazmi, Shahana Urooj; Khan, Naveed Ahmed

    2013-01-01

    Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30%) in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics. PMID:23865073

  15. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.

    PubMed Central

    Buckner, F S; Verlinde, C L; La Flamme, A C; Van Voorhis, W C

    1996-01-01

    A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli beta-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red beta-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 microM in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with beta-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified. PMID:8913471

  16. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse\\/toxicology screening in emergency medicine

    Microsoft Academic Search

    Matthew D Krasowski; Anthony F Pizon; Mohamed G Siam; Spiros Giannoutsos; Manisha Iyer; Sean Ekins

    2009-01-01

    BACKGROUND: Laboratory tests for routine drug of abuse and toxicology (DOA\\/Tox) screening, often used in emergency medicine, generally utilize antibody-based tests (immunoassays) to detect classes of drugs such as amphetamines, barbiturates, benzodiazepines, opiates, and tricyclic antidepressants, or individual drugs such as cocaine, methadone, and phencyclidine. A key factor in assay sensitivity and specificity is the drugs or drug metabolites that

  17. Comparison of rapid screening assays using organic chemicals

    SciTech Connect

    Beach, S.A.; Robideau, R.R. [3M Environmental Lab., St. Paul, MN (United States)

    1994-12-31

    In a continuation of a study presented last year using metals, the sensitivity of short term toxicity tests is examined using common organic chemicals. In toxicity testing, the focus has shifted from the traditional long-term studies utilizing the mortality of complex, multicellular eukaryotic organisms as the endpoint towards short-term studies in which transformation of biochemical pathways are monitored. The relative sensitivity of aquatic screening techniques are compared to the standardized 48-hr Daphnia magna and Ceriodaphnia dubia, 96-hr fathead minnow and 96-hr algal acute assays. The short-term test procedures investigated are: dehydrogenase enzyme activity assays utilizing triphenyltetrazolium chloride (TTC) and resazurin as the calorimetric indicators; TOXI-Chromotest, inhibition of {beta}-galactosidase; reduction in bioluminescence output utilizing the Microtox{reg_sign} test; nitrification inhibition assays with a commercial preparation of nitrifying bacteria (Nitroseed{trademark}) and municipal activated sludge; respiration inhibition assays with a commercial preparation of heterotrophic bacteria (Polytox{reg_sign}) and activated sludge; inhibition of root growth in terrestrial plants; and galactosidase inhibition through the use of a fluorometrically tagged substrate with the Daphnia magna IQ{trademark} test. Toxicity values generated by this laboratory on commonly used organic chemicals are compared.

  18. Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni

    PubMed Central

    2013-01-01

    Background Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Methods Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. Results The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. Conclusion NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode. PMID:23941505

  19. A Differential Drug Screen for Compounds That Select Against Antibiotic Resistance

    E-print Network

    Kishony, Roy

    A Differential Drug Screen for Compounds That Select Against Antibiotic Resistance Remy Chait1, Massachusetts, United States of America Abstract Antibiotics increase the frequency of resistant bacteria of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit

  20. Disagreement between Human Papillomavirus Assays: An Unexpected Challenge for the Choice of an Assay in Primary Cervical Screening

    PubMed Central

    Ejegod, Ditte Møller; Rygaard, Carsten; Lynge, Elsebeth; Bonde, Jesper

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41% tested positive on all four. Agreement was lower in women aged ?30 years (30%, vs. 49% at <30 years), in primary screening samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30–65 years (n?=?2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women. PMID:24466262

  1. An enzyme-linked immunosorbent assay for urinary screening of fentanyl citrate abuse.

    PubMed

    Makowski, G S; Richter, J J; Moore, R E; Eisma, R; Ostheimer, D; Onoroski, M; Wu, A H

    1995-01-01

    An enzyme-linked immunosorbent assay (ELISA) for quantitation of urinary fentanyl was evaluated as a screening tool for detecting abuse of this potent narcotic. The assay was found to have reproducible calibration curves from 0.5 to 10 ng/mL and a limit of detection of 0.5 ng/mL. Interference by proteins, glucose, or pH was negligible. The assay was specific for fentanyl with little cross-reactivity against despropionyl fentanyl and norfentanyl metabolites, other analgesics and common drugs of abuse. To evaluate its use in humans, urines were collected from 57 normal individuals, 48 patients seen in the Emergency Department, and 18 surgical patients receiving either low (50 micrograms) or moderate fentanyl dosage (200 and 250 micrograms) for routine anesthesia. In patients receiving 50 micrograms (a dose consistent with early abuse), urinary fentanyl was detectable for 3 to 10 h post administration. In patients receiving 200 or 250 micrograms (a dose more consistent with addiction), urinary fentanyl was detectable for longer time periods (> 24 h). These results indicate that the ELISA is sensitive for the detection of recent fentanyl exposure under conditions likely to mimic those in abuse and addiction. The assay is simple to perform, reliable, and can be used to screen urine specimens prior to gas chromatography/mass spectrometry (GC/MS) confirmation. PMID:7785965

  2. Label-free biosensor assays in GPCR screening.

    PubMed

    Grundmann, Manuel; Kostenis, Evi

    2015-01-01

    About one third of currently marketed drugs target G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins in the human proteome. GPCRs are ubiquitously expressed throughout the human body and play a pivotal role in a vast number of physiological and pathophysiological processes. Because of their intriguing complexity, their relevance, and yet unexploited potential in the treatment of diseases, GPCRs are studied intensively by both academic and industrial research labs.Classical biochemical and molecular biology techniques, including traditional second messenger assays, took biomedical research to the next level and represent the fascinating power of in vitro pharmacology. While extremely efficient in capturing one clearly defined cellular readout, those methods do not authentically portray the events taking place in living cells as a whole; hence the process of drug discovery runs the risk to lose sight of a wider context already in early stages. Label-free cell-based assays hold the promise to overcome these shortcomings by considering cellular processes holistically. If combined with diligent assay adjustments, dynamic mass redistribution (DMR) technology is an excellent tool to investigate GPCR signaling. In this article we aim to provide guidance for scientists seeking for information on how to set up and optimize DMR assays with the objective to establish a knowledge base on deciphering integrated cellular readouts. For this reason we focus on a basic DMR protocol for the investigation of the long-chain fatty acid FFA1 receptor as a model family A GPCR and complement it with information that allow a sophisticated approach to more specialized scientific questions with the use of this comparatively novel method. PMID:25563186

  3. A systems approach for analysis of high content screening assay data with topic modeling

    PubMed Central

    2013-01-01

    Background High Content Screening (HCS) has become an important tool for toxicity assessment, partly due to its advantage of handling multiple measurements simultaneously. This approach has provided insight and contributed to the understanding of systems biology at cellular level. To fully realize this potential, the simultaneously measured multiple endpoints from a live cell should be considered in a probabilistic relationship to assess the cell's condition to response stress from a treatment, which poses a great challenge to extract hidden knowledge and relationships from these measurements. Method In this work, we applied a text mining method of Latent Dirichlet Allocation (LDA) to analyze cellular endpoints from in vitro HCS assays and related to the findings to in vivo histopathological observations. We measured multiple HCS assay endpoints for 122 drugs. Since LDA requires the data to be represented in document-term format, we first converted the continuous value of the measurements to the word frequency that can processed by the text mining tool. For each of the drugs, we generated a document for each of the 4 time points. Thus, we ended with 488 documents (drug-hour) each having different values for the 10 endpoints which are treated as words. We extracted three topics using LDA and examined these to identify diagnostic topics for 45 common drugs located in vivo experiments from the Japanese Toxicogenomics Project (TGP) observing their necrosis findings at 6 and 24 hours after treatment. Results We found that assay endpoints assigned to particular topics were in concordance with the histopathology observed. Drugs showing necrosis at 6 hour were linked to severe damage events such as Steatosis, DNA Fragmentation, Mitochondrial Potential, and Lysosome Mass. DNA Damage and Apoptosis were associated with drugs causing necrosis at 24 hours, suggesting an interplay of the two pathways in these drugs. Drugs with no sign of necrosis we related to the Cell Loss and Nuclear Size assays, which is suggestive of hepatocyte regeneration. Conclusions The evidence from this study suggests that topic modeling with LDA can enable us to interpret relationships of endpoints of in vitro assays along with an in vivo histological finding, necrosis. Effectiveness of this approach may add substantially to our understanding of systems biology. PMID:24267543

  4. Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin ?1-antitrypsin Z

    Microsoft Academic Search

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

    2010-01-01

    The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small

  5. Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase)

    Microsoft Academic Search

    T. Schultis; J. W. Metzger

    2004-01-01

    In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed. With the LYES-assay the estrogenic activities of natural (17?-estradiol

  6. Confirmation of drugs of abuse in urine drug screens by direct exposure probe mass spectrometry.

    PubMed

    Papa, V M; Ng, P S; Robbins, E F; Ou, D W

    1988-10-01

    A direct exposure probe (DEP) mass spectrometric method was developed to confirm the presence of cocaine (C), phencyclidine (P), benzoyl ecognine (BE) and 11-norcarboxy tetrahydrocannabinol (11-nor-c-THC) in previously screened urine specimens. Essentially, a urine sample is lyophilized overnight and reconstituted in 30 microliters of a 3:1 mixture of ethyl acetate:methanol. One microliter is placed on a rhenium filament and left to air dry. The specimen was analyzed by negative ion chemical ionization DEP using ammonia as reagent gas at 0.20 Torr with an ion source temperature of 100 degrees C. An electronics setting of 1700 V (EM), 0.30 mA filament current, and 100 eV with scanning at m/z 100-650 (1 sec/SCAN) resulted in simple spectra with easily identifiable molecular ions for C, BE, P and 11-nor-c-THC. The sensitivity of the assay was 1 ng for the drugs of abuse. The method was validated by analyzing 50 urine samples that have been previously screened and confirmed for drugs at the University of Illinois Toxicology Laboratory. The results showed that the DEP method confirmed 87%, 71%, 100%, and 85% of the BE, C, P and 11-nor-c-THC. In summary, a rapid and sensitive DEP method for the confirmation of drugs of abuse in urine has been developed which can serve as a useful adjunct to gas chromatographic/mass spectrometric confirmation. PMID:2853983

  7. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    NASA Astrophysics Data System (ADS)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  8. The validation of an invitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions

    SciTech Connect

    Keating, Christopher, E-mail: C.Keating@sheffield.ac.u [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom); Martinez, Vicente; Ewart, Lorna [Department of Safety Pharmacology, Safety Assessment UK, AstraZeneca, Alderley Park (United Kingdom); Gibbons, Stephen; Grundy, Luke [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom); Valentin, Jean-Pierre [Department of Safety Pharmacology, Safety Assessment UK, AstraZeneca, Alderley Park (United Kingdom); Grundy, David [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom)

    2010-06-15

    Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a 'blinded' evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs assessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.

  9. Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

    PubMed Central

    Cherry, Jonathan J.; Kobayashi, Dione T.; Lynes, Maureen M.; Naryshkin, Nikolai N.; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.

    2014-01-01

    Abstract Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of ?-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ?1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. PMID:25147906

  10. Comparison of SYBR Green I-, PicoGreen-, and [3H]-hypoxanthine-based assays for in vitro antimalarial screening of plants from Nigerian ethnomedicine.

    PubMed

    Abiodun, Oyindamola O; Gbotosho, Grace O; Ajaiyeoba, Edith O; Happi, Christian T; Hofer, Sandra; Wittlin, Sergio; Sowunmi, Akin; Brun, Reto; Oduola, Ayoade M J

    2010-03-01

    The standard method for in vitro antimalarial drug screening is based on the isotopic assay which is expensive and utilizes radioactive materials with limited availability, safety, and disposal problems in developing countries. The use of non-radioactive DNA stains SYBR Green I (SG) and PICO green (PG) for antimalarial screening had been reported. However, the use of the two DNA stains for antimalarial screening of medicinal plants has not been compared. Thus, this study compared SG, PG with the [(3)H]-hypoxanthine (HP) incorporation assays for in vitro antimalarial screening of medicinal plants. The 50% inhibitory concentration (IC(50)) values obtained using the three methods for antimalarial activity of medicinal plants and standard antimalarial drugs were similar. Data generated from this study suggests that the non-radioactive micro-flourimetric assay is sufficiently sensitive to reproducibly identify plant extracts with antimalarial activity from those lacking activity. The HP-based assay exhibited the most robust signal-to-noise ratio of 100, compared with signal-to-noise ratios of 7 for SG and 8 for PG. The SG-based assay is less expensive than the PG- and HP-based assays. SG appears to be a cost-effective alternative for antimalarial drug screening and a viable technique that may facilitate antimalarial drug discovery process especially in developing countries. PMID:20165881

  11. Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin ? and ?.

    PubMed

    Madoux, Franck; Tredup, Claudia; Spicer, Timothy P; Scampavia, Louis; Chase, Peter S; Hodder, Peter S; Fields, Gregg B; Becker-Pauly, Christoph; Minond, Dmitriy

    2014-09-01

    Zinc metalloproteinases meprin ? and meprin ? are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin ? and meprin ? and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n?=?1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ?. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ? active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ? reported to the date. These results demonstrate the ability of meprin ? and ? assays to identify selective compounds and discard artifacts of primary screening. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 396-406, 2014. PMID:25048711

  12. Versatile Assays for High Throughput Screening for Activators or Inhibitors of Intracellular Proteases and Their Cellular Regulators

    PubMed Central

    Hayashi, Hideki; Cuddy, Michael; Shu, Vincent Chih-Wen; Yip, Kenneth W.; Madiraju, Charitha; Diaz, Paul; Matsuyama, Toshifumi; Kaibara, Muneshige; Taniyama, Kohtaro; Vasile, Stefan; Sergienko, Eduard; Reed, John C.

    2009-01-01

    Background 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 & Findings 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. Conclusions 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. PMID:19876397

  13. Microplate Alamar Blue Assay versus BACTEC 460 System for High-Throughput Screening of Compounds against Mycobacterium tuberculosis and Mycobacterium avium

    Microsoft Academic Search

    LISA A. COLLINS; SCOTT G. FRANZBLAU

    1997-01-01

    In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared

  14. Screening Test Finds Drugs That Show Promise Against Ebola

    MedlinePLUS

    ... Screening Test Finds Drugs That Show Promise Against Ebola Researchers uncover 53 potential treatments; all are already ... 2015) Wednesday, December 17, 2014 Related MedlinePlus Pages Ebola Medicines WEDNESDAY, Dec. 17, 2014 (HealthDay News) -- A ...

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

    PubMed Central

    Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

    2011-01-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

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

    PubMed

    Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

    2011-09-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility. PMID:21725152

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

    PubMed Central

    2011-01-01

    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. PMID:21702939

  18. High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening

    E-print Network

    Gelb, Michael

    High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening Zdenek Spacil,1 Haribabu: There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the avail- ability spots from 58 random newborns and lysosomal storage disease­affected patients showed that the assay

  19. Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens

    PubMed Central

    Sugui, Janyce A.; Fothergill, Annette; Southall, Noel; Shinn, Paul; McKew, John C.; Kwon-Chung, Kyung J.; Zheng, Wei; Williamson, Peter R.

    2013-01-01

    A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens. PMID:23990907

  20. A FRET-based assay for screening SIRT5 specific modulators.

    PubMed

    Li, Yan; Huang, Wenfei; You, Ling; Xie, Ting; He, Bin

    2015-04-15

    A fluorogenic assay for SIRT5 has been developed to screen their small molecule modulators based on the recent discovery that SIRT5 is a demalonylase and desuccinylase. However, this assay uses a fluorogenic peptide containing 7-amino-4-methylcoumarin (AMC), which becomes the cause of false positive hits from the screening. To overcome this, we have developed an alternative method called a FRET-based assay, which will be reliable and useful for screening SIRT5 modulators in a high-throughput format since no AMC group present in this assay. PMID:25818461

  1. Comparative drug pair screening across multiple glioblastoma cell lines reveals novel drug-drug interactions

    PubMed Central

    Schmidt, Linnéa; Kling, Teresia; Monsefi, Naser; Olsson, Maja; Hansson, Caroline; Baskaran, Sathishkumar; Lundgren, Bo; Martens, Ulf; Häggblad, Maria; Westermark, Bengt; Forsberg Nilsson, Karin; Uhrbom, Lene; Karlsson-Lindahl, Linda; Gerlee, Philip; Nelander, Sven

    2013-01-01

    Background Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers. Methods We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling. Results Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses. Conclusion We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases. PMID:24101737

  2. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

    PubMed Central

    Kouznetsova, Jennifer; Sun, Wei; Martínez-Romero, Carles; Tawa, Gregory; Shinn, Paul; Chen, Catherine Z; Schimmer, Aaron; Sanderson, Philip; McKew, John C; Zheng, Wei; García-Sastre, Adolfo

    2014-01-01

    In light of the current outbreak of Ebola virus disease, there is an urgent need to develop effective therapeutics to treat Ebola infection, and drug repurposing screening is a potentially rapid approach for identifying such therapeutics. We developed a biosafety level 2 (BSL-2) 1536-well plate assay to screen for entry inhibitors of Ebola virus-like particles (VLPs) containing the glycoprotein (GP) and the matrix VP40 protein fused to a beta-lactamase reporter protein and applied this assay for a rapid drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. We report here the identification of 53 drugs with activity of blocking Ebola VLP entry into cells. These 53 active compounds can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel antagonists, and anticancer/antibiotics. Several of these compounds, including microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in animal model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compounds in drug development for the treatment of Ebola virus infection.

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

    PubMed

    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. PMID:24265131

  4. Application of ORAL.screen saliva drug test for the screening of methamphetamine, MDMA, and MDEA incorporated in hair.

    PubMed

    Miki, Akihiro; Katagi, Munehiro; Shima, Noriaki; Tsuchihashi, Hitoshi

    2004-03-01

    By the use of a one-step immunoassay drug test for oral fluid, a convenient and fairly sensitive screening method has been devised for methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) incorporated in hair. These drugs, in a 10-mg portion of hair, were extracted into 5M HCl/methanol (1:20, v/v), and the extract reconstituted in 100 micro L water was assayed with the saliva drug test ORAL.screen trade mark. The limits of detection were 0.5 ng/mg hair for d-MA, 0.8 ng/mg for dl-MDMA, and 1.0 ng/mg for dl-MDEA. The results are in good agreement with those of gas chromatography-mass spectrometry (GC-MS) determination. Although all positive results must be confirmed by either GC-MS or a specific alternative methodology, this method provided a simple screening, suitable for drug enforcement purposes, while requiring only a 10-mg hair specimen. PMID:15068568

  5. Dynamic optical tweezers based assay for monitoring early drug resistance

    NASA Astrophysics Data System (ADS)

    Wu, Xiaojing; Zhang, Yuquan; Min, Changjun; Zhu, Siwei; Feng, Jie; Yuan, X.-C.

    2013-06-01

    In this letter, a dynamic optical tweezers based assay is proposed and investigated for monitoring early drug resistance with Pemetrexed-resistant non-small cell lung cancer (NSCLC) cell lines. The validity and stability of the method are verified experimentally in terms of the physical parameters of the optical tweezers system. The results demonstrate that the proposed technique is more convenient and faster than traditional techniques when the capability of detecting small variations of the response of cells to a drug is maintained.

  6. Automated Neurite Extraction Using Dynamic Programming for High-Throughput Screening of Neuron-Based Assays

    PubMed Central

    Zhang, Yong; Zhou, Xiaobo; Degterev, Alexei; Lipinski, Marta; Adjeroh, Donald; Yuan, Junying; Wong, Stephen T.C.

    2007-01-01

    High throughput screening (HTS) of cell-based assays has recently emerged as an important tool of drug discovery. The analysis and modeling of HTS microscopy neuron images, however, is particularly challenging. In this paper we present a novel algorithm for extraction and quantification of neurite segments from HTS neuron images. The algorithm is designed to be able to detect and link neurites even with complex neuronal structures and of poor imaging quality. Our proposed algorithm automatically detects initial seed points on a set of grid lines and estimates the ending points of the neurite by iteratively tracing the centerline points along the line path representing the neurite segment. The live-wire method is then applied to link the seed points and the corresponding ending points using dynamic programming techniques, thus enabling the extraction of the centerlines of the neurite segments accurately and robustly against noise, discontinuity, and other image artifacts. A fast implementation of our algorithm using dynamic programming is also provided in the paper. Any thin neurite and its segments with low intensity contrast can be well preserved by detecting the starting and ending points of the neurite. All these properties make the proposed algorithm attractive for high-throughput screening of neuron-based assays. PMID:17363284

  7. Microfluidics-assisted in vitro drug screening and carrier production

    PubMed Central

    Tsui, Jonathan H.; Lee, Woohyuk; Pun, Suzie H.; Kim, Jungkyu; Kim, Deok-Ho

    2013-01-01

    Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development. PMID:23856409

  8. Quantum dot approaches for target-based drug screening and multiplexed active biosensing

    NASA Astrophysics Data System (ADS)

    Kovtun, Oleg; Arzeta-Ferrer, Xochitl; Rosenthal, Sandra J.

    2013-11-01

    Biomolecule detection using quantum dots (Qdots), nanometer-sized semiconductor crystals, effectively addresses the limitations associated with conventional optical and biochemical techniques, as Qdots offer several key advantages over traditional fluorophores. In this minireview, we discuss the role of Qdots as a central nanoscaffold for the polyvalent assembly of multifunctional biomolecular probes and describe recent advances in Qdot-based biorecognition. Specifically, we focus on Qdot applications in target-based, drug screening assays and real-time active biosensing of cellular processes.

  9. [In vitro screening of psychoactive drugs].

    PubMed

    Satoh, Kanako; Ogata, Akio

    2013-01-01

    Prior to the designation of illegal drugs (psychoactive drugs) by prefectural regulations, the Tokyo Metropolitan Government conducts surveys on the risk of drugs, reports the results to the governor through the Tokyo Metropolitan Government Advisory Committee on Illegal Drugs, an affiliated organization, and provides the central government with information. The Tokyo Metropolitan Institute of Public Health conducts identification of the constituents of drugs and biological effect tests to help the committee analyze and assess information on the risks of drugs. Narcotics and stimulants increase the concentrations of dopamine, serotonin, and norepinephrine, i.e., neurotransmitters, in the presynaptic clefts, exerting an excitatory effect. In the postsynaptic region, these neurotransmitters are considered to directly combine with the receptors and activate guanine nucleotide-binding proteins, causing activation. We have developed nine types (categorized into three groups) of simple, high-throughput measurement systems and examined their measurement methods. The systems are designed to assess the following properties of drugs: effects of: 1) inhibiting reuptake; 2) stimulating the release of neurotransmitters in the presynaptic region; and 3) activating G proteins in the postsynaptic region. The systems provide useful information in that they allow searches for the effects of a variety of psychoactive substances that are expected to become widespread, e.g., designer drugs, hallucinogenic plants and synthetic cannabinoids; they also allow you to conduct a test using micrograms of a drug, facilitating testing even when it is not available in a large quantity. PMID:23292015

  10. High-throughput screening assays for the identification of chemical probes

    Microsoft Academic Search

    Ronald L Johnson; Anton Simeonov; Menghang Xia; Wei Zheng; Christopher P Austin; Douglas S Auld; James Inglese

    2007-01-01

    High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for

  11. Screening for Small Molecules' Bilayer-Modifying Potential Using a Gramicidin-Based Fluorescence Assay

    PubMed Central

    Ingólfsson, Helgi I.

    2010-01-01

    Abstract Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides a possible mechanism for “off-target” drug effects. We have previously shown that channels formed by the linear gramicidins are suitable probes for changes in lipid bilayer properties, as experienced by bilayer-spanning proteins. We now report a gramicidin-based fluorescence assay for changes in bilayer properties. The assay is based on measuring the time course of fluorescence quenching in fluorophore-loaded large unilamellar vesicles, due to entry of a gramicidin channel-permeable quencher. The method is scalable and suitable for both mechanistic studies and high-throughput screening for bilayer-perturbing, potential off-target effects, which we illustrate using capsaicin (Cap) and other compounds. PMID:20233091

  12. Robust ridge regression estimators for nonlinear models with applications to high throughput screening assay data.

    PubMed

    Lim, Changwon

    2015-03-30

    Nonlinear regression is often used to evaluate the toxicity of a chemical or a drug by fitting data from a dose-response study. Toxicologists and pharmacologists may draw a conclusion about whether a chemical is toxic by testing the significance of the estimated parameters. However, sometimes the null hypothesis cannot be rejected even though the fit is quite good. One possible reason for such cases is that the estimated standard errors of the parameter estimates are extremely large. In this paper, we propose robust ridge regression estimation procedures for nonlinear models to solve this problem. The asymptotic properties of the proposed estimators are investigated; in particular, their mean squared errors are derived. The performances of the proposed estimators are compared with several standard estimators using simulation studies. The proposed methodology is also illustrated using high throughput screening assay data obtained from the National Toxicology Program. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25490981

  13. A Different Approach to Validating Screening Assays for Developmental Toxicity

    EPA Science Inventory

    BACKGROUND: There continues to be many efforts around the world to develop assays that are shorter than the traditional embryofetal developmental toxicity assay, or use fewer or no mammals, or use less compound, or have all three attributes. Each assay developer needs to test th...

  14. High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

    PubMed

    Yoshii, Yukie; Furukawa, Takako; Waki, Atsuo; Okuyama, Hiroaki; Inoue, Masahiro; Itoh, Manabu; Zhang, Ming-Rong; Wakizaka, Hidekatsu; Sogawa, Chizuru; Kiyono, Yasushi; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2015-05-01

    Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells. PMID:25771018

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

    PubMed

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and ?-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system. PMID:25563179

  16. Production of drug metabolites by immobilised Cunninghamella elegans: from screening to scale up.

    PubMed

    Quinn, Laura; Dempsey, Rita; Casey, Eoin; Kane, Ayla; Murphy, Cormac D

    2015-05-01

    Cunninghamella elegans is a fungus that has been used extensively as a microbial model of mammalian drug metabolism, whilst its potential as a biocatalyst for the preparative production of human drug metabolites has been often proposed, little effort has been made to enable this. Here, we describe a workflow for the application of C. elegans for the production of drug metabolites, starting from well-plate screening assays leading to the preparative production of drug metabolites using fungus immobilised either in alginate or as a biofilm. Using 12- and 96-well plates, the simultaneous screening of several drug biotransformations was achieved. To scale up the biotransformation, both modes of immobilisation enabled semi-continuous production of hydroxylated drug metabolites through repeated addition of drug and rejuvenation of the fungus. It was possible to improve the productivity in the biofilm culture for the production of 4'-hydroxydiclofenac from 1 mg/l h to over 4 mg/l h by reducing the incubation time for biotransformation and the number of rejuvenation steps. PMID:25665503

  17. Screening for alcohol and drug use during pregnancy.

    PubMed

    Chang, Grace

    2014-06-01

    The use of alcohol and other substances is not infrequent during pregnancy and may be associated with adverse effects on pregnancy outcome. Many pregnant women may continue these practices throughout pregnancy and even after delivery, unless they are recognized and assessed. Screening may be one way to achieve consistent and early identification. Prenatal health care providers may wish to screen all pregnant patients for their use of alcohol and other drugs using an approach that works best in their setting. A positive screen is an opportunity for the clinician and patient to discuss health practices and behaviors. PMID:24845485

  18. Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

    PubMed Central

    Chudy, Michael; Kress, Julia; Halbauer, Jochen; Heiden, Margarethe; Funk, Markus B.; Nübling, C. Micha

    2014-01-01

    Summary Background Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). Methods Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. Results Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. Conclusion HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants. PMID:24659947

  19. Comparison of SYBR Green I-, PicoGreen-, and [ 3 H]-hypoxanthine-based assays for in vitro antimalarial screening of plants from Nigerian ethnomedicine

    Microsoft Academic Search

    Oyindamola O. Abiodun; Grace O. Gbotosho; Edith O. Ajaiyeoba; Christian T. Happi; Sandra Hofer; Sergio Wittlin; Akin Sowunmi; Reto Brun; Ayoade M. J. Oduola

    2010-01-01

    The standard method for in vitro antimalarial drug screening is based on the isotopic assay which is expensive and utilizes\\u000a radioactive materials with limited availability, safety, and disposal problems in developing countries. The use of non-radioactive\\u000a DNA stains SYBR Green I (SG) and PICO green® (PG) for antimalarial screening had been reported. However, the use of the two\\u000a DNA stains

  20. The Discovery Channel: microfluidics and microengineered systems in drug screening.

    PubMed

    Moraes, Christopher

    2015-03-01

    Approaches to drug development have changed significantly over the last decade in response to the continued decline in productivity of the current discovery model. Here, we highlight exciting findings and promising approaches in the recent literature in which researchers integrate advanced micro-engineering, design, and analytical strategies to improve the relevance and utility of high-throughput screening in the drug discovery pipeline. PMID:25677245

  1. Predicting the presence of plasma heparin using neural networks to analyze coagulation screening assay optical profiles.

    PubMed

    Givens, T B; Braun, P; Fischer, T J

    1996-11-01

    A method for predicting the presence of heparin from coagulation screening assays is described and data are presented. This method incorporates the use of a multilayer perceptron trained through an error back-propagation algorithm in analyzing clotting optical data profiles. This method may lead to the identification of abnormalities from screening assays that might otherwise go undetected, or require additional testing to isolate. PMID:8997540

  2. Comparative drug screening in NUT midline carcinoma

    PubMed Central

    Beesley, A H; Stirnweiss, A; Ferrari, E; Endersby, R; Howlett, M; Failes, T W; Arndt, G M; Charles, A K; Cole, C H; Kees, U R

    2014-01-01

    Background: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed. Methods: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts. Results: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease. Conclusions: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease. PMID:24518598

  3. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 ?g /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  4. A new fluorescent based screening system for high throughput screening of drugs targeting HBV-core and HBsAg interaction.

    PubMed

    Suresh, V; Krishnakumar, K A; Asha, V V

    2015-03-01

    The existing screening systems for anti-hepatitis B virus (anti-HBV) drug discovery is time-consuming mainly due to the laborious detection system it is using. A new fluorescence based screening system for high throughput anti-HBV drug discovery was created by tagging hepatitis B surface antigen (HBsAg) with monomeric red fluorescent protein and hepatitis B virus (HBV) core protein with enhanced green fluorescent protein. The two constructs were co-transfected on to Hep3B cells and the transfection was stabilized by fluorescent activated cell sorter (FACS). The fusion proteins expressed through the secretory protein pathway as evidenced by localization with ER-Tracker and tubulin tracker. The new system has given analogues results like that of conventional enzyme-linked immunosorbent assay (ELISA). Hence it can be of very high potential for large scale drug screening systems. PMID:25776516

  5. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    Microsoft Academic Search

    Caroline Vanparys; Sophie Depiereux; Stéphanie Nadzialek; Johan Robbens; Ronny Blust; Patrick Kestemont; Wim De Coen

    2010-01-01

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated

  6. Activity of Clinically Relevant Antimalarial Drugs on Plasmodium falciparum Mature Gametocytes in an ATP Bioluminescence “Transmission Blocking” Assay

    PubMed Central

    Lozano, Sonia; Miguel, Celia; Franco, Virginia; Leroy, Didier; Herreros, Esperanza

    2012-01-01

    Background Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. Methods and Findings Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV–V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. Conclusions The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways. PMID:22514702

  7. CELL-FREE NEUROCHEMICAL SCREENING ASSAYS TO PREDICT ADVERSE EFFECTS IN MAMMALS, FISH, AND BIRDS

    EPA Science Inventory

    This work will result in the establishment of a high-throughput screening assay that can be used to predict reproductive impairment across multiple ecologically relevant species (birds, fish, mammals). Resources exist to adapt this platform to screen 1,000s of toxicants. It...

  8. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  9. Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening

    E-print Network

    Gelb, Michael

    ­9). There is excellent doc- umentation that enzyme replacement therapy for Gaucher disease [acid -glucocerebrosidase (ABGDirect Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening Yijun Li,1 C. Gelb1,5* Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry

  10. Robust regression for high throughput drug screening.

    PubMed

    Fomenko, Igor; Durst, Mark; Balaban, David

    2006-04-01

    Effective analysis of high throughput screening (HTS) data requires automation of dose-response curve fitting for large numbers of datasets. Datasets with outliers are not handled well by standard non-linear least squares methods, and manual outlier removal after visual inspection is tedious and potentially biased. We propose robust non-linear regression via M-estimation as a statistical technique for automated implementation. The approach of finding M-estimates by Iteratively Reweighted Least Squares (IRLS) and the resulting optimization problem are described. Initial parameter estimates for iterative methods are important, so self-starting methods for our model are presented. We outline the software implementation, done in Matlab and deployed as an Excel application via the Matlab Excel Builder Toolkit. Results of M-estimation are compared with least squares estimates before and after manual editing. PMID:16556471

  11. Gold nanoparticles based colorimetric assay for bacterial enzyme identification and inhibitors screening

    Microsoft Academic Search

    Rongrong Liu; Bengang Xing

    2010-01-01

    We present a simple and specific colorimetric assay for real time identification of bacterial enzymes and inhibitors screening. In the presence of ??-lactamases, the lactam amide bond in cephem nucleus was cleaved following the release of thiol modified linker which induce the cross-link of gold nanoparticles. Upon aggregation, gold nanoparticles changed color from red to purple-blue significantly. The colorimetric assay

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

    EPA Science Inventory

    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 fluores...

  13. Reduction of Diagnostic Window by New Fourth-Generation Human Immunodeficiency Virus Screening Assays

    Microsoft Academic Search

    BERNARD WEBER; ANNEMARIE BERGER; HANS WILHELM; Laboratoires Reunis

    1998-01-01

    In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomerieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared

  14. Development of a high-throughput screening method for LIM kinase 1 using a luciferase-based assay of ATP consumption

    PubMed Central

    Mezna, Mokdad; Wong, Ai Ching; Ainger, Margaret; Scott, Rebecca W; Hammonds, Tim; Olson, Michael F

    2014-01-01

    Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed ATP-binding pockets make ideal targets for small molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of a HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases. PMID:22156225

  15. Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish

    PubMed Central

    Bruni, Giancarlo; Lakhani, Parth; Kokel, David

    2014-01-01

    Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish. PMID:25104936

  16. Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

    SciTech Connect

    Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2009-05-11

    The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

  17. Bioluminescence Resonance Energy Transfer as a Screening Assay: Focus on Partial and Inverse Agonism

    Microsoft Academic Search

    Lisbeth Elster; Christian Elling; Anders Heding

    2007-01-01

    The reported data for compound screening with the bioluminescence resonance energy transfer (BRET2) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven &bgr;2 adrenergic receptor (&bgr;2-AR) agonists were tested for full or partial agonism in an improved version of the receptor\\/&bgr;-arrestin2 BRET2 assay and in 2 cyclic adenosine monophosphate (cAMP) assays (column cAMP

  18. siRNA Genome Screening Approaches to Therapeutic Drug Repositioning

    PubMed Central

    Perwitasari, Olivia; Bakre, Abhijeet; Tompkins, S. Mark; Tripp, Ralph A.

    2013-01-01

    Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome. PMID:24275945

  19. Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease

    PubMed Central

    Bhinder, Bhavneet; Antczak, Christophe; Shum, David; Radu, Constantin; Mahida, Jeni P.; Liu-Sullivan, Nancy; Ibáñez, Glorymar; Raja, Balajee Somalinga; Calder, Paul A.; Djaballah, Hakim

    2014-01-01

    Memorial Sloan-Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution’s commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator’s research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia. PMID:24661215

  20. Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay

    PubMed Central

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

    2012-01-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of FDA approved drugs, drug-like molecules and natural products extracts we identified several lead compounds that are promising candidates for medicinal chemistry. PMID:22644268

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

    PubMed

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

    2012-09-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry. PMID:22644268

  2. Multiple animal studies for medical chemical defense program in soldier/patient decontamination and drug development on task 88-36: Development of in vitro screening assays for candidate pretreatment and treatment compounds. Final report, 1 July 1988-1 July 1989

    SciTech Connect

    Joiner, R.; Dill, G.; Hobson, D.; Blank, J.

    1990-03-01

    A task was instituted at the Medical Research and Evaluation Facility (MREF) to develop in vitro assays to screen pretreatment and treatment compounds for their ability to protect or reverse the toxic effects of organophosphates and vesicants. Four vesicant assays and three nerve agent assays were developed. Two of the vesicant assays were for cell viability of keratinocyte, one in the presence of distilled mustard and one lewisite. One assay determines the effect of vesicants on keratinocyte reproduction and the other the effect of distilled mustard on cellular coenzyme nicotinamide adenine dinucleotide content. The organophosphate assays measure the effects on acetylcholinesterase of selected compounds measured by ability to reactivate, effect on aging rate, and directly. In vitro screen; HD; L; Cellular NAD+ cellular viability; GA; GD; VX; Acetylcholinesterase inhibition; Reactivators; RA 5; Aging rate; Keratinocytes; Treatment and pretreatments; Assaying; Tabun (GA); Sarin (GB); Soman (GD); Organoarsenic; Organophosphates; Chemical Surety Material (CSM); Blisters; Toxicity; Toxic agents; Nerve agents; Chemical warfare agents; G Agents; V Agents; Vesicants; Mustard agents.

  3. Phospholipid Vesicle-Based Permeation Assay and EpiSkin(®) in Assessment of Drug Therapies Destined for Skin Administration.

    PubMed

    Engesland, André; Škalko-Basnet, Nataša; Flaten, Gøril Eide

    2015-03-01

    Cost-effective and efficient methods for permeability screening are crucial during early development of drugs, drug formulations, and cosmeceuticals. Alternatives to animal experiments are impelled for both economical and ethical reasons. The aim of this study was to determine the ability of the phospholipid vesicle-based permeation assay (PVPA) to assess the effect of different formulations on drug permeability and thus establish its utility in formulation development. Three model drugs were tested in solutions and as liposomal formulations. The permeability results for the PVPA models were compared with the results for the reconstructed human skin model, EpiSkin(®) . The drugs were ranked based on their estimated penetration potentials, and the results were in accordance with what was expected considering the physicochemical properties of the drugs. PVPAs (E-80, ceramide, cholesterol, cholesteryl sulfate, and palmitic acid) was able to distinguish between drug solutions and liposomal formulations; however, EpiSkin(®) detected only small differences between the drugs in solution and formulations. In contrast with EpiSkin(®) , which is limited by a 3-day testing window, PVPA barriers can be stored frozen for up to 2 weeks or even up to 16 months, depending on their compositions. The PVPA models are thus more cost effective and efficient than the EpiSkin(®) model for permeability screening during early drug development. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1119-1127, 2015. PMID:25558045

  4. Small molecule screening in zebrafish: swimming in potential drug therapies.

    PubMed

    Tamplin, Owen J; White, Richard M; Jing, Lili; Kaufman, Charles K; Lacadie, Scott A; Li, Pulin; Taylor, Alison M; Zon, Leonard I

    2012-01-01

    Phenotype-driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well-characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA-approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials. PMID:23801494

  5. Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

    PubMed Central

    Mathias, Jonathan R.; Coothankandaswamy, Veena; Xie, Xiayang; Distel, Martin; Köster, Reinhard W.; Parsons, Michael J.; Bhalla, Kapil N.; Saxena, Meera T.; Mumm, Jeff S.

    2012-01-01

    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. PMID:22238673

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

    SciTech Connect

    Tani, Hidenori [Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Akimitsu, Nobuyoshi [Radioisotope Center, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Fujita, Osamu; Matsuda, Yasuyoshi [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Miyata, Ryo [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Tsuneda, Satoshi [Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Igarashi, Masayuki [Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan); Sekiguchi, Yuji [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Noda, Naohiro [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan)], E-mail: noda-naohiro@aist.go.jp

    2009-02-20

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

  7. An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

    PubMed Central

    Melnick, Laurence; Yang, Shiow-Shong; Rossi, Rick; Zepp, Charlie; Heefner, Donald

    1998-01-01

    We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity. PMID:9835523

  8. Drug discovery for human African trypanosomiasis: identification of novel scaffolds by the newly developed HTS SYBR Green assay for Trypanosoma brucei.

    PubMed

    Faria, Joana; Moraes, Carolina B; Song, Rita; Pascoalino, Bruno S; Lee, Nakyung; Siqueira-Neto, Jair L; Cruz, Deu John M; Parkinson, Tanya; Ioset, Jean-Robert; Cordeiro-da-Silva, Anabela; Freitas-Junior, Lucio H

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR Green assay was developed for T. brucei in a 384-well format. This semi-automated assay is cost- and time-effective, robust, and reproducible. The SYBR Green assay was compared to the resazurin assay by screening a library of 4000 putative kinase inhibitors, revealing a superior performance in terms of assay time, sensitivity, simplicity, and reproducibility, and resulting in a higher hit confirmation rate. Although the resazurin assay allows for comparatively improved detection of slow-killing compounds, it also has higher false-positive rates that are likely to arise from the assay experimental conditions. The compounds with the most potent antitrypanosomal activity were selected in both screens and grouped into 13 structural clusters, with 11 new scaffolds as antitrypanosomal agents. Several of the identified compounds had IC50 <1 µM coupled with high selectivity toward the parasite. The core structures of the scaffolds are shown, providing promising new starting points for drug discovery for HAT. PMID:25342146

  9. A 1536-well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

    PubMed Central

    Minuesa, Gerard; Antczak, Christophe; Shum, David; Radu, Constantin; Bhinder, Bhavneet; Li, Yueming; Djaballah, Hakim; Kharas, Michael G.

    2014-01-01

    RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10?M. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs. PMID:24912481

  10. Using the zebrafish photomotor response for psychotropic drug screening.

    PubMed

    Kokel, David; Peterson, Randall T

    2011-01-01

    Because psychotropic drugs affect behavior, we can use changes in behavior to discover psychotropic drugs. The original prototypes of most neuroactive medicines were discovered in humans, rodents and other model organisms. Most of these discoveries were made by chance, but the process of behavior based drug discovery can be made more systematic and efficient. Fully automated platforms for analyzing the behavior of embryonic zebrafish capture digital video recordings of animals in each individual well of a 96-well plate before, during, and after a series of stimuli. To analyze systematically the thousands of behavioral recordings obtained from a large-scale chemical screen, we transform these behavioral recordings into numerical barcodes, providing a concise and interpretable summary of the observed phenotypes in each well. Systems-level analysis of these behavioral phenotypes generate testable hypotheses about the molecular mechanisms of poorly understood drugs and behaviors. By combining the in vivo relevance of behavior-based phenotyping with the scale and automation of modern drug screening technologies, systematic behavioral barcoding represents a means of discovering psychotropic drugs and provides a powerful, systematic approach for unraveling the complexities of vertebrate behavior. PMID:21951545

  11. Establishment of a panel of reference Trypanosoma evansi and Trypanosoma equiperdum strains for drug screening.

    PubMed

    Gillingwater, K; Büscher, P; Brun, R

    2007-09-01

    The animal pathogenic protozoan, Trypanosoma evansi, leads to a wasting disease in equines, cattle and camels, commonly known as Surra. It is extensively distributed geographically with a wide range of mammalian hosts and causes great economical loss. Trypanosoma equiperdum causes a venereal disease called Dourine in horses and donkeys. Chemotherapy appears to be the most effective form of control for T. evansi, whereas infections caused by T. equiperdum are considered incurable. Due to emerging drug resistance, efficient control of T. evansi is severely threatened, emphasising the urgent need to find new alternative drugs. A drug profile for a panel of T. evansi and T. equiperdum strains has been established for the four standard drugs currently used in treatment. The (3)H-hypoxanthine incorporation assay was used to obtain 50% inhibitory concentration (IC(50)) values for each standard drug against the various strains. The results indicate the presence (and in some cases, the emergence) of drug resistance in several strains. This panel of characterised strains with known drug sensitivities and resistances will be of great value for the screening of new active compounds, in comparison with the four standard drugs currently available. PMID:17624671

  12. Assessment of the Microscreen phage-induction assay for screening hazardous wastes

    SciTech Connect

    Houk, V.S.; DeMarini, D.M.

    1987-09-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

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

    PubMed Central

    Frick, Amber; Fedoriw, Yuri; Richards, Kristy; Damania, Blossom; Parks, Bethany; Suzuki, Oscar; Benton, Cristina S; Chan, Emmanuel; Thomas, Russell S; Wiltshire, Tim

    2015-01-01

    Background Interpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin. Methods Splenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 ?M (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6. Results Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.

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

    PubMed

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

    2012-09-01

    In the pharmaceutical industry, new drugs are tested to find appropriate compounds for therapeutic purposes for contemporary diseases. Unfortunately, novel compounds emerge at expensive prices and current target evaluation processes have limited throughput, thus leading to an increase of cost and time for drug development. This work shows the development of the novel inkjet-based deposition method for assembling a miniature drug-screening platform, which can realistically and inexpensively evaluate biochemical reactions in a picoliter-scale volume at a high speed rate. As proof of concept, applying a modified Hewlett Packard model 5360 compact disc printer, green fluorescent protein expressing Escherichia coli cells along with alginate gel solution have been arrayed on a coverslip chip under a repeatable volume of 180% ± 26% picoliters per droplet; subsequently, different antibiotic droplets were patterned on the spots of cells to evaluate the inhibition of bacteria for antibiotic screening. The proposed platform was compared to the current screening process, validating its effectiveness. The viability and basic function of the printed cells were evaluated, resulting in cell viability above 98% and insignificant or no DNA damage to human kidney cells transfected. Based on the reduction of investment and compound volume used by this platform, this technique has the potential to improve the actual drug discovery process at its target evaluation stage. PMID:22728820

  15. A novel mixed phospholipid functionalized monolithic column for early screening of drug induced phospholipidosis risk.

    PubMed

    Zhao, XiangLong; Chen, WeiJia; Liu, ZhengHua; Guo, JiaLiang; Zhou, ZhengYin; Crommen, Jacques; Moaddel, Ruin; Jiang, ZhengJin

    2014-11-01

    Drug-induced phospholipidosis (PLD) is characterized by the excessive accumulation of phospholipids, resulting in multilamellar vesicle structure within lysosomes. In the present study, a novel mixed phospholipid functionalized monolithic column was developed for the first time through a facile one-step co-polymerization approach. The phospholipid composition of the monolith can be adjusted quantitatively and accurately to mimic the mixed phospholipid environment of different biomembranes on a solid matrix. The mixed phospholipid functionalized monolith as a promising immobilized artificial membrane technique was used to study drug-phospholipid interaction. Scanning electron microscopy, elemental analysis, FT-IR spectra, ?-potential analysis and micro-HPLC were carried out to characterize the physicochemical properties and separation performance of the monolith. Mechanism studies revealed that both hydrophobic and electrostatic interactions play an important role in the retention of analytes. The ratio of their contributions to retention can be easily manipulated by adjusting the composition of the mixed phospholipids, in order to better mimic the interaction between drugs and cell membrane. The obtained mixed phospholipid functionalized monolithic columns were applied to the screening of drug-induced PLD potency. Data from 79 drugs on the market demonstrated that the chromatographic hydrophobicity index referring to the mixed phospholipid functionalized monolith at pH 7.4 (CHI IAM7.4) for the selected drugs were highly correlated with the drug-induced PLD potency data obtained from other in vivo or in vitro assays. Moreover, the effect of the acidic phospholipid phosphatidylserine proportion on prediction accuracy was also investigated. The monolith containing 20% phosphatidylserine and 80% phosphatidylcholine exhibited the best prediction ability for the drug-induced PLD potency of the tested compounds. This research has led to the successful development of a novel and facile approach to prepare a mixed phospholipids functionalized monolith, which offers a reliable, cost-effective and high-throughput screening tool for early prediction of the PLD potency of drug candidates. PMID:25294294

  16. Screening for Drug Abuse Among College Students: Modification of the Michigan Alcoholism Screening Test

    ERIC Educational Resources Information Center

    Cannell, M. Barry; Favazza, Armando R.

    1978-01-01

    Modified version of the Michigan Alcoholism Screening Test was anonymously given to 245 college students on two Midwestern university campuses. Cutoff score for suspected drug abuse was set at five points. The percent of students scoring five or more points was 25 and 22 from campuses A and B respectively. (Author)

  17. A survey of yeast genomic assays for drug and target discovery

    PubMed Central

    Smith, Andrew M.; Ammar, Ron; Nislow, Corey; Giaever, Guri

    2010-01-01

    Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach. PMID:20546776

  18. Resazurin-based assay for screening bacteria for radiation sensitivity.

    PubMed

    Hudman, Deborah A; Sargentini, Neil J

    2013-12-01

    We report a simple and efficient colorimetric method to screen large numbers of bacterial strains for UV- and X-radiation sensitivity. We used reference radiation-sensitive and control strains of Escherichia coli K-12 to compare our colorimetric method to a standard clonogenic plating method. Our colorimetric method was as accurate as the standard method and was superior in terms of savings in supplies and man-hours. PMID:23483236

  19. Fluorescent Cellular Assay for Screening Agents Inhibiting Pseudomonas aeruginosa Adherence

    PubMed Central

    Nosková, Libuše; Kubí?ková, Božena; Vašková, Lucie; Bláhová, Barbora; Wimmerová, Michaela; Stiborová, Marie; Hodek, Petr

    2015-01-01

    Antibodies against Pseudomonas aeruginosa (PA) lectin, PAIIL, which is a virulence factor mediating the bacteria binding to epithelium cells, were prepared in chickens and purified from egg yolks. To examine these antibodies as a prophylactic agent preventing the adhesion of PA we developed a well plate assay based on fluorescently labeled bacteria and immortalized epithelium cell lines derived from normal and cystic fibrosis (CF) human lungs. The antibodies significantly inhibited bacteria adhesion (up to 50%) in both cell lines. In agreement with in vivo data, our plate assay showed higher susceptibility of CF cells towards the PA adhesion as compared to normal epithelium. This finding proved the reliability of the developed experimental system. PMID:25602268

  20. A Melanophore-Based Screening Assay for Erythropoietin Receptors

    Microsoft Academic Search

    Michael D. Carrithers; Louis A. Marotti; Akihiko Yoshimura; Michael R. Lerner

    1999-01-01

    A rapid, functional assay in frog melanophore cells for the erythropoietin receptor (EPOR), a member of the cytokine receptor family, is demonstrated. A chimeric receptor that comprised the extracellular portion of the murine EPOR and the transmembrane and intracellular domains of the human epidermal growth factor receptor (EGFR) was subcloned into the expression vector pJG3.6. When the full-length EGFR was

  1. Development of a high throughput screening assay for inhibitors of small ubiquitin-like modifier proteases

    PubMed Central

    Yang, Wei; Wang, Liangli; Paschen, Wulf

    2013-01-01

    Small ubiquitin-like modifier (SUMO1-3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways, and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high throughput screening-compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z’ factor was 0.83±0.04, confirming the suitability for high throughput screening applications. PMID:23470489

  2. An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes

    PubMed Central

    Genovesio, Auguste; Ayong, Lawrence; Freitas-Junior, Lucio H.

    2012-01-01

    Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria. PMID:22952585

  3. Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen.

    PubMed

    Beaudet, L; Bédard, J; Breton, B; Mercuri, R J; Budarf, M L

    2001-04-01

    AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 microL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping. PMID:11282975

  4. Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening.

    PubMed

    Yu, Linfen; Ni, Cynthia; Grist, Samantha M; Bayly, Carmen; Cheung, Karen C

    2015-04-01

    We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index. PMID:25681969

  5. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine

    PubMed Central

    Krasowski, Matthew D; Pizon, Anthony F; Siam, Mohamed G; Giannoutsos, Spiros; Iyer, Manisha; Ekins, Sean

    2009-01-01

    Background Laboratory tests for routine drug of abuse and toxicology (DOA/Tox) screening, often used in emergency medicine, generally utilize antibody-based tests (immunoassays) to detect classes of drugs such as amphetamines, barbiturates, benzodiazepines, opiates, and tricyclic antidepressants, or individual drugs such as cocaine, methadone, and phencyclidine. A key factor in assay sensitivity and specificity is the drugs or drug metabolites that were used as antigenic targets to generate the assay antibodies. All DOA/Tox screening immunoassays can be limited by false positives caused by cross-reactivity from structurally related compounds. For immunoassays targeted at a particular class of drugs, there can also be false negatives if there is failure to detect some drugs or their metabolites within that class. Methods Molecular similarity analysis, a computational method commonly used in drug discovery, was used to calculate structural similarity of a wide range of clinically relevant compounds (prescription and over-the-counter medications, illicit drugs, and clinically significant metabolites) to the target ('antigenic') molecules of DOA/Tox screening tests. These results were compared with cross-reactivity data in the package inserts of immunoassays marketed for clinical testing. The causes for false positives for phencyclidine and tricyclic antidepressant screening immunoassays were investigated at the authors' medical center using gas chromatography/mass spectrometry as a confirmatory method. Results The results illustrate three major challenges for routine DOA/Tox screening immunoassays used in emergency medicine. First, for some classes of drugs, the structural diversity of common drugs within each class has been increasing, thereby making it difficult for a single assay to detect all compounds without compromising specificity. Second, for some screening assays, common 'out-of-class' drugs may be structurally similar to the target compound so that they account for a high frequency of false positives. Illustrating this point, at the authors' medical center, the majority of positive screening results for phencyclidine and tricyclic antidepressants assays were explained by out-of-class drugs. Third, different manufacturers have adopted varying approaches to marketed immunoassays, leading to substantial inter-assay variability. Conclusion The expanding structural diversity of drugs presents a difficult challenge for routine DOA/Tox screening that limit the clinical utility of these tests in the emergency medicine setting. PMID:19400959

  6. Quantum dot approaches for target-based drug screening and multiplexed active biosensing.

    PubMed

    Kovtun, Oleg; Arzeta-Ferrer, Xochitl; Rosenthal, Sandra J

    2013-12-21

    Biomolecule detection using quantum dots (Qdots), nanometer-sized semiconductor crystals, effectively addresses the limitations associated with conventional optical and biochemical techniques, as Qdots offer several key advantages over traditional fluorophores. In this minireview, we discuss the role of Qdots as a central nanoscaffold for the polyvalent assembly of multifunctional biomolecular probes and describe recent advances in Qdot-based biorecognition. Specifically, we focus on Qdot applications in target-based, drug screening assays and real-time active biosensing of cellular processes. PMID:23946011

  7. Screening for noise in gene expression identifies drug synergies.

    PubMed

    Dar, Roy D; Hosmane, Nina N; Arkin, Michelle R; Siliciano, Robert F; Weinberger, Leor S

    2014-06-20

    Stochastic fluctuations are inherent to gene expression and can drive cell-fate specification. We used such fluctuations to modulate reactivation of HIV from latency-a quiescent state that is a major barrier to an HIV cure. By screening a diverse library of bioactive small molecules, we identified more than 80 compounds that modulated HIV gene-expression fluctuations (i.e., "noise"), without changing mean expression. These noise-modulating compounds would be neglected in conventional screens, and yet, they synergized with conventional transcriptional activators. Noise enhancers reactivated latent cells significantly better than existing best-in-class reactivation drug combinations (and with reduced off-target cytotoxicity), whereas noise suppressors stabilized latency. Noise-modulating chemicals may provide novel probes for the physiological consequences of noise and an unexplored axis for drug discovery, allowing enhanced control over diverse cell-fate decisions. PMID:24903562

  8. Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs

    PubMed Central

    Kittanakom, Saranya; Arnoldo, Anthony; Brown, Kevin R.; Wallace, Iain; Kunavisarut, Tada; Torti, Dax; Heisler, Lawrence E.; Surendra, Anuradha; Moffat, Jason; Giaever, Guri; Nislow, Corey

    2013-01-01

    The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields “hits” that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl?positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug’s activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a “minipool” composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug?target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug?target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings. PMID:23797109

  9. Optimization and Validation of Two Miniaturized Glucocerebrosidase Enzyme Assays for High-Throughput Screening

    PubMed Central

    Urban, Daniel J.; Zheng, Wei; Goker-Alpan, Ozlem; Jadhav, Ajit; LaMarca, Mary E.; Inglese, James; Sidransky, Ellen; Austin, Christopher P.

    2009-01-01

    Glucocerebrosidase (GC) catalyzes the hydrolysis of ?-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High-throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl ?-D-glucopyranoside and resorufin ?-D-glucopyranoside, have Km values of 768 ?M and 33 ?M, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value. PMID:19075603

  10. Drug abuse in the workplace: employee screening techniques

    SciTech Connect

    Buzzeo, R.W.

    1984-07-01

    Recent studies show that as many as three to five percent of the employees of a medium- to large-sized plant may be dependent on drugs as a way of life. The detrimental effects of drug abuse in the workplace can be measured in lost productivity, poor quality control and other areas at an annual cost to the American economy of $30 billion. However, a price tag cannot be attached to the lives affected by this unrelenting problem. The purpose of this paper is to provide an overview of the employee screening and hiring techniques available to industry to detect and eliminate potentially dangerous or fatal situations involving drug abuse in the workplace. The techniques are universal and can be effectively applied by the nuclear industry as well as other businesses to ensure that its work force is a reputable and reliable one.

  11. Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells

    PubMed Central

    Xu, Xiao-hong; Zhong, Zhong

    2013-01-01

    With the general decline of pharmaceutical research productivity, there are concerns that many components of the drug discovery process need to be redesigned and optimized. For example, the human immortalized cell lines or animal primary cells commonly used in traditional drug screening may not faithfully recapitulate the pathological mechanisms of human diseases, leading to biases in assays, targets, or compounds that do not effectively address disease mechanisms. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells. In this article, we will review the progress made to date on cellular disease models using human stem cells, with a focus on patient-specific iPSCs for neurological diseases. We will discuss the key challenges and the factors that associated with the success of using stem cell models for drug discovery through examples from monogenic diseases, diseases with various known genetic components, and complex diseases caused by a combination of genetic, environmental and other factors. PMID:23685955

  12. Evaluation of the suitability of recombinant yeast-based estrogenicity assays as a pre-screening tool in environmental samples

    Microsoft Academic Search

    Rikke Brix; Tania-Noelia Noguerol; Benjamin Piña; Jan Balaam; Anja Julie Nilsen; Knut-Erik Tollefsen; Walkiria Levy; Karl-Werner Schramm; Damià Barceló

    2010-01-01

    This paper presents a study evaluating the suitability of recombinant yeast-based estrogenicity assays as a pre-screening tool for monitoring of the chemical status of water bodies in support of the Water Framework Directive (WFD). Three different recombinant yeast-based assays were evaluated; the Yeast Estrogen Screen (YES), the Recombinant Yeast Assay (RYA) and the Rikilt Estrogen bioAssay (REA), of which the

  13. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

    Scanlon, Thomas C.; Dostal, Sarah M.; Griswold, Karl E.

    2014-01-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  14. A high-throughput screen for antibiotic drug discovery.

    PubMed

    Scanlon, Thomas C; Dostal, Sarah M; Griswold, Karl E

    2014-02-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ?25?pL in volume, and can be synthesized and sorted at rates exceeding 3,000?drops/s. Using this technique, we have achieved screening throughputs exceeding 5?million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  15. Development of a Screening Assay for Microbial Community Profiling

    NASA Astrophysics Data System (ADS)

    Miracle, A. L.; Tilton, F.; Bonheyo, G. T.; McDermott, J.

    2010-12-01

    Remediation of subsurface contaminant plumes has been challenging in the aspects of site characterization, design for treatability, and monitoring of treatment efficacy, to name a few. Characterization of physical and geochemical properties can be achieved through advances in sensor technologies, modeling, and well placement. However, the biotic composition within the subsurface is also an important component that adds an additional biochemical contribution that is not currently being assessed. Changes in the environment have impacts to the composition of microbial communities at this solid/fluid phase interface. The introduction of a remediative treatment may provide an abundant food source for microorganisms in the subsurface and alter the community dynamics. Such changes to the microbial community composition may have dramatic effects on bulk community biochemistry, which in turn may affect the quality of the remediative treatment in terms of effectiveness and transport through alteration of the environment. A screening array is being developed based on DNA sequence information from indigenous microorganisms within target sediments to be used to assess microbial community changes throughout remediative treatments and through time. Integration of physical, chemical, and biotic community information will be assessed to determine efficacy of treatment before, during, and after treatment to assess success of treatment, and measure any post-treatment changes.

  16. Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels.

    PubMed

    Corstjens, Paul L A M; De Dood, Claudia J; Kornelis, Dieuwke; Fat, Elisa M Tjon Kon; Wilson, R Alan; Kariuki, Thomas M; Nyakundi, Ruth K; Loverde, Philip T; Abrams, William R; Tanke, Hans J; Van Lieshout, Lisette; Deelder, André M; Van Dam, Govert J

    2014-12-01

    The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring. PMID:24932595

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

    PubMed Central

    Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

    2015-01-01

    Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

  18. Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

    PubMed

    Axerio-Cilies, Peter; Castañeda, Ivan P; Mirza, Amin; Reynisson, Jóhannes

    2009-03-01

    A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate. PMID:18692938

  19. Survey of estrogenic activity in fish feed by yeast estrogen-screen assay

    Microsoft Academic Search

    Takeru Matsumoto; Makito Kobayashi; Toshihisa Moriwaki; Shin'ichiro Kawai; Shugo Watabe

    2004-01-01

    Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the

  20. DEVELOPMENT OF A DETACHED LEAF ASSAY FOR STRIPE RUST RESISTANCE SCREENING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stripe rust (Puccinia striiformis F. sp. Tritici) is one of the major diseases of wheat (Triticum aestivum L. em Thell.) worldwide causing significant yield and quality loss. Detached leaf assay experiments have been developed to screen for resistance to Fusarium head blight, Stagonospora nodorum an...

  1. MAMMALIAN SCREENING ASSAYS FOR THE DETECTION OF POTENTIAL ENDOCRINE DISRUPTING CHEMICALS WITH AN EMPHASIS ON MALES

    EPA Science Inventory

    MAMMALIAN SCREENING ASSAYS FOR THE DETECTION OF POTENTIAL ENDOCRINE DISRUPTING CHEMICALS WITH AN EMPHASIS ON MALES. Authors: L E Gray 1 , J Furr 1 , M G Price 2 , C J Wolf 3 and J S Ostby 1 Institutions: 1. Endocrinology Branch, Reproductive Toxicology Division, NH...

  2. Magnetically optimized SERS assay for rapid detection of trace drug-related biomarkers in saliva and fingerprints.

    PubMed

    Yang, Tianxi; Guo, Xiaoyu; Wang, Hui; Fu, Shuyue; Wen, Ying; Yang, Haifeng

    2015-06-15

    New developments in the fields of human healthcare and social security call for the exploration of an easy and on-field method to detect drug-related biomarkers. In this paper, Au nanoparticles dotted magnetic nanocomposites (AMN) modified with inositol hexakisphosphate (IP6) were used as surface-enhanced Raman scattering (SERS) substrate to quickly monitor trace drug-related biomarkers in saliva and to on-site screen a trace drug biomarker in fingerprints. Due to inducing with an external magnet, such substrate presented a huge SERS activity, which has met the sensitivity requirement for assay to detect the drug biomarkers in saliva from the U.S. Substance Abuse and Mental Health Services Administration, and also the limit of detection for drug biomarker in fingerprint reached 100nM. In addition, this AMN-based SERS assay was successfully conducted using a portable Raman spectrometer, which could be used to on-site and accurately differentiate between the smokers and drug addicts in near future. PMID:25603400

  3. Loop-mediated isothermal amplification assays for screening of bacterial integrons

    PubMed Central

    2014-01-01

    Background The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. Results In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 ?L, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. Conclusions The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons. PMID:25418445

  4. Development of selective blockers for Ca2+-activated Cl channel using Xenopus laevis oocytes with an improved drug screening strategy

    Microsoft Academic Search

    Soo-Jin Oh; Jung Hwan Park; Sungyu Han; Jae Kyun Lee; Eun Joo Roh; C Justin Lee

    2008-01-01

    BACKGROUND: Ca2+-activated Cl- channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in Xenopus laevis oocytes and two-electrode voltage-clamp

  5. Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.

    PubMed Central

    Kellam, P; Larder, B A

    1994-01-01

    Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates. Images PMID:8141575

  6. A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas

    PubMed Central

    Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Kurnick, James T.

    2012-01-01

    Abstract Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells. PMID:22085019

  7. A screening assay to identify agents that enhance T-cell recognition of human melanomas.

    PubMed

    Haggerty, Timothy J; Dunn, Ian S; Rose, Lenora B; Newton, Estelle E; Kurnick, James T

    2012-04-01

    Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells. PMID:22085019

  8. Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol

    PubMed Central

    Zhang, Jun; Cai, Sutang; Peterson, Blake R.; Kris-Etherton, Penny M.

    2011-01-01

    Abstract Reverse cholesterol transport is the process by which extrahepatic cells, including macrophage-derived foam cells in arterial atherosclerotic plaque, transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Cholesterol efflux from peripheral cells is the first step in this process, and finding drugs and interventions that promote this event is an important endeavor. Radioisotope-labeled cholesterol traditionally has been employed in measuring efflux efficiency, but this reagent has limitations for high-throughput screening. We developed an alternative method to measure cholesterol efflux in macrophage-derived foam cells using a novel fluorescent cholesterol mimic comprising the Pennsylvania Green fluorophore, attached by a linker containing a glutamic acid residue, to a derivative of N-alkyl-3?-cholesterylamine. Compared with the traditional radioisotope-based assay, this fluorescence-based assay gave similar results in the presence of known modulators of cholesterol efflux, such as cyclic AMP, and different cholesterol acceptors. When the fluorescent probe was employed in a high-throughput screening format, a variety of chemicals and bioactive compounds with known and unknown effects on cholesterol efflux could be tested simultaneously by plate-reader in a short period of time. Treatment of THP-1-derived macrophages with inhibitors of the membrane transporter ATP-binding cassette A1, such as glyburide or a specific antibody, significantly reduced the export of this fluorescent compound, indicating that ATP-binding cassette A1 represents the primary mediator of its cellular efflux. This fluorescent mimic of cholesterol provides a safe, sensitive, and reproducible alternative to radioactive assays in efflux experiments and has great potential as a valuable tool when incorporated into a drug discovery program. PMID:21050070

  9. A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

    PubMed Central

    Madrid, Peter B.; Chopra, Sidharth; Manger, Ian D.; Gilfillan, Lynne; Keepers, Tiffany R.; Shurtleff, Amy C.; Green, Carol E.; Iyer, Lalitha V.; Dilks, Holli Hutcheson; Davey, Robert A.; Kolokoltsov, Andrey A.; Carrion, Ricardo; Patterson, Jean L.; Bavari, Sina; Panchal, Rekha G.; Warren, Travis K.; Wells, Jay B.; Moos, Walter H.; Burke, RaeLyn L.; Tanga, Mary J.

    2013-01-01

    Background The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. Methodology/Principal Findings A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. Conclusions/Significance The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses. PMID:23577127

  10. Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs.

    PubMed

    Porter, J; O'Loan, N; Bell, B; Mahoney, J; McGarrity, M; McConnell, R I; Fitzgerald, S P

    2012-07-01

    Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71-135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output. PMID:22566198

  11. Automated high-throughput Vibrio fischeri assay for (eco)toxicity screening: application to ionic liquids.

    PubMed

    Pinto, Paula C A G; Costa, Susana P F; Lima, José L F C; Saraiva, M Lúcia M F S

    2012-06-01

    An automated high-throughput Vibrio fischeri assay was developed and further applied to the evaluation of ionic liquids (ILs) (eco)toxicity. The assay was based on the reduction of bacterial bioluminescence in the presence of test compounds and the results were presented as EC(50). The assays were performed with eight commercially available ILs with distinct cationic head groups, alkyl side chains and anions. EC(50) values between 6.5 and 691.9 mmol L(-1) were obtained for the tested ILs, being hmim [Cl] the most toxic and bmim [Cl] the less toxic ones, confirming the influence of the different structural elements. Moreover, all the tested ILs exhibited a (eco)toxicity lower than Cu(II), used as a positive control during the optimization and analysis steps. The automated assay assured the precise control of the contact time between V. fischeri and test compound by means of a simple protocol that guaranteed adequate aspiration and handling of the solutions as well as the precise implementation of a computer controlled stop period. Furthermore, a significant reduction of the assay costs was achieved through automation mainly by a drastic reduction of the volume of bacterial suspension and test compound. The methodology was validated by comparison with a microplate assay; it was stated that the results, obtained after a 3min contact time, changed proportionally relatively to Cu(II) in both assays. This confirmed the applicability of the methodology as an (eco)toxicity screening assay, with reduction of time and increase of robustness and repeatability (n=10; rsd<1.1%). It is expected that due to its simplicity and reduced cost the developed assay can be integrated in the early stage of development of new compounds as a rapid screening test. PMID:22417674

  12. Screening and quantitative determination of drugs of abuse in diluted urine by UPLC-MS/MS.

    PubMed

    Hegstad, Solfrid; Hermansson, Sigurd; Betnér, Ingvar; Spigset, Olav; Falch, Berit Margrethe Hasle

    2014-02-01

    The purpose of this work was to develop and evaluate a fast, robust and specific UPLC-MS/MS screening platform for the determination and quantification of a variety of commonly used drugs of abuse in urine, i.e. a high-throughput quantitative analysis. Substances in the drug classes opioids, central nervous system stimulants and benzodiazepines and related agents were included in addition to cannabis and pregabalin, a total of 35 different analytes. Based on the concentrations and the physico-chemical properties of the substances, three UPLC-MS/MS methods were developed in parallel. Prior to analysis, sample preparation consisted of two different simple dilutions with 60 and 100 ?L urine, respectively, using a Tecan Freedom Evo pipetting robot platform. A Waters Xevo TQ-S tandem quadrupole mass spectrometer coupled to a Waters I-class UPLC was used for quantitative analysis of one quantitative and one qualifying MRM transition for each analyte, except for tramadol for which the metabolite O-desmethyl-tramadol was included in the MRM method to confirm tramadol identity. Deuterated analogs were included as internal standards. The between-assay relative standard deviations varied from 2% to 11% and the limits of quantification were in the range 1-200 ng/mL for the various analytes. After development and initial testing, the method has been successfully implemented and routinely used at our hospital for quantitative screening of drugs of abuse in more than 35,000 urinary samples. PMID:24413020

  13. Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

    PubMed Central

    Creek, Darren J.; Nijagal, Brunda; Kim, Dong-Hyun; Rojas, Federico; Matthews, Keith R.

    2013-01-01

    In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; ? = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening. PMID:23571546

  14. Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents

    PubMed Central

    Karapetyan, Yervand Eduard; Sferrazza, Gian Franco; Zhou, Minghai; Ottenberg, Gregory; Spicer, Timothy; Chase, Peter; Fallahi, Mohammad; Hodder, Peter; Weissmann, Charles; Lasmézas, Corinne Ida

    2013-01-01

    Prion diseases such as Creutzfeldt–Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP–FRET-enabled high throughput assay (PrP–FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood–brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform. PMID:23576755

  15. Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding: Evaluation of a Novel, High-Throughput Screening Assay

    PubMed Central

    Boyd, Windy A.; McBride, Sandra J.; Freedman, Jonathan H.

    2007-01-01

    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 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 µM. 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 environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems. PMID:18060055

  16. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

    PubMed Central

    Collins, L; Franzblau, S G

    1997-01-01

    In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria. PMID:9145860

  17. Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen ).

    PubMed

    Peppard, Jane; Glickman, Fraser; He, Yang; Hu, Shou-Ih; Doughty, John; Goldberg, Ronald

    2003-04-01

    Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report. PMID:12844435

  18. Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

    PubMed

    Martins Alho, Miriam A; Marrero-Ponce, Yovani; Barigye, Stephen J; Meneses-Marcel, Alfredo; Machado Tugores, Yanetsy; Montero-Torres, Alina; Gómez-Barrio, Alicia; Nogal, Juan J; García-Sánchez, Rory N; Vega, María Celeste; Rolón, Miriam; Martínez-Fernández, Antonio R; Escario, José A; Pérez-Giménez, Facundo; Garcia-Domenech, Ramón; Rivera, Norma; Mondragón, Ricardo; Mondragón, Mónica; Ibarra-Velarde, Froylán; Lopez-Arencibia, Atteneri; Martín-Navarro, Carmen; Lorenzo-Morales, Jacob; Cabrera-Serra, Maria Gabriela; Piñero, Jose; Tytgat, Jan; Chicharro, Roberto; Arán, Vicente J

    2014-03-01

    Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses. PMID:24513185

  19. Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection

    PubMed Central

    Veljkovic, Veljko; Loiseau, Philippe M.; Figadere, Bruno; Glisic, Sanja; Veljkovic, Nevena; Perovic, Vladimir R.; Cavanaugh, David P.; Branch, Donald R.

    2015-01-01

    The ongoing Ebola virus epidemic has presented numerous challenges with respect to control and treatment because there are no approved drugs or vaccines for the Ebola virus disease (EVD). Herein is proposed simple theoretical criterion for fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. We performed a repurposing screen of 6438 drugs from DrugBank using this criterion and selected 267 approved and 382 experimental drugs as candidates for treatment of EVD including 15 anti-malarial drugs and 32 antibiotics. An open source Web server allowing screening of molecular libraries for candidate drugs for treatment of EVD was also established. PMID:25717373

  20. Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection.

    PubMed

    Veljkovic, Veljko; Loiseau, Philippe M; Figadere, Bruno; Glisic, Sanja; Veljkovic, Nevena; Perovic, Vladimir R; Cavanaugh, David P; Branch, Donald R

    2015-01-01

    The ongoing Ebola virus epidemic has presented numerous challenges with respect to control and treatment because there are no approved drugs or vaccines for the Ebola virus disease (EVD). Herein is proposed simple theoretical criterion for fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. We performed a repurposing screen of 6438 drugs from DrugBank using this criterion and selected 267 approved and 382 experimental drugs as candidates for treatment of EVD including 15 anti-malarial drugs and 32 antibiotics. An open source Web server allowing screening of molecular libraries for candidate drugs for treatment of EVD was also established. PMID:25717373

  1. An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

    PubMed Central

    Dong, Huina; Liu, Yongfei; Zu, Xin; Li, Ning; Li, Feiran; Zhang, Dawei

    2015-01-01

    Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more). PMID:25816248

  2. A Male and Female Gametocyte Functional Viability Assay To Identify Biologically Relevant Malaria Transmission-Blocking Drugs

    PubMed Central

    Ruecker, A.; Mathias, D. K.; Straschil, U.; Churcher, T. S.; Dinglasan, R. R.; Leroy, D.; Sinden, R. E.

    2014-01-01

    Malaria elimination will require interventions that prevent parasite transmission from the human host to the mosquito. Experimentally, this is usually determined by the expensive and laborious Plasmodium falciparum standard membrane feeding assay (PfSMFA), which has limited utility for high-throughput drug screening. In response, we developed the P. falciparum dual gamete formation assay (PfDGFA), which faithfully simulates the initial stages of the PfSMFA in vitro. It utilizes a dual readout that individually and simultaneously reports on the functional viability of male and female mature stage V gametocytes. To validate, we screen the Medicines for Malaria Venture (MMV) Malaria Box library with the PfDGFA. Unique to this assay, we find compounds that target male gametocytes only and also compounds with reversible and irreversible activity. Most importantly, we show that compound activity in the PfDGFA accurately predicts activity in PfSMFAs, which validates and supports its adoption into the transmission-stage screening pipeline. PMID:25267664

  3. A rapid, inexpensive yeast-based dual-fluorescence assay of programmed--1 ribosomal frameshifting for high-throughput screening.

    PubMed

    Rakauskaite, Rasa; Liao, Pei-Yu; Rhodin, Michael H J; Lee, Kelvin; Dinman, Jonathan D

    2011-08-01

    Programmed -1 ribosomal frameshifting (-1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5' direction that is utilized by many RNA viruses. Importantly, rates of -1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, -1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of -1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor -1 PRF have evolved since the initial discovery of -1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of -1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of -1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics. PMID:21602263

  4. A rapid, inexpensive yeast-based dual-fluorescence assay of programmed—1 ribosomal frameshifting for high-throughput screening

    PubMed Central

    Rakauskait?, Rasa; Liao, Pei-Yu; Rhodin, Michael H. J.; Lee, Kelvin; Dinman, Jonathan D.

    2011-01-01

    Programmed ?1 ribosomal frameshifting (?1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5? direction that is utilized by many RNA viruses. Importantly, rates of ?1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, ?1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of ?1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor ?1 PRF have evolved since the initial discovery of ?1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of ?1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of ?1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics. PMID:21602263

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

    PubMed

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

    2014-01-01

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD. PMID:25405319

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

    PubMed Central

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

    2014-01-01

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD. PMID:25405319

  7. The human tumor cloning assay in cancer drug development

    Microsoft Academic Search

    Peter Agre; Thomas E. Williams

    1983-01-01

    Animal studies with transplantable tumor cell lines suggested that the sensitivities of the in vitro tumor cultures to certain anticancer drugs agreed with the drug sensitivities of the same tumors in vivo. Soft agar cloning techniques have been established for human myeloma and ovarian cancer cells. Refinement of the techniques now permits cloning of most human malignancies. Drug sensitivity studies

  8. Screening for personality disorder in drug and alcohol dependence.

    PubMed

    Gonzalez, Carlos

    2014-06-30

    Comorbidity of personality disorders in addiction is common, and there is a need for efficient detection methods. This study describes the use of two quick screening instruments: the self-reported versions of the Iowa Personality Disorder Screen (IPDS-SR) and the Standardised Assessment of Personality Abbreviated Scale (SAPAS-SR). The sample included 53 inpatients dependent on alcohol and/or drugs, with a 42% prevalence of any DSM-IV personality disorder. The Personality Assessment Schedule (PAS) was used as gold standard. Receiver-Operant-Characteristic (ROC) was used for analysis. The Area Under the Curve for the IPDS-SR was 0.84 (95% CI 0.72-0.93) and for the SAPAS-SR was 0.82 (95% CI 0.70-0.93). An IPDS-SR score of 5 or more correctly classified 77.4% of patients, with a sensitivity of 86.4% and a specificity of 71%. A SAPAS-SR score of 4 or more correctly classified 73.6% of patients, with a sensitivity of 81.8% and a specificity of 67.7%. Both instruments were quick, easy to administer, and acceptable to use by this population. They can be implemented in routine clinical practice in busy substance misuse departments. However further research into the implications of positive screenings is required. PMID:24680874

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

    PubMed

    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 ?(E) is critical for maintenance of the cell envelope. Because ?(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 ?(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a ?(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 ?(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify ?(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds ?(E), inhibits RNA polymerase holoenzyme formation, and inhibits ?(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 ?(E) pathway. PMID:25331704

  10. A combined HPLC-immunoenzymatic comprehensive screening for suspected drug poisoning in the emergency department

    PubMed Central

    Fabbri, A; Ruggeri, S; Marchesini, G; Vandelli, A

    2004-01-01

    Objective: To review the results of a comprehensive drug screening as first line diagnostic tool in patients attending an emergency department for suspected drug poisoning. Methods: A comprehensive drug screening was carried out in plasma or urine, or both, of 310 patients combining an HPLC multidrug profiling system and a fluorescence polarisation immunoassay. Results: In 64.2% of cases the screening confirmed the diagnosis of drug poisoning, in 13.9% suspected drugs were measurable at non-toxic concentrations, and in 21.9% no drugs were found. The suspected drugs were fully confirmed in a minority of cases, (symptomatic patients: 28.2% compared with asymptomatic: 16.5%). Symptomatic patients were less likely to have at least one suspected drug (29.6% compared with 57.7%; p<0.001), and more likely to have at least one unsuspected drug found at analysis (17.4% compared with 3.1%; p = 0.005). In 5% of patients, asymptomatic when first observed, one or more unsuspected drugs were found. In 6 of 29 patients, with suspected poisoning of an unspecified drug, the screening identified the specific drug and excluded acute intoxication in the remaining cases. Conclusion: A rapid comprehensive drug screening adds to the diagnosis of patients with suspected drug poisoning, identifying unsuspected drugs in symptomatic patients and excluding drugs in asymptomatic subjects. PMID:15107370

  11. A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin.

    PubMed

    Li, Li; Du, Yuhong; Chen, Wei; Fu, Haian; Harrison, David G

    2011-09-01

    Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels. PMID:21693765

  12. Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

    PubMed

    Krug, Anne K; Balmer, Nina V; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel

    2013-12-01

    Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants. PMID:23670202

  13. Unveiling new biological relationships using shared hits of chemical screening assay pairs

    PubMed Central

    Liu, Xueping; Campillos, Monica

    2014-01-01

    Motivation: Although the integration and analysis of the activity of small molecules across multiple chemical screens is a common approach to determine the specificity and toxicity of hits, the suitability of these approaches to reveal novel biological information is less explored. Here, we test the hypothesis that assays sharing selective hits are biologically related. Results: We annotated the biological activities (i.e. biological processes or molecular activities) measured in assays and constructed chemical hit profiles with sets of compounds differing on their selectivity level for 1640 assays of ChemBank repository. We compared the similarity of chemical hit profiles of pairs of assays with their biological relationships and observed that assay pairs sharing non-promiscuous chemical hits tend to be biologically related. A detailed analysis of a network containing assay pairs with the highest hit similarity confirmed biological meaningful relationships. Furthermore, the biological roles of predicted molecular targets of the shared hits reinforced the biological associations between assay pairs. Contact: monica.campillos@helmholtz-muenchen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161250

  14. Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

    PubMed Central

    Ritter, C.; Lucke, K.; Sirgel, F. A.; Warren, R. W.; van Helden, P. D.; Böttger, E. C.

    2014-01-01

    The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens. PMID:24403306

  15. Establishment of an In Vitro Assay for Assessing the Effects of Drugs on the Liver Stages of Plasmodium vivax Malaria

    PubMed Central

    Chattopadhyay, Rana; Velmurugan, Soundarapandian; Chakiath, Chinnamma; Andrews Donkor, Lucy; Milhous, Wilbur; Barnwell, John W.; Collins, William E.; Hoffman, Stephen L.

    2010-01-01

    Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages. PMID:21151554

  16. A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

    PubMed

    Figueroa-López, Alejandro Miguel; Cordero-Ramírez, Jesús Damián; Quiroz-Figueroa, Francisco Roberto; Maldonado-Mendoza, Ignacio Eduardo

    2014-07-01

    A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates). PMID:23787812

  17. iRFP Is a Real Time Marker for Transformation Based Assays in High Content Screening

    PubMed Central

    Paulus-Hock, Viola; Cheung, Eric C.; Roxburgh, Patricia; Vousden, Karen H.; Hock, Andreas K.

    2014-01-01

    Anchorage independent growth is one of the hallmarks of oncogenic transformation. Here we show that infrared fluorescent protein (iRFP) based assays allow accurate and unbiased determination of colony formation and anchorage independent growth over time. This protocol is particularly compatible with high throughput systems, in contrast to traditional methods which are often labor-intensive, subjective to bias and do not allow further analysis using the same cells. Transformation in a single layer soft agar assay could be documented as early as 2 to 3 days in a 96 well format, which can be easily combined with standard transfection, infection and compound screening setups to allow for high throughput screening to identify therapeutic targets. PMID:24887316

  18. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (inventors)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  19. Optical oxygen sensing systems for drug discovery applications: Respirometric Screening Technology (RST)

    NASA Astrophysics Data System (ADS)

    Papkovsky, Dmitri B.; Hynes, James; Fernandes, Richard

    2005-11-01

    Quenched-fluorescence oxygen sensing allows non-chemical, reversible, real-time monitoring of molecular oxygen and rates of oxygen consumption in biological samples. Using this approach we have developed Respirometric Screening Technology (RST); a platform which facilitates the convenient analysis of cellular oxygen uptake. This in turn allows the investigation of compounds and processes which affect respiratory activity. The RST platform employs soluble phosphorescent oxygen-sensitive probes, which may be assessed in standard microtitter plates on a fluorescence plate reader. New formats of RST assays and time-resolved fluorescence detection instrumentation developed by Luxcel provide improvements in assay sensitivity, miniaturization and overall performance. RST has a diverse range of applications in drug discovery area including high throughput analysis of mitochondrial function; studies of mechanisms of toxicity and apoptosis; cell and animal based screening of compound libraries and environmental samples; and, sterility testing. RST has been successfully validated with a range of practical targets and adopted by several leading pharmaceutical companies.

  20. A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

    PubMed

    Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

    2014-08-01

    For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion. PMID:24788205

  1. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Toxicology Test Systems § 862.3645 Neuroleptic drugs radioreceptor assay test system. (a)...

  2. A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay.

    PubMed

    Zheng, Jianyong; Fu, Xianfeng; Ying, Xiangxian; Zhang, Yinjun; Wang, Zhao

    2014-05-01

    A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments. PMID:24525041

  3. Validation of a High-Content Screening Assay Using Whole-Well Imaging of Transformed Phenotypes

    PubMed Central

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

    2011-01-01

    Abstract 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 (PDGFR?) 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 PDGFR? inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFR? 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. PMID:21182456

  4. A TR-FRET based functional assay for screening activators of CARM1

    PubMed Central

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T.; Sbardella, Gianluca; Hoffmann, F. Michael; Bi, Kun; Xu, Wei

    2013-01-01

    Epigenome is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator Associated Arginine (R) Methyltransferase 1 (CARM1) is a coactivator of estrogen receptor ? (ER?), the main target in human breast cancer. We previously showed that overexpression of CARM1 by two-fold in MCF7 breast cancer cells increased the expression of ER?-target genes involved in differentiation and reduced cell proliferation, leading to the hypothesis that activating CARM1 by chemical activators may be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved Förster resonance energy transfer (TR-FRET) assay using poly (A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen® TR-FRET assay utilizes MCF7 cells expressing GFP-PABP1 fusion protein via BacMam gene delivery system, methyl-PABP1 specific antibody and terbium-labeled secondary antibody. This assay has been validated to reflect the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  5. Continuous colorimetric assay that enables high-throughput screening of N-acetylamino Acid racemases.

    PubMed

    Sánchez-Carrón, Guiomar; Fleming, Toni; Holt-Tiffin, Karen E; Campopiano, Dominic J

    2015-04-01

    N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli l-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-d-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an l-amino acid acylase and its subsequent oxidation by an FAD-dependent l-amino acid oxidase (l-AAO). Cofactor recycling of the l-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-d-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates. PMID:25716802

  6. Current status of drug screening and disease modelling in human pluripotent stem cells

    PubMed Central

    Rajamohan, Divya; Matsa, Elena; Kalra, Spandan; Crutchley, James; Patel, Asha; George, Vinoj; Denning, Chris

    2013-01-01

    The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost. PMID:22886688

  7. Difference spectrophotometric assay of 1,2-diphenolic drugs in pharmaceutical formulations II: Germanium dioxide reagent.

    PubMed

    Davidson, A G

    1984-11-01

    A rapid, difference UV spectrophotometric assay of formulated drugs containing a 1,2-diphenolic group is described. In this assay are utilized the bathochromic shift of the absorption band of 1,2-diphenolic substances from approximately 280 to approximately 287 nm and the concomitant hyperchromic effect induced by the addition of germanium dioxide to an aqueous solution of the drug buffered at pH 6. The absorbance of the solution of the complexed drug relative to that of an equimolar solution of the free drug, which is maximum at approximately 292 nm, is proportional to the concentration of the drug and is unaffected by the presence of other UV-absorbing substances in the formulations that lack the 1,2-diphenolic moiety. Applications of the assay are described for formulations containing epinephrine, isoetharine, isoproterenol, levodopa, and methyldopa. PMID:6520761

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

    PubMed

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

    2014-01-01

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

  9. Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

    PubMed Central

    2010-01-01

    Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity < 30% of normal control was 20.3% and a prevalence of severe deficiency that would predispose to primaquine-induced hemolysis (WHO Class I-II) of 6.9%. Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration. PMID:20684792

  10. Screening of natural and synthetic drugs against Trypanosoma cruzi. 1. Establishing a structure/activity relationship.

    PubMed

    de Castro, S L; Pinto, M C; Pinto, A V

    1994-01-01

    The activity of 45 compounds against bloodstream forms of Trypanosoma cruzi was investigated. The aim was to consider new agents which might subsequently be assayed for chemoprophylaxis in donated blood. In a preliminary screening the drugs were assayed (50 to 1,000 microM at 29 degrees C) and those active against bloodstream forms at concentrations below 600 microM were selected for further assays under blood-bank conditions (4 degrees C/24 h). Three compounds isolated from natural sources and six synthetic agents were selected. The active compounds of plant origin included purpurin, a member of the trihydroxylated anthraquinone group, which is known to exhibit trypanocidal activity. Among the active synthetic compounds, five displayed a common structural feature in that they were potentially one-electron acceptors, via reductive functional groups. All five compounds form tricentered C or N intermediates, joined in a hypothetical 'Y' radical pattern. It is possible that the trypanocidal mechanisms initiated by these compounds are similar to those found with crystal violet, since this dye, which is already used in endemic areas for the treatment of banked blood, also conforms to this general Y structural pattern. PMID:8047025

  11. High-throughput Assay to Identify New Cancer Drugs

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Pharmacology seeks parties interested in collaborative research to evaluate or commercialize a diagnostic tool that can identify new drugs that increase chromosome instability.

  12. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    SciTech Connect

    Taxvig, Camilla, E-mail: camta@food.dtu.dk; Olesen, Pelle Thonning; Nellemann, Christine

    2011-02-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  13. Screening for drugs of abuse in hair with ion spray LC-MS-MS

    Microsoft Academic Search

    Robert Kronstrand; Ingrid Nystrom; Joakim Strandberg; Henrik Druid

    Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high. The aim of this study was to develop an LC-MS-MS method for the simultaneous analysis of several drugs of abuse in

  14. Evaluation of a Fluorescence-Based Method for Antibabesial Drug Screening

    PubMed Central

    Guswanto, Azirwan; Sivakumar, Thillaiampalam; Rizk, Mohamed Abdo; Elsayed, Shimaa Abd Elsalam; Youssef, Mohamed Ahmed; ElSaid, ElSaid El Shirbini; Yokoyama, Naoaki

    2014-01-01

    In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined by the fluorescence-based method (408 nM and 8.13 ?M, respectively) and microscopy (400.3 nM and 9.4 ?M, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 ?M) was similar to the recently described microscopy-based value (21.7 ?M) for B. bovis. Additionally, the Z? factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay. PMID:24914124

  15. High-Throughput Screening for RecA Inhibitors Using a Transcreener Adenosine 5?-O-Diphosphate Assay

    PubMed Central

    Peterson, Eliza J.R.; Janzen, William P.; Kireev, Dmitri

    2012-01-01

    Abstract The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5?-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener® adenosine 5?-O-diphosphate [ADP]2 fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z?=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC50 values ?10??M and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays. PMID:22192312

  16. An in vitro model for screening antileishmanial drugs: the human leukaemia monocyte cell line, THP-1.

    PubMed

    Gebre-Hiwot, A; Tadesse, G; Croft, S L; Frommel, D

    1992-08-01

    Standard anti-leishmanial drugs were tested for their ability to inhibit the growth of intracellular amastigotes of Leishmania aethiopica, L. donovani and L. infantum in the human leukemia monocyte THP-1 cell line. Sodium stibogluconate and meglumine antimoniate were active against L. donovani with ED50 values of 8.9 micrograms SbV/ml and 2.9 micrograms SbV/ml, respectively. L. aethiopica was less sensitive to sodium stibogluconate with an ED50 value of 25.3 micrograms SbV/ml while pentamidine had an ED50 value of 0.6 microM. Both L. donovani (ED50, 9.3 microM), and L. aethiopica (ED50, 6.4 microM), were sensitive to aminosidine sulphate. An L. infantum isolate, clinically resistant to meglumine antimoniate treatment, had an ED50 of 22.2 micrograms SbV/ml. The toxic level of drugs on host cells was determined by colorimetric Methyl Tetrazolium (MTT) assay prior to activity tests. The results obtained with the THP-1 in vitro drug screening model were similar to those obtained in the mouse peritoneal macrophage model. PMID:1359751

  17. Predicting in vivo phospholipidosis-inducing potential of drugs by a combined high content screening and in silico modelling approach.

    PubMed

    Bauch, Caroline; Bevan, Samantha; Woodhouse, Heather; Dilworth, Clive; Walker, Paul

    2015-04-01

    Drug induced phospholipidosis (PLD) is an adverse side effect which can affect registration of new drug entities. Phospholipids can accumulate in lysosomes, organelles essential in cellular biogenesis and if compromised can lead to cellular toxicity. Drug accumulation in lysosomes (lysosomotropism) is a known mechanism leading to PLD, however phospholipidosis can also occur indirectly by altering synthesis and processing of phospholipids. Drug induced PLD can be measured in vitro using High Content Screening (HCS) approaches, by either determining accumulation of phospholipids conjugated to dyes in cells or by determining accumulation of drugs within lysosomes, by competitive loss of lysosomal dye uptake. In this study we validate two in vitro assays using HepG2 and H9c2 cells in conjunction with in silico models based on physico-chemical properties using 56 compounds (28 phospholipidogenic, 25 non-phospholipidogenic and three kidney specific). Using HCS to determine PLD and lysosomal trapping in HepG2 cells in combination with in silico modelling increase the overall prediction of PLD in vivo with a sensitivity of 96%, specificity of 92% and overall accuracy of 94%. The findings of this study demonstrate the applicability of in vitro and in silico approaches to understand the mechanism underlying PLD and the utility of these approaches as a screening strategy in the pharmaceutical industry to select drug candidates with a low in vivo PLD liability. PMID:25668432

  18. Screening American Indian Youth for Referral to Drug Abuse Prevention and Intervention Services

    ERIC Educational Resources Information Center

    Winters, Ken C.; Dewolfe, Jerome; Graham, Donald

    2006-01-01

    The development and psychometric properties of a brief screening tool for use with American Indian youth suspected of abusing substances is described. The Indian Health Service-Personal Experience Screening Questionnaire (IHS-PESQ) is a brief questionnaire that screens for drug abuse problem severity, response distortion tendencies, and…

  19. Drug screening of patients who deliberately harm themselves admitted to the emergency department.

    PubMed

    Skelton, H; Dann, L M; Ong, R T; Hamilton, T; Ilett, K F

    1998-02-01

    This study was a retrospective analysis of drugs present in blood and urine samples taken from patients (n = 200) admitted to the emergency department of a major teaching hospital with a provisional diagnosis of deliberate self-harm. The aim was to assess the current limited drug screening strategy to see whether it needed to be changed in any way. Drugs present in blood and urine were identified by immunoassay or chromatography, categorized, and concentration-toxicity effects evaluated when practicable. For each case, the various drugs/drug classes detected were correlated with those reported by the patient. A questionnaire evaluation of doctor's perceptions of the influence of the primary blood drug screen on patient destinations was administered. The rapid primary drug screen using a blood/plasma sample detected some 46% of all drugs identified. The doctors considered that it was influential in deciding on immediate patient destination, and therefore, it is likely to be a cost-effective measure. In addition, the screen detected toxic concentrations of drugs in a significant proportion of patients who did not report their ingestion correctly. A primary drug screen using a urine sample detected opiates, cannabinoids, and amphetamines but such detection was considered unlikely to alter short-term treatment. A high-performance liquid chromatography and gas chromatography-mass spectroscopy secondary screen using blood and urine detected a significant number of additional drugs, but was slow, costly, and not likely to alter short-term treatment. The authors conclude that the primary screen for alcohol, benzodiazepines, paracetamol, salicylate, and tricyclic antidepressants remains the optimal drug screening strategy. Quantitative or qualitative estimation of patient-reported drugs such as quinine, theophylline, verapamil, and antiepileptics may be justifiable in individual patients. PMID:9485563

  20. A genome scale overexpression screen to reveal drug activity in human cells

    PubMed Central

    2014-01-01

    Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before. PMID:24944581

  1. Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections.

    PubMed

    Long, A; Polinski, M P; Call, D R; Cain, K D

    2012-06-01

    It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme-linked immunosorbent assay (ELISA), a membrane-filtration fluorescent antibody test (MF-FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF-FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron-limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF-FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub-clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme. PMID:22486267

  2. Identification of antifungal niphimycin from Streptomyces sp. KP6107 by screening based on adenylate kinase assay.

    PubMed

    Kim, Hye Yoon; Kim, Jeong Do; Hong, Jin Sung; Ham, Jong Hyun; Kim, Beom Seok

    2013-07-01

    Microbial culture extracts are used for natural product screening to find antifungal lead compounds. A microbial culture extract library was constructed using 343 actinomycete isolates to examine the value of the adenylate kinase (AK) assay for screening to identify antifungal metabolites that disrupt cell integrity in plant pathogenic fungi. A culture extract of Streptomyces sp. strain KP6107 lysed cells of Fusarium oxysporum f.sp. lycopersici which resulted in high AK activity. The active ingredient N-1 was purified from the culture extract using various chromatographic procedures and identified to be the guanidyl-polyol macrolide antibiotic, niphimycin, which is a potent fungal cell membrane disruptor. Niphimycin showed broad-spectrum antifungal activity against Alternaria mali, Aspergillus oryzae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Cercospora canescens, Cylindrocarpon destructans, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. lycopersici, and Rhizoctonia solani at concentrations of 8-64?µg?ml(-1). Anthracnose development in pepper plants was completely inhibited by treatment with 50 µg?ml(-1) niphimycin, which was as effective as chlorothalonil. These results show that the AK assay is an efficient and selective tool in screening for cell membrane/wall disruptors of plant pathogenic fungi. PMID:22915202

  3. In Vitro Reporter Assays for Screening of Chemicals That Disrupt Androgen Signaling

    PubMed Central

    Paul Khurana, S. M.

    2014-01-01

    Endocrine disruptive chemicals (EDCs) modulate hormone signaling and cause developmental and reproductive anomalies. Today, there is a global concern regarding endocrine disruption effects, particularly those mediated by the androgen receptor (AR). Androgen or male hormones are critical for the development and maintenance of male characteristics and numerous EDCs exist in the environment with the potential to disrupt androgen action. The threat is more during critical developmental windows when there is increased sensitivity to these compounds. Timely screening and detection of the EDCs is essential to minimize deleterious effects produced by these toxic chemicals. As a first line of screening, in vitro transcription assays are very useful due to their speed, convenience, and cost effectiveness. In this paper, recent in vitro reporter assays for detecting androgenic or antiandrogenic activity of EDCs have been reviewed. Two important cell systems used for this purpose, namely, the mammalian or yeast cell systems, have been discussed. Use of reporter genes such as bacterial luciferase (lux) and green fluorescent protein (gfp) has significantly improved speed and sensitivity of detection. Also, many of the current reporter assay systems can be used in a high throughput format allowing speedy evaluation of multiple potential EDCs at a lower price. PMID:25435875

  4. Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

    PubMed

    Blackford, John A; Brimacombe, Kyle R; Dougherty, Edward J; Pradhan, Madhumita; Shen, Min; Li, Zhuyin; Auld, Douglas S; Chow, Carson C; Austin, Christopher P; Simons, S Stoney

    2014-07-01

    Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful. PMID:24850414

  5. Two high throughput screening assays for Aberrant RNA-protein interactions in Myotonic Dystrophy Type-1

    PubMed Central

    Chen, Catherine Z.; Sobczak, Krzysztof; Hoskins, Jason; Southall, Noel; Marugan, Juan J.; Zheng, Wei; Thornton, Charles A.; Austin, Christopher P.

    2012-01-01

    Myotonic dystrophy type-1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3? untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG repeat RNA. The resulting loss of MBNL1 activity causes mis-regulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high throughput screen (HTS) suitable assays using both homogenous time-resolved fluorescence energy transfer (HTRF) and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)12 RNA. These assays are homogenous and successfully miniaturized to 1536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z’ factors. PMID:22218462

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

    PubMed Central

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

    2011-01-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

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

    PubMed

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

    2011-07-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

  8. Drug screening of hair by liquid chromatography-tandem mass spectrometry.

    PubMed

    Hegstad, S; Khiabani, H Z; Kristoffersen, L; Kunøe, N; Lobmaier, P P; Christophersen, A S

    2008-06-01

    Hair has become an important matrix for drug analysis, complementary to blood and urine as a matrix. A prolonged detection window makes hair analysis suitable for the detection of exposure to illegal and medicinal drugs for periods up to 12 months. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for drug screening in hair was developed and validated. To 20 mg of hair, 0.45 mL of acetonitrile/25 mM formic acid (5:95 v/v) and 50 microL of deuterated internal standards were added, and the sample was incubated in a water bath at 37 degrees C for 18 h. LC separation was achieved with a Zorbax SB-Phenyl column (2.1 x 100 mm, 3.5-microm particle). Mass detection was performed by positive ion mode electrospray LC-MS-MS and included the following drugs/metabolites: nicotine, cotinine, morphine, 6-monoacetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymeth-amphetamine, cocaine, benzoylecgonine, 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, oxazepam, diazepam, alprazolam, zopiclone, zolpidem, carisoprodol, meprobamate, buprenorphine, and methadone. Within- and between-assay relative standard deviations varied from 2.0% to 12% and 2.7% to 15%, respectively. The accuracies were in the range of -24% to 16%, and recoveries ranged from 25% to 100%. The LC-MS-MS method proved to be simple and robust for the determination of drugs in hair. It has been used for authentic samples in our laboratory in the past year. PMID:18544222

  9. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    NASA Astrophysics Data System (ADS)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-10-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  10. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    PubMed Central

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-01-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

  11. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry.

    PubMed

    Gergov, M; Robson, J N; Ojanperä, I; Heinonen, O P; Vuori, E

    2001-09-15

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the simultaneous screening and quantitation of 18 antihistamine drugs in blood samples. Sample pretreatment involved liquid-liquid extraction of the basic antihistamines followed by a second extraction of the acidic antihistamines. The recoveries were 43-113% for basic drugs and 23-66% for acidic drugs. The combined extracts were run by LC on C(18) reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The mass spectrometric analysis was performed with a triple stage quadrupole mass analyzer. Screening was performed using multiple reaction monitoring (MRM) and any compounds tentatively identified as antihistamine drugs were then automatedly verified by their Product Ion Spectra in a subsequent MS/MS run. Quantitation was based on the MRM data from the screening step. In validation tests, the method showed good linearity at the relevant concentrations. The attained limits of quantitation varied between 0.0005 and 0.01mg/l in blood and were lower than the therapeutic concentrations (C(max)). The limits for identification by Product Ion Spectra were also lower than C(max), except for clemastine, which has exceptionally low concentrations in blood. The intra-assay relative standard deviations were better than 10% and the inaccuracy varied between 39% for levocabastine and 5% for cyclizine, the majority of the values being <20%. PMID:11516895

  12. Detection and prevalence of drug use in arrested drivers using the Dräger Drug Test 5000 and Affiniton DrugWipe oral fluid drug screening devices.

    PubMed

    Logan, Barry K; Mohr, Amanda L A; Talpins, Stephen K

    2014-09-01

    The use of oral fluid (OF) drug testing devices offers the ability to rapidly obtain a drug screening result at the time of a traffic stop. We describe an evaluation of two such devices, the Dräger Drug Test 5000 and the Affiniton DrugWipe, to detect drug use in a cohort of drivers arrested from an investigation of drug impaired driving (n = 92). Overall, 41% of these drivers were ultimately confirmed positive by mass spectrometry for the presence of one or more drugs. The most frequently detected drugs were cannabinoids (30%), benzodiazepines (11%) and cocaine (10%). Thirty-nine percent of drivers with blood alcohol concentrations >0.08 g/100 mL were found to be drug positive. Field test results obtained from OF samples were compared with collected OF and urine samples subsequently analyzed in the laboratory by gas or liquid chromatography-mass spectrometry. The Dräger Drug Test 5000 (DDT5000) and DrugWipe returned overall sensitivities of 51 and 53%, and positive predictive values of 93 and 63%, respectively. The most notable difference in performance was the DDT5000's better sensitivity in detecting marijuana use. Both devices failed to detect benzodiazepine use. Oral fluid proved to be a more effective confirmatory specimen, with more drugs being confirmed in OF than urine. PMID:24894458

  13. Drug and bioactive molecule screening based on a bioelectrical impedance cell culture platform

    PubMed Central

    Ramasamy, Sakthivel; Bennet, Devasier; Kim, Sanghyo

    2014-01-01

    This review will present a brief discussion on the recent advancements of bioelectrical impedance cell-based biosensors, especially the electric cell-substrate impedance sensing (ECIS) system for screening of various bioactive molecules. The different technical integrations of various chip types, working principles, measurement systems, and applications for drug targeting of molecules in cells are highlighted in this paper. Screening of bioactive molecules based on electric cell-substrate impedance sensing is a trial-and-error process toward the development of therapeutically active agents for drug discovery and therapeutics. In general, bioactive molecule screening can be used to identify active molecular targets for various diseases and toxicity at the cellular level with nanoscale resolution. In the innovation and screening of new drugs or bioactive molecules, the activeness, the efficacy of the compound, and safety in biological systems are the main concerns on which determination of drug candidates is based. Further, drug discovery and screening of compounds are often performed in cell-based test systems in order to reduce costs and save time. Moreover, this system can provide more relevant results in in vivo studies, as well as high-throughput drug screening for various diseases during the early stages of drug discovery. Recently, MEMS technologies and integration with image detection techniques have been employed successfully. These new technologies and their possible ongoing transformations are addressed. Select reports are outlined, and not all the work that has been performed in the field of drug screening and development is covered. PMID:25525360

  14. Indeterminate Human Immunodeficiency Virus Western Blot Profiles in Ethiopians with Discordant Screening-Assay Results

    PubMed Central

    Meles, Hailu; Wolday, Dawit; Fontanet, Arnaud; Tsegaye, Aster; Tilahun, Tesfaye; Aklilu, Mathias; Sanders, Eduard; De Wit, Tobias F. Rinke

    2002-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB reactivity among Ethiopians are hardly known. Here, we describe the profiles of indeterminate WB reactivity in Ethiopians with discordant screening assays. Between 1996 and 2000, a total of 12,124 specimens were tested for HIV-1 antibodies. Overall, 1,437 (11.9%) were positive for HIV-1 antibody. Ninety-one (?0.8%) gave equivocal results because of discordant results among the various screening assays and indeterminate WB profiles by the American Red Cross (ARC) criteria. Most (30.4%) of these indeterminate WB results were due to p24 reactivity. However, 12 samples (13.2%) displayed reactivity to p24 and gp41 or to p24 and gp120/160 proteins (positive by Centers for Disease Control and Prevention [CDC] criteria). Only two samples (2.2%) were reactive to both env glycoproteins gp41 and gp120/160 (positive by the World Health Organization [WHO] criteria). Of 31 WB assays initially indeterminate by the ARC criteria and with follow-up samples, 29 (93.5%) became negative when retested subsequently while 2 (6.5%) remained indeterminate for more than a year and were thus considered negative. Using CDC and WHO criteria, 6 (19.4%) and 2 (6.5%), respectively, of these WB assays would have been considered falsely positive. In addition, 17 indeterminate samples were negative when assessed by a nucleic acid-based amplification assay for HIV-1 viremia. In general, there was 97.8% concordance between the ARC and WHO criteria and 85.7% concordance between the ARC and CDC criteria for an indeterminate WB result. The ARC criteria best met the specified objectives for diagnosis in our setting. PMID:11777847

  15. Eco-genotoxicity of six anticancer drugs using comet assay in daphnids.

    PubMed

    Parrella, Alfredo; Lavorgna, Margherita; Criscuolo, Emma; Russo, Chiara; Isidori, Marina

    2015-04-01

    The eco-genotoxicity of six anti-neoplastic drugs, 5-fluorouracil, capecitabine, cisplatin, doxorubicin, etoposide, and imatinib, belonging to five classes of anatomical therapeutic classification (ATC), was studied applying the in vivo comet assay on cells from whole organisms of Daphnia magna and Ceriodaphnia dubia. For the first time, this test was performed in C. dubia. In addition, to have a wider genotoxic/mutagenic profile of the anticancer drugs selected, SOS chromotest and Salmonella mutagenicity assay were performed. The comet results showed that all drugs induced DNA damage, in both Cladocerans, with environmental concern; indeed Doxorubicin induced DNA damage in the order of tens of ngL(-1) in both crustaceans, as well as 5-flurouracil in C. dubia and cisplatin in D. magna. In the SOS Chromotest all drugs, except imatinib, were able to activate the repair system in Escherichia coli PQ37 while in the Salmonella mutagenicity assay, doxorubicin was the only drug able to cause direct and indirect frameshift and base-pair substitution mutations. Comet assay was the most sensitive tool of genotoxic exposure assessment, able to detect in vivo the adverse effects at concentration lower than those evaluated in vitro by bacterial assays. PMID:25638790

  16. A First Application of Enzyme-Linked Immunosorbent Assay for Screening Cyclodiene Insecticides in Ground Water

    USGS Publications Warehouse

    Dombrowski, T.R.; Thurman, E.M.; Mohrman, G.B.

    1996-01-01

    A commercially available enzyme-linked immunosorbent assay (ELISA) plate kit for screening of cyclodiene insecticides (aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor) was evaluated for sensitivity, cross reactivity, and overall performance using groundwater samples from a contaminated site. Ground-water contaminants included several pesticide compounds and their manufacturing byproducts, as well as many other organic and inorganic compounds. Cross-reactivity studies were carried out for the cyclodiene compounds, and results were compared to those listed by the manufacturer. Data obtained were used to evaluate the sensitivity of the ELISA kit to the cyclodiene compounds in ground water samples with a contaminated matrix. The method quantitation limit for the ELISA kit was 15 ??g/L (as chlordane). Of the 56 ground-water samples analyzed using the ELISA plate kits, more than 85% showed cyclodiene insecticide contamination. The ELISA kit showed excellent potential as a screening tool for sites with suspected groundwater contamination by insecticides.

  17. Modular real-time PCR screening assay for common European animal families.

    PubMed

    Naue, J; Lutz-Bonengel, S; Sänger, T; Schlauderer, N; Schmidt, U

    2014-01-01

    A screening assay based on real-time PCR and melt curve analysis was developed to detect DNA from nine common European animal families/species and human. The assay consists of a 10-cycle universal pre-amplification followed by specific nested PCR and was designed to exploit the different melting temperatures (T m) of family/species-specific 12S ribosomal ribonucleic acid and cytochrome b fragments, which are amplified in duplex reactions. Case-related modular application is possible. Beyond determination of the animal family and discrimination from human DNA, evaluation of the melt curve in some cases additionally allows for species determination (e.g. cat vs. lynx). The method presents a quick, flexible and sample-saving approach to assess non-human DNA at low expenses, and it is especially useful in resolution of DNA mixtures. PMID:23613031

  18. An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells.

    PubMed

    Graves, R; Davies, R; Owen, P; Clynes, M; Cleary, I; O'Beirne, G

    1997-06-01

    We have developed an homogeneous assay to measure the uptake and efflux of [14C]adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates. The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR). The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of [14C]thymidine. The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents. PMID:9314096

  19. Adaptation of High-Throughput Screening in Drug Discovery—Toxicological Screening Tests

    PubMed Central

    Szyma?ski, Pawe?; Markowicz, Magdalena; Mikiciuk-Olasik, El?bieta

    2012-01-01

    High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study. PMID:22312262

  20. Development of a p38 Kinase Binding Assay for High Throughput Screening.

    PubMed

    Warrior; Chiou; Sheets; Sciotti; Parry; Simmer; Surber; Burns; Beutel; Mollison; Djuric; Trevillyan

    1999-01-01

    p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors. PMID:10838421

  1. Ecient Database Screening for Rational Drug Design Using Pharmacophore-Constrained Conformational Search

    E-print Network

    Pratt, Vaughan

    EÃ?cient Database Screening for Rational Drug Design Using Pharmacophore-Constrained Conformational Stanford, CA, USA Abstract Computational tools have greatly expedited the pharma- ceutical drug design in drug design that ligand binding is due primarily to the interaction of some features of the ligand

  2. Screening of crude drug extracts for prolyl endopeptidase inhibitory activity.

    PubMed

    Tezuka, Y; Fan, W; Kasimu, R; Kadota, S

    1999-07-01

    Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes. Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition. Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var. trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity. In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition. PMID:10439485

  3. Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.

    PubMed

    Marjuki, Henju; Mishin, Vasiliy P; Sleeman, Katrina; Okomo-Adhiambo, Margaret; Sheu, Tiffany G; Guo, Lizheng; Xu, Xiyan; Gubareva, Larisa V

    2013-11-01

    The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens. PMID:23917311

  4. A Drug Screening Method Based on the Autophagy Pathway and Studies of the Mechanism of Evodiamine against Influenza A Virus

    PubMed Central

    Dai, Jian-Ping; Li, Wei-Zhong; Zhao, Xiang-Feng; Wang, Ge-Fei; Yang, Jia-Cai; Zhang, Lin; Chen, Xiao-Xuan; Xu, Yan-Xuan; Li, Kang-Sheng

    2012-01-01

    In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation - fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-?, IL-1?, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound. PMID:22900043

  5. Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells

    PubMed Central

    2011-01-01

    Background Tumor Necrosis Factor-? Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis. Methods FDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis. Results Fourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 ?M. At these low concentrations, mitoxantrone demonstrated selectivity toward malignant cells over normal pancreatic epithelial cells. Conclusions The identification of a number of FDA-approved drugs as TRAIL sensitizers can expand chemotherapeutic options for combination treatments in prostate and pancreatic cancer diseases. PMID:22044796

  6. A High-throughput Screening Assay using Krabbe Disease Patient Cells

    PubMed Central

    Ribbens, Jameson; Whiteley, Grace; Furuya, Hirokazu; Southall, Noel; Hu, Xin; Marugan, Juan; Ferrer, Marc; Maegawa, Gustavo H.B.

    2013-01-01

    Globoid-cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by ?-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD as they are more prone to cross the blood-brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen showed to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from a SV40-transformed GLD patient fibroblast was measurable in high-dense microplates plates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration-response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live-GLD patient cells to identify therapeutic agents that can be potentially be used for the treatment of this progressive neurodegenerative disease. PMID:23138179

  7. Development of a new immunochromatographic assay using gold nanoparticles for screening of IgA deficiency.

    PubMed

    Goudarzi, Saeid; Ahmadi, Anita; Farhadi, Mohammad; Kamrava, Seyed Kamran; Saghafi, Shiva; Omidfar, Kobra

    2015-02-01

    A new competitive immunochromatography (ICG) strip test based on polyclonal antibody (pAb) conjugated with gold nanoparticles (NPs) was developed and its applications for primary screening of immunoglobulin (Ig) A in serum were evaluated. Nanocolloidal gold as the detection reagent, with an average particle diameter of 20 nm, was synthesized and labelled pAb. The antibody-nanocolloidal gold probe was applied on the conjugate pad, and human IgA was immobilized on a nitrocellulose membrane as the capture reagent to prepare the ICG strip test. It took only 10 minutes to accomplish a semi-quantitative detection of serum IgA in this assay. In the optimized investigational conditions, the ICG strip test could distinguish human serum IgA in the range from 1 to 270 ng/mL with a detection limit of 5 ng/mL. The reliability of testing procedures was examined by performing the ICG strip test with 11 serum samples and comparing the results with those obtained via enzyme-linked immunosorbent assay (ELISA). The ICG strip was sufficiently sensitive and accurate for a rapid screening of IgA in human serum. PMID:25530146

  8. A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

    PubMed Central

    Iwano, Hidetomo; Kawai, Kazuhiro; Ohta, Takehiro; Obayashi, Tetsu; Hirose, Kazuhiko; Ito, Nobuhiko; Yokota, Hiroshi; Tamura, Yutaka; Nagahata, Hajime

    2011-01-01

    A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms. PMID:21586880

  9. A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle.

    PubMed

    Higuchi, Hidetoshi; Iwano, Hidetomo; Kawai, Kazuhiro; Ohta, Takehiro; Obayashi, Tetsu; Hirose, Kazuhiko; Ito, Nobuhiko; Yokota, Hiroshi; Tamura, Yutaka; Nagahata, Hajime

    2011-06-01

    A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms. PMID:21586880

  10. Developing highER-throughput zebrafish screens for in-vivo CNS drug discovery

    PubMed Central

    Stewart, Adam Michael; Gerlai, Robert; Kalueff, Allan V.

    2015-01-01

    The high prevalence of brain disorders and the lack of their efficient treatments necessitate improved in-vivo pre-clinical models and tests. The zebrafish (Danio rerio), a vertebrate species with high genetic and physiological homology to humans, is an excellent organism for innovative central nervous system (CNS) drug discovery and small molecule screening. Here, we outline new strategies for developing higher-throughput zebrafish screens to test neuroactive drugs and predict their pharmacological mechanisms. With the growing application of automated 3D phenotyping, machine learning algorithms, movement pattern- and behavior recognition, and multi-animal video-tracking, zebrafish screens are expected to markedly improve CNS drug discovery. PMID:25729356

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

    PubMed Central

    2010-01-01

    Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU PMID:20637080

  12. Detection of resistance to second-line antituberculosis drugs by use of the genotype MTBDRsl assay: a multicenter evaluation and feasibility study.

    PubMed

    Ignatyeva, Olga; Kontsevaya, Irina; Kovalyov, Alexander; Balabanova, Yanina; Nikolayevskyy, Vladislav; Toit, Kadri; Dragan, Anda; Maxim, Daniela; Mironova, Svetlana; Kummik, Tiina; Muntean, Ionela; Koshkarova, Ekaterina; Drobniewski, Francis

    2012-05-01

    The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement. PMID:22378910

  13. Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs

    PubMed Central

    Zustiak, Silviya; Nossal, Ralph; Sackett, Dan L.

    2013-01-01

    It is now well understood that the cell microenvironment, including the surrounding matrix, profoundly affects cell fate. This is especially true for solid tumors where, for example, matrix stiffness is believed to be an important factor in tumorogenesis. Our hypothesis is that since matrix stiffness affects cell fate, it may also be important in drug resistance. To test this hypothesis, we designed and built a multiwell polyacrylamide (PA) gel-based stiffness assay, in which the gels were coated with collagen in order to facilitate cell attachment and proliferation. This PA-based assay was used to examine the effect of stiffness on cultured cell responsiveness to cytotoxic drugs. In particular, we tested multiple cancer cell lines and their susceptibility to paclitaxel, a microtubule-targeting agent. By assessing cell proliferation, morphology, and the IC50 of the drug, we were able to establish that the stiffness affects responsiveness to cytotoxic drugs in a cell dependent manner. PMID:24018833

  14. A qualitative and quantitative high-throughput assay for screening of gluconate high-yield strains by Aspergillus niger.

    PubMed

    Shi, Fei; Tan, Jun; Chu, Ju; Wang, Yonghong; Zhuang, Yingping; Zhang, Siliang

    2015-02-01

    A novel two-step high-throughput strategy was developed for screening of gluconate high-yield strains by Aspergillus niger. The first step was fast qualitative assay according to the indicator color change, the second step was quantitative assay according to the absorbance of chelate formed with Cu(2+) at 810nm. The accuracy of high-throughput assay was comparable to that of high-performance liquid chromatography (HPLC). The correlation coefficient between CuSO4 assay and HPLC assays was exceeding 0.99 by statistical analysis. As a result, 3 high-yield mutants were screened out from 1000 viable single colonies, the mutants II-2-A1, IV-7-C6, and V-11-C5 were further validated in 5L of bioreactor. The average production rates were 15.5%, 32.8%, and 12.1% higher than that of the parental strain, respectively. PMID:25498457

  15. Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-? transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

    PubMed

    Lee, Hae Kyung; Kim, Tae Sung; Kim, Chang Yeong; Kang, Il Hyun; Kim, Mi Gyeong; Jung, Ki Kyung; Kim, Hyung Sik; Han, Soon Young; Yoon, Hae Jung; Rhee, Gyu Seek

    2012-01-01

    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17?-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ER? of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17?-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ER? binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors. PMID:22467034

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

    PubMed Central

    2014-01-01

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

  17. Are drunk-driving offenders referred for screening accurately reporting their drug use?

    Microsoft Academic Search

    Sandra C Lapham; Janet C'de Baca; Iyiin Chang; William C Hunt; Lawrence R Berger

    2002-01-01

    Several studies report that a substantial percentage of offenders arrested for impaired driving test positive for drugs of abuse besides alcohol. Current guidelines recommend screening offenders for both alcohol and other drug use, yet little is known about the accuracy of self-reports of drug use in this population. We compared drug abuse and dependence DSM-III-R diagnoses from an initial, court-ordered

  18. A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

    PubMed Central

    Liu, Xin; Chen, Yu; Fierke, Carol A.

    2014-01-01

    Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5? end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5? end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P. PMID:25249623

  19. A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P.

    PubMed

    Liu, Xin; Chen, Yu; Fierke, Carol A

    2014-11-10

    Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5' end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5' end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P. PMID:25249623

  20. Screening assay for small-molecule inhibitors of synaptojanin 1, a synaptic phosphoinositide phosphatase.

    PubMed

    McIntire, Laura Beth J; Lee, Kyu-In; Chang-Ileto, Belle; Di Paolo, Gilbert; Kim, Tae-Wan

    2014-04-01

    Elevation of amyloid ?-peptide (A?) is critically associated with Alzheimer disease (AD) pathogenesis. A?-induced synaptic abnormalities, including altered receptor trafficking and synapse loss, have been linked to cognitive deficits in AD. Recent work implicates a lipid critical for neuronal function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], in A?-induced synaptic and behavioral impairments. Synaptojanin 1 (Synj1), a lipid phosphatase mediating the breakdown of PI(4,5)P2, has been shown to play a role in synaptic vesicle recycling and receptor trafficking in neurons. Heterozygous deletion of Synj1 protected neurons from A?-induced synaptic loss and restored learning and memory in a mouse model of AD. Thus, inhibition of Synj1 may ameliorate A?-associated impairments, suggesting Synj1 as a potential therapeutic target. To this end, we developed a screening assay for Synj1 based on detection of inorganic phosphate liberation from a water-soluble, short-chain PI(4,5)P2. The assay displayed saturable kinetics and detected Synj1's substrate preference for PI(4,5)P2 over PI(3,4,5)P3. The assay will enable identification of novel Synj1 inhibitors that have potential utility as chemical probes to dissect the cellular role of Synj1 as well as potential to prevent or reverse AD-associated synaptic abnormalities. PMID:24186361

  1. Study on microvisualizing assay of delivered drug infiltration using 2-color optical coherence dosigraphy

    NASA Astrophysics Data System (ADS)

    Nakamichi, Yu; Saeki, Souichi; Saito, Takashi; Hiro, Takafumi; Matsuzaki, Masunori

    2009-02-01

    Recently, clinical treatments applying drug delivery system (DDS) have been being developed. However, it is quite difficult to in vivo diagnose spatiotemporal distribution of drug infiltration, so the validation study should be too insufficient to progress the DDS development. In this study, we propose a visualizing assay of DDS, namely 2-Color Optical Coherence Dosigraphy (2C-OCD). 2C-OCD is based on optical coherence tomography using two waveband "2-Color" light sources having different optical absorbance of drug. This can simultaneously provide microscale tomographic images of scatterer density and drug concentration. In order to evaluate the efficacy of this technique, this was applied to drug-diffusion phenomena in microchannel and lipidrich plaques of rabbit with drug administration, respectively. As a result of diffusion experiment, it was confirmed that 2C-OCD can visualize a cross-sectional map of drug concentration, with spatial resolution 5 micro m × 10 ?m and accuracy plus-minus 13.0 ?M. In ex vivo animal experiment, the enhancement of absorptivity could be observed inside lipidrich plaques, in which DDS drug could be therein uptaken by drug administration. The absorption maps corresponding to drug concentration were calculated, comparing with their histological images. Consequently, they had good coincidence with histological examinations, therefore, it was concluded that 2C-OCD could visualize drug infiltration in biological tissue with almost the same spatial resolution as OCT system.

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

    PubMed Central

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

    2009-01-01

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

  3. A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN) vectors and multi-colour analyses

    PubMed Central

    2013-01-01

    Background Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs. PMID:23286882

  4. Assessment of ion-selective optical nanosensors for drug screening applications

    E-print Network

    Yun, Hannah

    2007-01-01

    Ion channels represent an important category of drug targets. They play a significant role in numerous physiological functions, from membrane excitation and signaling to fluid absorption and secretion. An ion-channel assay ...

  5. Screening for drugs of abuse in hair with ion spray LC–MS–MS

    Microsoft Academic Search

    Robert Kronstrand; Ingrid Nyström; Joakim Strandberg; Henrik Druid

    2004-01-01

    Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high.The aim of this study was to develop an LC–MS–MS method for the simultaneous analysis of several drugs of abuse in human

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

    Microsoft Academic Search

    Alida A. M. Stolker; Ruud J. B. Peters; Richard Zuiderent; Joseph M. DiBussolo; Cláudia P. B. Martins

    2010-01-01

    There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs\\u000a with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied\\u000a for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging\\u000a to seven different classes were selected

  7. A novel assay to assess the effectiveness of antiangiogenic drugs in human breast cancer.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cytotoxic drugs maintain antiangiogenic properties, but there are no human, tumor-based assays to evaluate their antiangiogenic potential. We used a fibrin-thrombin clot-based angiogenesis model to evaluate the angiogenic response of human breast cancer to various cytotoxic agents commonly used...

  8. High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

    PubMed Central

    Patel, Dhara A.; Patel, Anand C.; Nolan, William C.; Zhang, Yong; Holtzman, Michael J.

    2012-01-01

    Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z?>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit. PMID:22574190

  9. A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

    PubMed Central

    Bhattacharyya, Nisan; Hu, Xin; Chen, Catherine Z.; Mathews Griner, Lesley A.; Zheng, Wei; Inglese, James; Austin, Christopher P.; Marugan, Juan J.; Southall, Noel; Neumann, Susanne; Northup, John K.; Ferrer, Marc; Collins, Michael T.

    2014-01-01

    Mis-sense mutations in the ?-subunit of the G-protein, Gs?, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gs? 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 Gs? protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gs? proteins (R201C and R201H). Stable cell lines with equivalent transfected Gs? 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. PMID:24667240

  10. Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

    PubMed

    Paape, Daniel; Bell, Andrew S; Heal, William P; Hutton, Jennie A; Leatherbarrow, Robin J; Tate, Edward W; Smith, Deborah F

    2014-12-01

    We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors. PMID:25522361

  11. Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

    PubMed Central

    Paape, Daniel; Bell, Andrew S.; Heal, William P.; Hutton, Jennie A.; Leatherbarrow, Robin J.; Tate, Edward W.; Smith, Deborah F.

    2014-01-01

    We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors. PMID:25522361

  12. A High-Content Biosensor Based Screen Identifies Cell Permeable Activators and Inhibitors of EGFR Function: Implications in Drug Discovery

    PubMed Central

    Antczak, Christophe; Mahida, Jeni P.; Bhinder, Bhavneet; Calder, Paul A.; Djaballah, Hakim

    2013-01-01

    Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates; due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell permeable modulators for development. For this purpose, we have used our recently optimized EGFR biosensor (EGFRB) assay to screen for modulators of EGFR activity. Here, we report on its validation under HTS conditions displaying a S/N ratio of 21 and a Z’ value of 0.56; attributes of a robust cell based assay. We performed a pilot screen against a library of 6,912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95 %, respectively. Follow up dose response studies revealed that 12 out of the 13 known EGFR inhibitors in the library confirmed as hits. ZM-306416, a VEGFR antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic. PMID:22573732

  13. Drug screening and confirmation by GC–MS: Comparison of EMIT II and Online KIMS against 10 drugs between US and England laboratories

    Microsoft Academic Search

    Natalie T. Lu; Bruce G. Taylor

    2006-01-01

    Drug screening through urinalysis is a widely accepted tool for rapid detection of potential drug use at a relatively low cost. It is, therefore, a potentially useful method for detecting and monitoring drug use in a variety of contexts such as the criminal justice system, pre-employment screening and a variety of treatment centers. This article explores the efficacy of two

  14. Defining Drug Targets in Yeast Haploinsufficiency Screens: Application to Human Translational Pharmacology

    NSDL National Science Digital Library

    Michel Roberge (University of British Columbia; Department of Biochemistry and Molecular Biology REV)

    2008-08-26

    A major challenge in drug discovery is to identify the cellular targets responsible for the pharmacological activity of drug candidates. In the yeast Saccharomyces cerevisiae, a heterozygous diploid mutant collection of ~6000 strains, in each of which one copy of a single gene is deleted, is commercially available. With this collection, it is possible to evaluate the role of each gene product in the response of cells to a drug. Drug-induced haploinsufficiency refers to the situation where a heterozygous diploid mutant is more sensitive to a drug than is the wild-type strain. Drug-induced haploinsufficiency screening has the potential to reveal pharmacological targets of drugs and those that contribute to undesired side effects, as well as gene products involved in drug transport, metabolism, or resistance. Using published studies, I present advantages and limitations of this technique and discuss its value for predicting drug targets in human cells.

  15. A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening.

    PubMed

    Skopelitou, Katholiki; Labrou, Nikolaos E

    2010-10-15

    Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)(2) with the released halogen ion of the substrate in the presence of Fe(3+). The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes. PMID:20529659

  16. Development of a high-throughput AlphaScreen assay for modulators of synapsin I phosphorylation in primary neurons.

    PubMed

    Chan, Betty; Cottrell, Jeffrey R; Li, Bing; Larson, Kelley C; Ashford, Crystle J; Levenson, Jonathan M; Laeng, Pascal; Gerber, David J; Song, Jianping

    2014-02-01

    Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders. PMID:24088370

  17. Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

    PubMed Central

    2012-01-01

    Background Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. Results Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. Conclusions The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens. PMID:22967317

  18. Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high throughput chemical screening

    PubMed Central

    Ghebremariam, Yohannes T; Erlanson, Daniel; Yamada, Keisuke; Cooke, John P

    2013-01-01

    Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/Dimethylarginine dimethylaminohydrolase (DDAH)/Asymmetric Dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by iNOS) could play a role in idiopathic pulmonary fibrosis (IPF), sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high throughput screen for DDAH modulators. PMID:22460174

  19. Olfactory receptor screening assay using nanovesicle-immobilized carbon nanotube transistor.

    PubMed

    Lim, Jong Hyun; Park, Juhun; Hong, Seunghun; Park, Tai Hyun

    2015-01-01

    Olfactory receptor (OR) genes are considered to be the largest superfamily of the mammalian genome, and in the case of humans, approximately 390 kinds of functional ORs play a role in perceiving odors. In spite of their significance in olfaction, the function of all ORs has not yet been fully revealed. In order to efficiently identify specific ligands of orphan ORs, methods that can generate olfactory signals in a reliable manner and that can convert the cellular signals into measurable responses are required. Here, we describe an OR screening assay method using olfactory sensors that are based on cell-derived nanovesicles combined with single-walled carbon nanotube field-effect transistors (SWNT-FETs). The nanovesicles contain ORs on their surface membrane and induce influx of calcium ions similar to olfactory signal transduction. This ion influx causes an electrical current change along the carbon nanotube, and then this change is measured by the SWNT-FET sensor. This technique facilitates the simple and rapid screening of OR functions. PMID:25563185

  20. Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease Specific Patterns of Cardiotoxicity

    PubMed Central

    Liang, Ping; Lan, Feng; Lee, Andrew S.; Gong, Tingyu; Sanchez-Freire, Veronica; Wang, Yongming; Diecke, Sebastian; Sallam, Karim; Knowles, Joshua W.; Wang, Paul J.; Nguyen, Patricia K.; Bers, Donald M.; Robbins, Robert C.; Wu, Joseph C.

    2013-01-01

    Background Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs). Conclusions We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays. PMID:23519760

  1. Efficacy of a Polyethylene Glycol Marker System in Urine Drug Screening in an Opiate Substitution Program

    Microsoft Academic Search

    Harald Jörn Schneider; Birgit Rühl; Kirsten Meyer; Ruprecht Keller; Markus Backmund

    2008-01-01

    Aims: Screening for concomitant drug consumption is necessary in opiate substitution therapy of opiate-dependent patients. Adulteration of samples is a common problem in this setting. A recently developed polyethylene glycol marker system allows reliable identification of urine samples. In this study, we aimed to compare the rates of drug detection in conventional and marker urine samples. Design: This cross-sectional evaluation

  2. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis

    PubMed Central

    Bowling, Tana; Mercer, Luke; Don, Robert; Jacobs, Robert; Nare, Bakela

    2012-01-01

    Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei, and the disease is fatal if untreated. There is an urgent need to develop new, safe and effective treatments for HAT because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the development and application of a cell-based resazurin reduction assay for high throughput screening and identification of new inhibitors of T. b. brucei as starting points for the development of new treatments for human HAT. Active compounds identified in primary screening of ?48,000 compounds representing ?25 chemical classes were titrated to obtain IC50 values. Cytotoxicity against a mammalian cell line was determined to provide indications of parasite versus host cell selectivity. Examples from hit series that showed selectivity and evidence of preliminary SAR were re-synthesized to confirm trypanocidal activity prior to initiating hit-to-lead expansion efforts. Additional assays such as serum shift, time to kill and reversibility of compound effect were developed and applied to provide further criteria for advancing compounds through the hit-to-lead phase of the project. From this initial effort, six distinct chemical series were selected and hit-to-lead chemistry was initiated to synthesize several key analogs for evaluation of trypanocidal activity in the resazurin-reduction assay for parasite viability. From the hit-to-lead efforts, a series was identified that demonstrated efficacy in a mouse model for T. b. brucei infection and was progressed into the lead optimization stage. In summary, the present study demonstrates the successful and effective use of resazurin-reduction based assays as tools for primary and secondary screening of a new compound series to identify leads for the treatment of HAT. PMID:24533287

  3. Rapid and Simple Kinetics Screening Assay for Electrophilic Dermal Sensitizers using Nitrobenzenethiol

    PubMed Central

    Chipinda, Itai; Ajibola, Risikat O.; Morakinyo, Moshood K.; Ruwona, Tinashe B.; Simoyi, Reuben H.; Siegel, Paul D.

    2010-01-01

    The need for alternatives to animal based skin sensitization testing has spurred research on the use of in-vitro, in silico and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped flow techniques and conventional UV spectrophotometric measurements enabled determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for 7 extreme, 5 strong, 7 moderate and 4 weak/non-sensitizers. 17 out of the 23 tested chemicals were pseudo-first order and 3 were second order. In 3 out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, diones and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid and inexpensive absorbance based method has great potential for use as a preliminary screening tool for skin allergens. PMID:20402462

  4. GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

    SciTech Connect

    Cronn, M.T.; Miyada, C.G.; Fucini, R.V. [Affymetrix, Santa Clara, CA (United States)] [and others

    1994-09-01

    GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by a sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.

  5. A high-throughput in vivo micronucleus assay for genome instability screening in mice.

    PubMed

    Balmus, Gabriel; Karp, Natasha A; Ng, Bee Ling; Jackson, Stephen P; Adams, David J; McIntyre, Rebecca E

    2015-01-01

    We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labor-intensive, microscopy-based techniques. Here we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 ?l of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability. PMID:25551665

  6. A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus

    PubMed Central

    Voisset, Cécile; Daskalogianni, Chrysoula; Contesse, Marie-Astrid; Mazars, Anne; Arbach, Hratch; Le Cann, Marie; Soubigou, Flavie; Apcher, Sébastien; Fåhraeus, Robin; Blondel, Marc

    2014-01-01

    Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8+ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers. PMID:24558096

  7. Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

    PubMed Central

    Zych, Courtney; Domling, Alexander; Ayyavoo, Velpandi

    2013-01-01

    Targeting protein–protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a “proof of concept.” Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner. PMID:23737660

  8. Validation of Miniaturized One-Step Reverse Transcription qPCR Assays for High-Throughput Screening and Comparison to a Reporter Gene Methodology.

    PubMed

    Bardelle, Catherine; McWilliams, Lisa; Mounfield, Susan; Wigglesworth, Mark; Rich, Kirsty

    2015-03-01

    Quantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types. PMID:25785772

  9. DISIS: Prediction of Drug Response through an Iterative Sure Independence Screening

    PubMed Central

    Zhang, Naiqian; Wang, Jun; Wang, Haiyun; Zheng, Xiaoqi

    2015-01-01

    Prediction of drug response based on genomic alterations is an important task in the research of personalized medicine. Current elastic net model utilized a sure independence screening to select relevant genomic features with drug response, but it may neglect the combination effect of some marginally weak features. In this work, we applied an iterative sure independence screening scheme to select drug response relevant features from the Cancer Cell Line Encyclopedia (CCLE) dataset. For each drug in CCLE, we selected up to 40 features including gene expressions, mutation and copy number alterations of cancer-related genes, and some of them are significantly strong features but showing weak marginal correlation with drug response vector. Lasso regression based on the selected features showed that our prediction accuracies are higher than those by elastic net regression for most drugs. PMID:25794193

  10. An in silico screen links gene expression signatures to drug response in glioblastoma stem cells.

    PubMed

    Riddick, G; Song, H; Holbeck, S L; Kopp, W; Walling, J; Ahn, S; Zhang, W; Fine, H A

    2014-12-01

    Cancer stem cells (CSCs) are thought to promote resistance to chemotherapeutic drugs in glioblastoma, the most common and aggressive primary brain tumor. However, the use of high-throughput drug screens to discover novel small-molecule inhibitors for CSC has been hampered by their instability in long-term cell culture. We asked whether predictive models of drug response could be developed from gene expression signatures of established cell lines and applied to predict drug response in glioblastoma stem cells. Predictions for active compounds were confirmed both for 185 compounds in seven established glioma cell lines and 21 compounds in three glioblastoma stem cells. The use of established cell lines as a surrogate for drug response in CSC lines may enable the large-scale virtual screening of drug candidates that would otherwise be difficult or impossible to test directly in CSCs.The Pharmacogenomics Journal advance online publication, 2 December 2014; doi:10.1038/tpj.2014.61. PMID:25446780

  11. DISIS: Prediction of Drug Response through an Iterative Sure Independence Screening.

    PubMed

    Fang, Yun; Qin, Yufang; Zhang, Naiqian; Wang, Jun; Wang, Haiyun; Zheng, Xiaoqi

    2015-01-01

    Prediction of drug response based on genomic alterations is an important task in the research of personalized medicine. Current elastic net model utilized a sure independence screening to select relevant genomic features with drug response, but it may neglect the combination effect of some marginally weak features. In this work, we applied an iterative sure independence screening scheme to select drug response relevant features from the Cancer Cell Line Encyclopedia (CCLE) dataset. For each drug in CCLE, we selected up to 40 features including gene expressions, mutation and copy number alterations of cancer-related genes, and some of them are significantly strong features but showing weak marginal correlation with drug response vector. Lasso regression based on the selected features showed that our prediction accuracies are higher than those by elastic net regression for most drugs. PMID:25794193

  12. Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

    PubMed Central

    Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

    2010-01-01

    Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 ?M IC50 and having no effect on human ACC1 at 100 ?M. PMID:20439761

  13. Drug quality screening in developing countries: establishment of an appropriate laboratory in Swaziland.

    PubMed

    Kenyon, T A; Kenyon, A S; Sibiya, T

    1994-01-01

    A simple, low-cost, accurate thin-layer chromatography (TLC) method has been used to establish the first drug quality screening laboratory in Swaziland. For this purpose, office space at the central medical stores was first converted into a simple laboratory. Basic equipment, supplies, and materials were purchased, existing manpower was trained to perform accurately the TLC procedure, and a system was established for the qualitative/quantitative screening of selected drugs purchased by the Ministry of Health prior to their distribution to user facilities. The TLC method described can be used to set up similar low-cost, drug quality screening laboratories in other developing countries where analytical chemistry expertise is lacking, resources are scarce, and sophisticated analytical laboratories to assess drug quality are not available. PMID:7923541

  14. Highly sensitive photoelectrochemical assay for DNA methyltransferase activity and inhibitor screening by exciton energy transfer coupled with enzyme cleavage biosensing strategy.

    PubMed

    Shen, Qingming; Han, Li; Fan, Gaochao; Abdel-Halim, E S; Jiang, Liping; Zhu, Jun-Jie

    2015-02-15

    Highly sensitive DNA methyltransferase (MTase) activity and inhibitor screening photoelectrochemical (PEC) assay was developed based on the exciton energy transfer (EET) effect coupled with site-specific cleavage of restriction endonuclease (HpaII). The assay was designed by integrating the Au nanoparticles (NPs) labeled probe DNA (pDNA-Au) with CdSe quantum dots (QDs). The strong EET effect between Au NPs and CdSe QDs resulted in the dramatic decrease of photocurrent signal. The pDNA carried a sensing region for specifically recognizing target DNA (tDNA) and hybridizing with it to form a DNA duplex. With the site-specific cleavage of HpaII, the DNA duplex could be cleaved and Au NPs would be released, which broke the EET and resulted in the restoration of photocurrent signal. However, when the DNA duplex was methylated by M.SssI MTase, this cleavage of HpaII was blocked, and therefore the unbroken EET effect kept the lower photocurrent signal. That was, the restored photocurrent was inversely proportional to the MTase activity. Based on this strategy, the PEC assay could determine as low as ~0.0042 U/mL of M.SssI MTase with a linear range from 0.01 to 150 U/mL. In addition, the assay could be used for the screening of the inhibitors of MTase. This PEC assay provides a promising platform for monitoring the activity and inhibition of DNA MTase, and thus shows a great potential in cancer diagnostics and anti-cancer drugs discovery. PMID:25282398

  15. NEW JERSEY INSTITUTE OF TECHNOLOGY DRUG SCREENING POLICY AND PROCEDURE

    E-print Network

    for substance and/or prescribed medication abuse, and/or unregistered but medically prescribed use of illegal or medically unauthorized controlled dangerous substances (herein "drugs"), and formally register prescribed medication, which may include drugs as the term is defined herein. No person can function well

  16. Research Paper Rapid Colorimetric Screening of Drug Interaction

    E-print Network

    Jelinek, Raz

    passive diffusion and active, carrier-mediated transport (3,5). The goal of this work is to develop transport; polydiacetylene. INTRODUCTION An essential task in identifying drug candidates is the assessment failure of promising pharmacological candidates as viable drugs can be traced to difficulties in transport

  17. High-Throughput Screening of Ototoxic and Otoprotective Pharmacological Drugs

    ERIC Educational Resources Information Center

    Kalinec, Federico

    2005-01-01

    Drug ototoxicity research has relied traditionally on animal models for the discovery and development of therapeutic interventions. More than 50 years of research, however, has delivered few--if any--successful clinical strategies for preventing or ameliorating the ototoxic effects of common pharmacological drugs such as aminoglycoside…

  18. Comparison of the Velogene Rapid MRSA Identification Assay, Denka MRSA-Screen Assay, and BBL Crystal MRSA ID System for rapid identification of methicillin-resistant Staphylococcus aureus.

    PubMed

    Arbique, J; Forward, K; Haldane, D; Davidson, R

    2001-01-01

    Early detection of methicillin-resistant S.aureus (MRSA) is critical for both the management of infected patients, and the timely institution of appropriate infection control measures. Although detection of the mecA gene by PCR remains the gold standard, this technology is inaccessible for many laboratories. Therefore, we sought to evaluate several new rapid identification systems and compare them to PCR. A total of 71 methicillin-susceptible S. aureus (MSSA), 25 borderline oxacillin-resistant S. aureus (BORSA), and 213 MRSA were selected for study. S.aureus was identified using standard methods. Initial screening was performed on a Mueller-Hinton agar plate with 6 mg/L of oxacillin. MRSA strains were identified using PCR with primers specific for the mecA gene. PCR was used as the reference method. All isolates were tested using the BBL Crystal MRSA ID System (Becton Dickinson Microbiology Systems, Maryland, USA), the MRSA-Screen Assay (Denka Seiken Co., Ltd., Tokyo, Japan), and the Velogene Rapid MRSA Identification Assay (ID Biomedical Corp, Vancouver, BC). With minor modifications, all assays were performed according to manufacturers' instructions. Overall, the 3 commercial assays performed well. The sensitivity and specificity of the BBL, Denka, and Velogene systems were 99%/100%, 99%/100%, and 96%/100% respectively. The advantages of the phenotypic tests-BBL Crystal Kit and Denka MRSA-Screen Assay include lower cost per test, shelf-life, ease of use, and rapid turn-around times. Advantages of the Velogene Rapid MRSA include ability to perform genotypic high-volume testing without the equipment requirements and technical complexity involved with PCR. Turn-around times ranged from 15 min for the Denka MRSA-Screen Assay, 2 h for the Velogene Rapid MRSA, and 4 h for the BBL Crystal. The BBL Crystal, Denka MRSA-Screen, and Velogene Rapid MRSA identification systems are rapid, easy to perform, and provide accurate identification of MRSA. These rapid kits offer an acceptable alternative for smaller, non-reference, laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation. PMID:11448557

  19. Screening and Brief Intervention for Unhealthy Drug Use: Little or No Efficacy

    PubMed Central

    Saitz, Richard

    2014-01-01

    Unhealthy drug use ranges from use that risks health harms through severe drug use disorders. This narrative review addresses whether screening and brief intervention (SBI), efficacious for risky alcohol use, has efficacy for reducing other drug use and consequences. Brief intervention among those seeking help shows some promise. Screening tools have been validated though most are neither brief nor simple enough for use in general health settings. Several randomized trials have tested the efficacy of brief intervention for unhealthy drug use identified by screening in general health settings (i.e., in people not seeking help for their drug use). Substantial evidence now suggests that efficacy is limited or non-existent. Reasons likely include a range of actual and perceived severity (or lack of severity), concomitant unhealthy alcohol use and comorbid mental health conditions, and the wide range of types of unhealthy drug use (e.g., from marijuana, to prescription drugs, to heroin). Although brief intervention may have some efficacy for unhealthy drug users seeking help, the model of SBI that has effects in primary care settings on risky alcohol use may not be efficacious for other drug use. PMID:25228887

  20. Drug quality assessment methods. I. Gas-liquid chromatographic assay and identification of seven barbiturates.

    PubMed

    Black, D B; Kolasinski, H; Lovering, E G; Watson, J R

    1982-09-01

    A gas-liquid chromatographic (GLC) procedure has been developed for the assay and identification of amobarbital, butabarbital, heptabarbital, mephobarbital, pentobarbital, phenobarbital, and secobarbital in single component capsule, elixir, injectable, suppository, and tablet formulations. After extraction into chloroform from an acidified aqueous mixture of the product, the drug is eluted isothermally from a methylphenylsilicone GLC column at 210 or 240 degrees C and quantitated relative to thiamylal internal standard. Results were in good agreement with those obtained using pharmacopeial assay methods. The method is suitable for the rapid assessment of commercial formulations. PMID:7130075

  1. Using adverse outcome pathway analysis to guide development of high-throughput screening assays for thyroid-disruptors

    EPA Science Inventory

    Using Adverse Outcome Pathway Analysis to Guide Development of High-Throughput Screening Assays for Thyroid-Disruptors Katie B. Paul1,2, Joan M. Hedge2, Daniel M. Rotroff4, Kevin M. Crofton4, Michael W. Hornung3, Steven O. Simmons2 1Oak Ridge Institute for Science Education Post...

  2. Operational application of a rapid antibody-based detection assay for first line screening of paralytic shellfish toxins in shellfish

    Microsoft Academic Search

    Chun-Kwan Wong; Patricia Hung; Edward A. L. Ng; Kellie L. H. Lee; Grace T. C. Wong; Kai-Man Kam

    2010-01-01

    Paralytic shellfish poisoning toxins (PSP-toxins) are potent neurotoxins associated with marine dinoflagellates and may accumulate in filter-feeding shellfish to cause food intoxication in human. Monitoring programs for PSP in shellfish rely heavily on the use of traditional mouse bioassay (MBA). Considerable progress has been made in developing a reliable, rapid and relatively convenient assay for mass screening of possible PSP

  3. Screening ToxCast? Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  4. Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology

    PubMed Central

    Watson, Venita Gresham; Drake, Katherine M.; Peng, Yu

    2013-01-01

    Abstract Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z?-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats. PMID:23679849

  5. Drug resistance is a major obstacle to the successful treatment of HIV-1 infection. Genotypic assays are used

    E-print Network

    Samudrala, Ram

    - resistant variants of the virus severely limit the long-term effectiveness of anti-HIV drugs [1]. In recentDrug resistance is a major obstacle to the successful treatment of HIV-1 infection. Genotypic assays are used widely to provide indirect evidence of drug resistance, but the performance

  6. Tale of Two Livers -- Developing an Innovative Drug Screening Tool

    MedlinePLUS

    ... implanted in a mouse. The implant forms a so-called human ectopic artificial liver (HEAL). Mice with HEALs ... as people who metabolize drugs at different rates (so-called slow versus rapid metabolizers ). A person’s metabolism rate ...

  7. Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

    PubMed

    Lerche, Mathilde H; Meier, Sebastian; Jensen, Pernille R; Hustvedt, Svein-Olaf; Karlsson, Magnus; Duus, Jens Ø; Ardenkjaer-Larsen, Jan H

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08?µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring. PMID:20862657

  8. High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

    Microsoft Academic Search

    Michael W. Pantoliano; Eugene C. Petrella; Joseph D. Kwasnoski; Victor S. Lobanov; James Myslik; Edward Graf; Ted Carver; Eric Asel; Barry A. Springer; F. R. Salemme

    2001-01-01

    More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling

  9. Differential effects of drugs upon hematopoiesis can be assessed in long-term bone marrow cultures established on nylon screens.

    PubMed

    Naughton, B A; Sibanda, B; Azar, L; San Roman, J

    1992-04-01

    Hematotoxicity is associated with exposure to chemotherapeutic drugs and numerous other agents. Most measurements of the hematopoietic effects of prospective therapeutic drugs and environmental agents have been made in animal models. We tested the influence of various drugs on hematopoiesis in long-term cultures of Long-Evans rat bone marrow cells. These cultures were established on nylon screen-bone marrow stromal cell templates that were suspended in liquid medium. Previous phenotypic analyses of adherent zone cells of suspended nylon screen bone marrow cultures (NSBMC) using monoclonal antibodies and flow cytometry indicated that they maintain a multilineage character for extended periods in culture and display continuous proliferation of hematopoietic progenitors (colony-forming unit culture [CFU-C]). NSBMC of various ages were incubated for 21 hr with several concentrations of beta-D-cytosine arabinofuranoside, 5-fluorouracil, cyclophosphamide, or methotrexate. Adherent zone cells were dissociated enzymatically, phenotyped by flow cytometry, and assayed for colony-forming unit culture content. beta-D-cytosine arabinofuranoside, 5-fluorouracil, and methotrexate treatment of bone marrow cultures resulted in a dose-related diminution in colony-forming unit culture numbers in the adherent zones of NSBMC. Phenotypic analyses revealed similar trends but certain of these drugs manifested lineage specificities. Toxicity was also related to cyclophosphamide dose, but the presence of bone marrow stroma was necessary to demonstrate this effect in vitro. A subpopulation of these cells was found to metabolize ethoxyfluorescein ethyl ester to fluorescein after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an effect which was quantified by flow cytometry. NSBMC may be used to ascertain lineage-specific toxicities and evaluate the effects of drugs on the proliferation of hematopoietic progenitor cells. PMID:1549628

  10. The BAX PCR assay for screening Listeria monocytogenes targets a partial putative gene lmo2234.

    PubMed

    Zhang, Wei; Hughes, Allison; Wilt, Grier; Knabel, Stephen J

    2004-07-01

    The BAX PCR for screening Listeria monocytogenes is a commercial PCR assay for specifically targeting L. monocytogenes, a foodborne pathogen that can contaminate a variety of foods and cause a potentially fatal disease, listeriosis, among high-risk populations. The high specificity (> 98%) of this PCR assay is achieved by targeting a species-specific genomic region (approximately 400 bp) presumably found only in L. monocytogenes. In this study, the identity of the BAX PCR-targeted genomic region was determined by using PCR cloning, DNA sequencing, and basic local alignment search tool (BLAST) analysis of the amplicon sequences of an L. monocytogenes serotype 1/2a strain. BLAST analysis identified the BAX PCR amplicon (GenBank accession no. AY364605) as a 423-bp genomic region between nucleotides 224,409 and 224,831 in the genome of L. monocytogenes (serotype 1/2a strain EGD-e), including a 145-bp noncoding region and a 278-bp partial coding sequence of a putative gene, lmo2234. The translated amino acid sequence (92 amino acids) of this partial coding region is highly conserved between L. monocytogenes and Listeria innocua (93% homology). Reverse-position-specific BLAST analysis identified a conserved domain in Lmo2234 that was similar (95.3% aligned, E value = 9E-18) to the consensus amino acid sequence of sugar phosphate isomerases/epimerases (National Center for Biotechnology Information conserved domain database accession no. COG 1082.1, IolE), indicating that Lmo2234 might be involved in bacterial carbohydrate transport and metabolism. PMID:15270511

  11. Identifying putative drug targets and potential drug leads: starting points for virtual screening and docking.

    PubMed

    Wishart, David S

    2015-01-01

    The availability of 3D models of both drug leads (small molecule ligands) and drug targets (proteins) is essential to molecular docking and computational drug discovery. This chapter describes a simple approach that can be used to identify both drug leads and drug targets using two popular Web-accessible databases: (1) DrugBank and (2) The Human Metabolome Database. First, it is illustrated how putative drug targets and drug leads for exogenous diseases (i.e., infectious diseases) can be readily identified and their 3D structures selected using only the genomic sequences from pathogenic bacteria or viruses as input. The second part illustrates how putative drug targets and drug leads for endogenous diseases (i.e., noninfectious diseases or chronic conditions) can be identified using similar databases and similar sequence input. This chapter is intended to illustrate how bioinformatics and cheminformatics can work synergistically to help provide the necessary inputs for computer-aided drug design. PMID:25330974

  12. Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

    PubMed Central

    Vandghanooni, Somayeh; Eskandani, Morteza

    2011-01-01

    Introduction Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods In the current study, we reviewed recent drug delivery researches related to SCGE. Results We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems. PMID:23678412

  13. New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

    PubMed Central

    2013-01-01

    Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with ?max of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (?max of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators. PMID:24159930

  14. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  15. A Cell-Based Pharmacokinetics Assay for Evaluating Tubulin-Binding Drugs

    PubMed Central

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

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels. PMID:24688312

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

    PubMed

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

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels. PMID:24688312

  17. Assessment of DNA damage in Japanese nurses handling antineoplastic drugs by the comet assay.

    PubMed

    Sasaki, Makiko; Dakeishi, Miwako; Hoshi, Shigeko; Ishii, Noriko; Murata, Katsuyuki

    2008-01-01

    To clarify genotoxic effects of occupational exposure to antineoplastic drugs in Japan, we examined DNA damage, assessed by the comet assay, in 121 female nurses and 46 female clerks working at three hospitals in the northeast of Japan. The comet assay is considered to be a sensitive and rapid method for DNA strand break detection in individual cells, and tail length and tail moment are used as the comet parameters. Concerning the basal characteristics, the 46 control subjects had higher rates of smoking and coffee-drinking habits and lower hemoglobin than the 121 nurses (p<0.05). The log-transformed tail length in the nurses was significantly longer than that in the control subjects after adjusting for possible covariates such as age and smoking habit (p<0.05). Also, the log-transformed tail length was significantly longer, in the 57 nurses who had handled antineoplastic drugs in the last six months, than that in the 46 control subjects (p<0.05); but, no significant difference in tail length or tail moment was seen between the two nurse groups with and without experience of handling hazardous drugs (p>0.05). These results suggest that Japanese nurses who have worked at hospitals using antineoplastic drugs may have a potential risk of DNA damage. To minimize this risk in Japan, use of biological safety cabinet and appropriate protective equipment, in addition to staff education and training, should be implemented in the healthcare environment. PMID:18285639

  18. A comparison of four serologic assays in screening for Brucella exposure in Hawaiian monk seals.

    PubMed

    Nielsen, O; Nielsen, K; Braun, R; Kelly, L

    2005-01-01

    A survey for Brucella spp. antibodies was undertaken on 164 serum samples from 144 Hawaiian monk seals (Monachus schauinslandi) from the northwestern Hawaiian Islands collected between 1995 and 2002. The buffered antigen plate agglutination test (BPAT), the indirect enzyme immunoassay (I-ELISA), the competitive enzyme immunoassay (C-ELISA), and the fluorescence polarization assay (FPA) were compared with regard to their ability in detecting antibodies to Brucella spp. in the serum samples. Overall, antibodies were detected in 28 (17.1%) animals, using the BPAT test, 25 (15.2%) by the C-ELISA, and 19 (11.6%) in the I-ELISA and the FPA test, using thresholds established for cattle. No evidence of gross pathology consistent with clinical brucellosis was noted in any of the seropositive animals tested. Although further work would be necessary to validate these tests for use with monk seals it appears that both the C-ELISA and the FPA tests would be appropriate as diagnostic screening tests for detection of antibodies to Brucella spp. in this species. PMID:15827218

  19. High-throughput screening assay for the environmental water samples using cellular response profiles.

    PubMed

    Pan, Tianhong; Li, Haoran; Khare, Swanand; Huang, Biao; Yu Huang, Dorothy; Zhang, Weiping; Gabos, Stephan

    2015-04-01

    Chemical and physical analyses are commonly used as screening methods for the environmental water. However, these methods can only look for the targeted substance but may miss unexpected toxicants. Furthermore, the synergistic effects of mixture cannot be detected. In order to set up the assay criteria for determining various biological activities at a cellular level that could potentially lead to toxicity of environmental water samples, a novel test based on cellular response by using Real-Time Cellular Analyzer (RTCA) is proposed in this study. First, the water sample is diluted to a series of strengths (80%, 60%, 40%, 30%, 20% and 10%) to get the multi-concentration cellular response profile. Then, the area under the cellular response profile (AUCRP) is calculated. Comparing to the normal cell growth of negative control, a new biological activity index named Percentage of Effect (PoE) has been presented which reflects the cumulative inhibitory activity of cell growth over the log-phase. Finally, a synthetical index PoE50 is proposed to evaluate the intensity of biological activities in water samples. The biological experiment demonstrates the effectiveness of the proposed method. PMID:25637748

  20. Combinatorial screening for specific drug solubilizers with switchable release profiles.

    PubMed

    Wieczorek, Sebastian; Vigne, Sara; Masini, Tiziana; Ponader, Daniela; Hartmann, Laura; Hirsch, Anna K H; Börner, Hans G

    2015-01-01

    Polymer-block-peptide conjugates are tailored to render hydrophobic small molecule drugs water soluble. The combinatorial strategy selects for bioconjugates that exhibit sequence-specific solubilization and switchable release profiles of the cargo through incorporation of a disulfide linker moiety into the peptide-library design. While the study focused on the photosensitizer m-THPC and reductive carrier cleavage, the approach is generic and might be expanded toward a broad range of poorly soluble small-molecule drugs and other selective cleavage mechanisms to disassemble a peptide binding domain of the bioconjugate-based solubilizer. PMID:25557786

  1. Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.

    PubMed

    Jones, Shuna A; Salter, Robert S; Goldsmith, Tim; Quintana, Julio; Rapnicki, Paul; Shuck, Karen; Wells, Jim E; Schneider, Marilyn J; Griffin, Dee

    2014-02-01

    A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption. PMID:24490924

  2. Indirect competitive assays on DVD for direct multiplex detection of drugs of abuse in oral fluids.

    PubMed

    Zhang, Lingling; Li, Xiaochun; Li, Yunchao; Shi, Xiaoli; Yu, Hua-Zhong

    2015-02-01

    On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods. PMID:25540088

  3. Preventing drug interactions by online prescription screening in community pharmacies and medical practices

    Microsoft Academic Search

    Hillel Halkin; Itzhak Katzir; Irena Kurman; Joseph Jan; Becky Ben-Oz Malkin

    2001-01-01

    Background: Drug interactions have been shown to be preventable by computerized prescription entry and screening only in hospitals and not in community-based practice.Methods: We retrospectively evaluated the effect of online prescription screening in community pharmacies and physician offices of one health maintenance organization, phased in during 3 consecutive 6-month periods in 1998 to 1999 (period I, system active only in

  4. A preliminary investigation into the use of biosensors to screen stomach contents for selected poisons and drugs.

    PubMed

    Redshaw, Natalie; Dickson, Stuart J; Ambrose, Vikki; Horswell, Jacqui

    2007-10-25

    The bioluminescence response of two genetically modified (lux-marked) bacteria to potentially toxic compounds (PTCs) in stomach contents was monitored using an in vitro assay. Cells of Escherichia coli HB101 and Salmonella typhimurium both carrying the lux light producing gene on a plasmid (pUDC607) were added to stomach contents containing various concentrations of organic and inorganic compounds. There was some variability in the response of the two biosensors, but both were sensitive to the herbicides glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); pentachlorophenol (PCP), and inorganic poisons arsenic and mercury at a concentration range likely to be found in stomach contents samples submitted for toxicological analysis. This study demonstrates that biosensor bioassays could be a useful preliminary screening tool in forensic toxicology and that such a toxicological screening should include more than one test organism to maximise the number of PTC's detected. The probability of false positive results from samples containing compounds that may interfere with the assay such as over-the-counter (OTC) drugs and caffeine in tea and coffee was also investigated. Of the substances tested only coffee has the potential to cause false positive results. PMID:17276023

  5. Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

    PubMed

    Basilicata, Pascale; Pieri, Maria; Settembre, Veronica; Galdiero, Alessandra; Della Casa, Elvira; Acampora, Antonio; Miraglia, Nadia

    2011-11-15

    According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred. PMID:21992470

  6. Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration

    NASA Astrophysics Data System (ADS)

    Kitova, Elena N.; El-Hawiet, Amr; Klassen, John S.

    2014-08-01

    A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (?103 M-1) ligands from moderate and high affinity (>105 M-1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

  7. A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples.

    PubMed

    Metcalfe, Chris D; Kleywegt, Sonya; Letcher, Robert J; Topp, Edward; Wagh, Purva; Trudeau, Vance L; Moon, Thomas W

    2013-06-01

    Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for EDCs in wastewater and other environmental matrixes. PMID:23542486

  8. Polymorphic screen and drug–excipient compatibility studies of the antichagasic benznidazole

    Microsoft Academic Search

    Flávia Pires Maximiano; Kátia Monteiro Novack; Maria Terezinha Bahia; Lívia Lira de Sá-Barreto; Marcílio Sérgio Soares da Cunha-Filho

    The purpose of this study was to investigate the polymorphism and compatibility of benznidazole (BNZ), a drug used in the\\u000a treatment of Chagas disease. This drug was subjected to a polymorphic screen using a number of solvents and precipitation\\u000a procedures to explore the possible existence of different crystal structures of BNZ. The compatibility of BNZ with selected\\u000a pharmaceutical excipients was

  9. The C3H/HeJ mouse and DEBR rat models for alopecia areata: review of preclinical drug screening approaches and results

    PubMed Central

    Sun, Jing; Silva, Kathleen A.; McElwee, Kevin J.; King, Lloyd E.; Sundberg, John P.

    2009-01-01

    The C3H/HeJ inbred mouse strain and the Dundee Experimental Bald Rat (DEBR) strain spontaneously develop adult onset alopecia areata (AA), a cell mediated disease directed against actively growing hair follicles. The low frequency of AA and the inability to predict the stage of AA as it evolves in the naturally occuring C3H/HeJ model of AA can be converted into a highly predictable system by grafting full thickness skin from AA affected mice to normal haired mice of the same strain. The rat DEBR model develops spontaneous AA at a higher frequency than in the mouse model but they are more expensive to use in drug studies due to their larger size. Regardless of the shortcomings of either model, these rodent models can be used succesfully to screen novel or approved drugs for efficacy to treat human AA. Since the pathogenesis of AA follows the canonical lymphocytic co-stimulatory cascade in the mouse AA model, it can be used to screen compounds potentially useful to treat a variety of cell mediated diseases. Efficacy of various agents can easily be screened by simply observing the presence, rate, and cosmetic acceptability of hair regrowth. More sophisticated assays can refine how the drugs induce hair regrowth and evaluate the underlying pathogenesis of AA. Some drugs commonly used to treat human AA patients work equally as well in both rodent models validating their usefulness as models for drug efficacy and safety for human AA. PMID:18798913

  10. Screening for antimycotic properties of 56 traditional Chinese drugs.

    PubMed

    Blaszczyk, T; Krzyzanowska, J; Lamer-Zarawska, E

    2000-05-01

    In the present study we screened extracts of 56 widely used dried Chinese medical plants or their parts (TCD) for their antimycotic properties against pathological phyla of Aspergillus fumigatus, Candida albicans, Geotrichum candidum and Rhodotorula rubra. The highest activity against Aspergillus fumigatus was shown by Carthamus tinctorius L. (flos) and Rheum palmatum L. (radix et rhizoma) and against Candida albicans, Scutellaria baicalensis Georgi (radix) had the highest activity. The highest activity against Geotrichum candidum was shown by Agastache rugosa (Fisch et Mey.) O. Ktze. Herba Menthae has moderate antimycotic properties. PMID:10815018

  11. A new automated fourth-generation HIV screening assay with sensitive antigen detection module and high specificity.

    PubMed

    Weber, Bernard

    2005-01-01

    New screening enzyme immunoassays, which permit the simultaneous detection of HIV antigens reduce the diagnostic window period between the time of immunodeficiency virus (HIV) infection and seroconversion. The VIDAS HIV DUO Ultra is an enzyme-linked fluorescent assay (ELFA) for the screening of HIV infection. It is performed with the fully automated VIDAS or mini-VIDAS instruments, which are so-called walk away systems. The detection limit is 3 pg of HIV-1 p24 Ag/mL serum. HIV antibody is detected with the same sensitivity as stand-alone third-generation antibody tests. The total incubation time is about 2 h. Results are calculated, interpreted, and printed by the VIDAS instrument. Usually, fourth-generation assays demand a special algorithm for the analysis of reactive samples. For the anti-HIV part of the assay, confirmation of reactivity should be done with an assay that lacks the p24-antigen detection module and when reactivity persists subsequently by immunoblot. For the p24-antigen part, confirmation of reactivity should be analyzed in an assay that lacks the anti-HIV detection part. PMID:16061981

  12. Application of a fish DNA damage assay as a biological toxicity screening tool for metal plating wastewater

    SciTech Connect

    Choi, K.; Zong, M.; Meier, P.G.

    2000-01-01

    The utility of a fish DNA damage assay as a rapid monitoring tool was investigated. Metal plating wastewater was chosen as a sample because it contains various genotoxic metal species. Fish DNA damage assay results were compared to data generated from the conventional whole effluent toxicity (WET) test procedure. The Microtox{reg_sign} assay (Azur Environmental, Carlsbad, CA, USA) using Vibrio fischeri was also employed. Eleven samples from two metal plating companies were collected for this evaluation. For the fish DNA damage assay, 7-d-old fathead minnow larvae, Pimephales promelas, were utilized. They were exposed to a series of dilutions at 20 C for 2 h. Whole effluent toxicity tests conducted in this study included two acute toxicity tests with Daphnia magna and fathead minnows and two chronic toxicity tests with Ceriodaphnia dubia and fathead minnows. The fish DNA damage assay showed good correlations with both the acute and chronic WET test results, especially with those obtained with fathead minnows. The kappa values, an index of agreement, between the fish DNA damage assay and WET tests were shown to be acceptable. These findings imply that this novel fish DNA damage assay has use as an expedient toxicity screening procedure since it produces comparable results to those of the acute and chronic fathead minnow toxicity tests.

  13. A rapid in vitro screening system for the identification and evaluation of anticancer drugs

    SciTech Connect

    Kao, J.W.; Collins, J.L. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-01-01

    We report the development of an in vitro screening system that can be used to identify new anticancer drugs that are specifically cytotoxic for dividing cells. The screening system takes advantage of the potential of many cell lines, including tumor cells, to stop dividing when they are plated at high cell density. The cytotoxic effects of anticancer drugs on dividing (i.e., cells plated at low cell density) and nondividing cells (i.e., cells plated at high cell density) is measured by the incorporation of 51Cr. This in vitro system was evaluated by measuring the cytotoxic effects of the anticancer drugs cisplatin, thiotepa, doxorubicin, methotrexate, and vinblastine on the cell lines B/C-N, ME-180, and MCF-7. In this in vitro system the concentrations of the anticancer drugs that produced significant cytotoxicity on only dividing cells are similar to the concentrations that are used clinically. The fact that this in vitro system is rapid, simple, applicable to many cell types, and able to predict effective concentrations of anticancer drugs should make it useful for the screening of new anticancer drugs and for the design of preclinical studies.

  14. Application of an ELISA-elution assay as a screening tool for dissociation of yolk antibody–antigen complexes

    Microsoft Academic Search

    Angela Kummer; Eunice C. Y Li-Chan

    1998-01-01

    A modified enzyme-linked immunosorbent assay termed ELISA-elution assay was used as a screening tool to compare the efficiency of eluents for the dissociation of hen yolk immunoglobulin IgY–bovine IgG complexes. The potential denaturing effects of the eluents were also monitored. Different buffers (pH 2.3–7.5), containing various types and concentrations of salts (NaCl, (NH4)2SO4 and MgCl2) as well as polyols (ethylene

  15. Rapid prototyping of concave microwells for the formation of 3D multicellular cancer aggregates for drug screening

    PubMed Central

    Tu, Ting-Yuan; Wang, Zhe; Bai, Jing; Sun, Wei; Peng, Weng Kung; Huang, Ruby Yun-Ju; Thiery, Jean-Paul; Kamm, Roger D.

    2014-01-01

    Microwell technology has revolutionized many aspects of in vitro cellular studies from 2-dimensional (2D) traditional cultures to 3-dimensional (3D) in vivo-like functional assays. However, existing lithography-based approaches are often costly and time-consuming. This study presents a rapid, low-cost prototyping method of CO2 laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells were investigated, and each produced distinctive microwell features. Among these three materials, PS cell culture dishes produced the optimal surface smoothness and roundness. A549 lung cancer cells were grown to form cancer aggregates of controllable size from ~40 to ~80 ?m in PS microwells. Functional assays of spheroids were performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening was investigated and revealed considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach. PMID:23983140

  16. Rapid prototyping of concave microwells for the formation of 3D multicellular cancer aggregates for drug screening.

    PubMed

    Tu, Ting-Yuan; Wang, Zhe; Bai, Jing; Sun, Wei; Peng, Weng Kung; Huang, Ruby Yun-Ju; Thiery, Jean-Paul; Kamm, Roger D

    2014-04-01

    Microwell technology has revolutionized many aspects of in vitro cellular studies from 2D traditional cultures to 3D in vivo-like functional assays. However, existing lithography-based approaches are often costly and time-consuming. This study presents a rapid, low-cost prototyping method of CO2 laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells are investigated, and each produces distinctive microwell features. Among these three materials, PS cell culture dishes produce the optimal surface smoothness and roundness. A549 lung cancer cells are grown to form cancer aggregates of controllable size from ?40 to ?80 ?m in PS microwells. Functional assays of spheroids are performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening is investigated and reveals considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach. PMID:23983140

  17. Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay

    PubMed Central

    Wiggers, Helton J.; Rocha, Josmar R.; Fernandes, William B.; Sesti-Costa, Renata; Carneiro, Zumira A.; Cheleski, Juliana; da Silva, Albérico B. F.; Juliano, Luiz; Cezari, Maria H. S.; Silva, João S.; McKerrow, James H.; Montanari, Carlos A.

    2013-01-01

    A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (Ki) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol?1 atom?1 (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization. PMID:23991231

  18. Efficacy of drug screening in forensic autopsy: Retrospective investigation of routine toxicological findings.

    PubMed

    Tominaga, Mariko; Michiue, Tomomi; Inamori-Kawamoto, Osamu; Hishmat, Asmaa Mohammed; Oritani, Shigeki; Takama, Masashi; Ishikawa, Takaki; Maeda, Hitoshi

    2015-05-01

    Toxicological analysis is indispensable in forensic autopsy laboratories, but often depends on the limitations of individual institutions. The present study reviewed routine drug screening data of forensic autopsy cases (n=2996) during an 18.5-year period (January 1996-June 2014) at our institute to examine the efficacy of the procedures and findings in autopsy diagnosis and interpretation. Drug screening was performed using on-site immunoassay screening devices and gas chromatography/mass spectrometry (GC/MS) in all cases, followed by re-examination using GC/MS and liquid chromatography/tandem mass spectrometry (LC/MS/MS) at a cooperating institute in specific cases in the last 4years. GC/MS detected drugs in 486 cases (16.2%), including amphetamines (n=160), major tranquilizers (n=72), minor tranquilizers (n=294), antidepressants (n=21), cold remedies (n=77), and other drugs (n=19). Among these cases, fatal intoxication (n=123) involved amphetamines (n=73), major tranquilizers (n=37), minor tranquilizers (n=86), antidepressants (n=3), and cold remedies (n=9); most cases involved self-administration, alleged suicide and accidental overdose, while homicide was not included. These drugs were also identified in other manners of death, including homicide (n=40/372), suicide (n=34/226), accidental falls (n=27/129), and natural death (n=72/514). In these cases, on-site immunoassay screening of drugs of abuse showed negative findings in 2440 cases (81.4% in all cases), while GC/MS detected other drugs in 218 cases (7.3% in all cases), including several antipsychotic drugs, acetaminophen and salicylic acid. Further analysis using LC/MS/MS detected low concentrations of benzodiazepines in 32 cases, and also anti-diabetic and hypertensive drugs in a case of fatal abuse. These observations indicate the efficacy of systematic routine toxicological analysis to investigate not only the cause of death but also the background of fatalities in forensic autopsy. The provision of extensive drug screening is needed for forensic and social risk management, considering the marked diversity of medical and illicit drugs. PMID:25637163

  19. Yeast three-hybrid screening for identifying anti-tuberculosis drug targets.

    PubMed

    Moser, Simone; Johnsson, Kai

    2013-11-25

    Mycobacterium goes yeast: Target deconvolution of anti-tuberculosis drugs can be a very challenging task. Here we report a yeast 3-hybrid system that allows promising small molecules to be screened for protein targets of a pathogen in nontoxic yeast cells. The system employs libraries of randomly fragmented bacterial DNA and offers a technically simple alternative approach for target identification. PMID:24133019

  20. Research paper Screen of FDA-approved drug library reveals compounds that protect hair cells

    E-print Network

    Rubel, Edwin

    Research paper Screen of FDA-approved drug library reveals compounds that protect hair cells from Accepted 9 August 2012 Available online xxx a b s t r a c t Loss of mechanosensory hair cells in the inner cause hair cell death as a side effect. These include amino- glycoside antibiotics, such as neomycin

  1. Docking and scoring in virtual screening for drug discovery: methods and applications

    Microsoft Academic Search

    Douglas B. Kitchen; Hélène Decornez; John R. Furr; Jürgen Bajorath

    2004-01-01

    Computational approaches that 'dock' small molecules into the structures of macromolecular targets and 'score' their potential complementarity to binding sites are widely used in hit identification and lead optimization. Indeed, there are now a number of drugs whose development was heavily influenced by or based on structure-based design and screening strategies, such as HIV protease inhibitors. Nevertheless, there remain significant

  2. Cervical cancer screening and abnormalities among women in a residential drug-rehabilitation program.

    PubMed

    Tilley, D M; Hristov, S; Templeton, D J; Sharp, N C; O'Connor, C C

    2012-01-01

    Women in a residential drug-rehabilitation program had lower rates of cervical screening attendance and higher rates ofdetected abnormalities than women attending a local Well Women's Clinic. As a result ofthis study we plan to include a more comprehensive sexual health history into routine women's health consultations. PMID:22951016

  3. Screening for anti-HIV drugs that can combine virucidal and virustatic activities synergistically

    Microsoft Academic Search

    A. N. Chakrabarty; Musfiqua Mookerjee; Sujata G. Dastidar

    2000-01-01

    Chlorcyclizine HCl and ciprofloxacin HCl were shown to have anti-HIV activity. They possess virustatic and virucidal activities against HIV, a murine retrovirus (RV) and several other RNA and DNA viruses. These drugs were screened from a large number of compounds on the basis of in vitro mutagenicity and antimetabolite detection tests. Subsequent studies were based on different exo vivo cell

  4. Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

    Microsoft Academic Search

    Alexandra Kroll; Christian Dierker; Christina Rommel; Daniela Hahn; Wendel Wohlleben; Christian Schulze-Isfort; Christian Göbbert; Matthias Voetz; Ferdinand Hardinghaus; Jürgen Schnekenburger

    2011-01-01

    Background  Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation\\u000a of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials\\u000a and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in

  5. Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted ?-Secretase Using Single-Molecule Imaging.

    PubMed

    Gunnarsson, Anders; Snijder, Arjan; Hicks, Jennifer; Gunnarsson, Jenny; Höök, Fredrik; Geschwindner, Stefan

    2015-04-21

    Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against ?-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds. PMID:25855499

  6. A High Content Drug Screen Identifies Ursolic Acid as an Inhibitor of Amyloid ? Protein Interactions with Its Receptor CD36*

    PubMed Central

    Wilkinson, Kim; Boyd, Justin D.; Glicksman, Marcie; Moore, Kathryn J.; El Khoury, Joseph

    2011-01-01

    A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid ? (A?) in the brain. A? binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of A? binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of A? binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled A? to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, A? concentration, time required for A? binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of A? binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of A? to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ?20 ?m, with a maximal inhibition of 64%. Ursolic acid also blocked binding of A? to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of A? to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block A?-CD36 interactions. PMID:21835916

  7. A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

    SciTech Connect

    Chang, Jerry [Vanderbilt University; Tomlinson, Ian [Oak Ridge National Laboratory (ORNL); Warnement, Michael [Vanderbilt University; Iwamoto, Hideki [Vanderbilt University

    2011-01-01

    The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

  8. Bridging the gap from screening assays to estrogenic effects in fish: potential roles of multiple estrogen receptor subtypes.

    PubMed

    Yost, Erin E; Lee Pow, Crystal; Hawkins, Mary Beth; Kullman, Seth W

    2014-05-01

    This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hER?. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hER? and hER?, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ER? subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ER? subtypes are critically involved in the teleost estrogenic response, with the ER?:ER? ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures. PMID:24422420

  9. Bridging the Gap From Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes

    PubMed Central

    2015-01-01

    This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hER?. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hER? and hER?, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ER? subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ER? subtypes are critically involved in the teleost estrogenic response, with the ER?:ER? ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures. PMID:24422420

  10. The US EPA's Endocrine Disruptor Screening Program: In VItro and In Vivo Mammalian Tier 1 Screening Assays

    EPA Science Inventory

    In response to emerging concerns that environmental chemicals may have adverse effects on human health by altering the function of the endocrine system, the Food Quality Protection Act mandated that the U.S. EPA develop and implement an endocrine disruptor screening program (EDSP...

  11. Drug screening in medical examiner casework by high-resolution mass spectrometry (UPLC-MSE-TOF).

    PubMed

    Rosano, Thomas G; Wood, Michelle; Ihenetu, Kenneth; Swift, Thomas A

    2013-10-01

    Postmortem drug findings yield important analytical evidence in medical examiner casework, and chromatography coupled with nominal mass spectrometry (MS) serves as the predominant general unknown screening approach. We report screening by ultra performance liquid chromatography (UPLC) coupled with hybrid quadrupole time-of-flight mass spectrometer (MS(E)-TOF), with comparison to previously validated nominal mass UPLC-MS and UPLC-MS-MS methods. UPLC-MS(E)-TOF screening for over 950 toxicologically relevant drugs and metabolites was performed in a full-spectrum (m/z 50-1,000) mode using an MS(E) acquisition of both molecular and fragment ion data at low (6 eV) and ramped (10-40 eV) collision energies. Mass error averaged 1.27 ppm for a large panel of reference drugs and metabolites. The limit of detection by UPLC-MS(E)-TOF ranges from 0.5 to 100 ng/mL and compares closely with UPLC-MS-MS. The influence of column recovery and matrix effect on the limit of detection was demonstrated with ion suppression by matrix components correlating closely with early and late eluting reference analytes. Drug and metabolite findings by UPLC-MS(E)-TOF were compared with UPLC-MS and UPLC-MS-MS analyses of postmortem blood in 300 medical examiner cases. Positive findings by all methods totaled 1,528, with a detection rate of 57% by UPLC-MS, 72% by UPLC-MS-MS and 80% by combined UPLC-MS and UPLC-MS-MS screening. Compared with nominal mass screening methods, UPLC-MS(E)-TOF screening resulted in a 99% detection rate and, in addition, offered the potential for the detection of nontargeted analytes via high-resolution acquisition of molecular and fragment ion data. PMID:23999055

  12. Cardiac Arrhythmia: In vivo screening in the zebrafish to overcome complexity in drug discovery

    PubMed Central

    MacRae, Calum A.

    2010-01-01

    Importance of the field Cardiac arrhythmias remain a major challenge for modern drug discovery. Clinical events are paroxysmal, often rare and may be asymptomatic until a highly morbid complication. Target selection is often based on limited information and though highly specific agents are identified in screening, the final efficacy is often compromised by unanticipated systemic responses, a narrow therapeutic index and substantial toxicities. Areas covered in this review Our understanding of complexity of arrhythmogenesis has grown dramatically over the last two decades, and the range of potential disease mechanisms now includes pathways previously thought only tangentially involved in arrhythmia. This review surveys the literature on arrhythmia mechanisms from 1965 to the present day, outlines the complex biology underlying potentially each and every rhythm disturbance, and highlights the problems for rational target identification. The rationale for in vivo screening is described and the utility of the zebrafish for this approach and for complementary work in functional genomics is discussed. Current limitations of the model in this setting and the need for careful validation in new disease areas are also described. What the reader will gain An overview of the complex mechanisms underlying most clinical arrhythmias, and insight into the limits of ion channel conductances as drug targets. An introduction to the zebrafish as a model organism, in particular for cardiovascular biology. Potential approaches to overcoming the hurdles to drug discovery in the face of complex biology including in vivo screening of zebrafish genetic disease models. Take home message In vivo screening in faithful disease models allows the effects of drugs on integrative physiology and disease biology to be captured during the screening process, in a manner agnostic to potential drug target or targets. This systematic strategy bypasses current gaps in our understanding of disease biology, but emphasizes the importance of the rigor of the disease model. PMID:20835353

  13. Screening drug target proteins based on sequence information.

    PubMed

    Wang, Jiao T; Liu, Wei; Tang, Hailin; Xie, Hongwei

    2014-06-01

    Identifying new drug target (DT) proteins is important in pharmaceutical and biomedical research. General machine learning method (GMLM) classifiers perform fairly well at prediction if the training dataset is well prepared. However, a common problem in preparing the training dataset is the lack of a negative dataset. To address this problem, we proposed two methods that can help GMLM better select the negative training dataset from the test dataset. The prediction accuracy was improved with the training dataset from the proposed strategies. The classifier identified 1797 and 227 potential DT proteins, some of which were mentioned in previous research, which added correlative weight to the new method. Practically, these two sets of potential DT proteins or their homologues are worth considering. PMID:24662273

  14. Chemosensitivity of Anaplastic Thyroid Cancer Based on a Histoculture Drug Response Assay

    PubMed Central

    Uruno, Takashi; Masaki, Chie; Akaishi, Junko; Matsuzu, Kenichi; Suzuki, Akifumi; Ohkuwa, Keiko; Shibuya, Hiroshi; Kitagawa, Wataru; Nagahama, Mitsuji; Sugino, Kiminori; Ito, Koichi

    2015-01-01

    The chemosensitivity of anaplastic thyroid cancer (ATC) to some cytotoxic agents was investigated by the histoculture drug response assay (HDRA). Thirty specimens from 22 patients with ATC were obtained from surgically resected subjects. The drugs tested were paclitaxel (PTX), docetaxel (DOC), adriamycin (ADM), nedaplatin (254-S), cisplatin (CDDP), carboplatin (CBDCA), etoposide (VP-16), 5-fluorouracil (5-FU), mitomycin C (MMC), and cyclophosphamide (CPA). PTX was the most effective agent, and 25 of 29 cases (86.2%) had high inhibition rates (IRs; over 70%), while DOC, another taxane, had lower IRs (median, 32.6%). 254-S had the second highest IR (median 68.1%), higher than other platins, CDDP (median 47.3%) and CBDCA (median 27.4%). The IR of 50% dose PTX (20??g/mL, median 30.6%) was markedly decreased, while that of 50% dose 254-S (10??g/mL, median 63.3%) still retained its inhibition effect compared to 100% dose. Most recurrent samples had higher IRs than primary lesions, but the IRs of different drugs differed between primary and recurrent lesions, even with samples from the same patients. PTX has a higher IR to ATC tissues in the HDRA, which suggests that it may be a key drug for the treatment of patients with ATC.

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

    PubMed Central

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

    2012-01-01

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

  16. NC-TEST: noncontact thermal emissions screening technique for drug and alcohol detection

    NASA Astrophysics Data System (ADS)

    Prokoski, Francine J.

    1997-01-01

    Drug abuse is highly correlated with criminal behavior. The typical drug-using criminal commits hundreds of crimes per year. The crime rate cannot be significantly reduced without a reduction in the percentage of the population abusing drugs and alcohol. Accurate and timely estimation of that percentage is important for policy decisions concerning crime control, public health measures, allocation of intervention resources for prevention and treatment, projections of criminal justice needs, and the evaluation of policy effectiveness. Such estimation is particularly difficult because self reporting is unreliable; and physical testing has to date required blood or urine analysis which is expensive and invasive, with the result that too few people are tested. MIKOS Ltd. has developed a non-contact, passive technique with the potential for automatic, real- time screening for drug and alcohol use. The system utilizes thermal radiation which is spontaneously and continuously emitted by the human body. Facial thermal patterns and changes in patterns are correlated with standardized effects of specific drugs and alcohol. A portable system incorporating the collection and analysis technique can be used episodically to collect data for estimating drug and alcohol use by general unknown populations such as crowds at airports, or it can be used for repetitive routine screening of specific known groups such as airline pilots, military personnel, school children, or persons on probation or parole.

  17. False-positive interferences of common urine drug screen immunoassays: a review.

    PubMed

    Saitman, Alec; Park, Hyung-Doo; Fitzgerald, Robert L

    2014-09-01

    Urine drug screen (UDS) immunoassays are a quick and inexpensive method for determining the presence of drugs of abuse. Many cross-reactivities exist with other analytes, potentially causing a false-positive result in an initial drug screen. Knowledge of these potential interferents is important in determining a course of action for patient care. We present an inclusive review of analytes causing false-positive interferences with drugs-of-abuse UDS immunoassays, which covers the literature from the year 2000 to present. English language articles were searched via the SciFinder platform with the strings 'false positive [drug] urine' yielding 173 articles. These articles were then carefully analyzed and condensed to 62 that included data on causes of false-positive results. The discussion is separated into six sections by drug class with a corresponding table of cross-reacting compounds for quick reference. False-positive results were described for amphetamines, opiates, benzodiazepines, cannabinoids, tricyclic antidepressants, phencyclidine, lysergic acid diethylamide and barbiturates. These false-positive results support the generally accepted practice that immunoassay positive results are considered presumptive until confirmed by a second independent chemical technique. PMID:24986836

  18. Screening, isolation and optimization of anti–white spot syndrome virus drug derived from terrestrial plants

    PubMed Central

    Ghosh, Upasana; Chakraborty, Somnath; Balasubramanian, Thangavel; Das, Punyabrata

    2014-01-01

    Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug. PMID:25183066

  19. Screening, isolation and optimization of anti–white spot syndrome virus drug derived from marine plants

    PubMed Central

    Chakraborty, Somnath; Ghosh, Upasana; Balasubramanian, Thangavel; Das, Punyabrata

    2014-01-01

    Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1?000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug. PMID:25183065

  20. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  1. Applicability of a blood-brain barrier specific artificial membrane permeability assay at the early stage of natural product-based CNS drug discovery.

    PubMed

    Könczöl, Arpád; Müller, Judit; Földes, Emília; Béni, Zoltán; Végh, Krisztina; Kéry, Agnes; Balogh, György T

    2013-04-26

    While numerous natural products (NPs) possess activity on central nervous system (CNS) targets, there has been no analytical approach to effectively identify compounds with high brain penetration potential in complex mixtures at the early stage of drug discovery. To overcome this issue, the performance of an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB) for natural products and for plant extracts has been validated and characterized. It was found that the PAMPA-BBB assay preserves its predictive power in the case of natural products and provides high phytochemical selectivity, which enables its use as a unique filtering tool in terms of selecting brain-penetrable compounds from plant extracts. Moreover, the present study has demonstrated that simple modifications in the assay design allow the direct use of PAMPA-BBB filtered samples in a dereplication process, as performed by NMR and LC-MS. The applicability of this procedure was demonstrated using extracts prepared from Tanacetum parthenium, Vinca major, Salvia officinalis, and Corydalis cava, representing different types of chemical diversity and complexity. Thus, the proposed protocol represents a potentially valuable strategy in the NP-based CNS drug discovery environment with a high-throughput screening platform. PMID:23565574

  2. Beyond reverse pharmacology: Mechanism-based screening of Ayurvedic drugs

    PubMed Central

    Lele, R. D.

    2010-01-01

    This paper reviews the pharmacology of Indian medicinal plants, starting with the historical background of European work on the subject beginning as early as the 17th century, and tracing its history through the work of Sen and Bose in the 1930‘s, and Vakhil’s historic 1949 paper on Sarpaghanda. The often crucial role of patient feedback in early discoveries is highlighted, as is the time lag between proof of pharmacological action and identification of the active principle, and subsequent elucidation of mechanism of action. In the case of Indian plants in the 20th century this process sometimes took almost 50 years. Reserpine and its mechanisms are given in detail, and its current relevance to public health discussed. The foundation of present day methods of pharmacology is briefly presented so the complexity of methods used to identify properties of Ayurveda derived drugs like forskolin and baicalein, and their bioavailability, may be better appreciated. Ayurveda derived anti-oxidants and their levels of action, immuno-modulators, particularly with respect to the NF-kB pathway and its implications for cancer control, are all considered. The example of curcumin derived from turmeric is explained in more detail, because of its role in cancer prevention. Finally, the paper emphasizes the importance of Ayurveda’s concepts of rasayana as a form of dietary chemo-prevention; the significance of ahar, diet, in Ayurveda’s aspiration to prevent disease and restore health thus becomes clear. Understood in this light, Ayurveda may transcend pharmacology as a treatment paradigm. PMID:21731372

  3. Evaluating the Predictivity of Virtual Screening for Abl Kinase Inhibitors to Hinder Drug Resistance

    PubMed Central

    Gani, Osman A B S M; Narayanan, Dilip; Engh, Richard A

    2013-01-01

    Virtual screening methods are now widely used in early stages of drug discovery, aiming to rank potential inhibitors. However, any practical ligand set (of active or inactive compounds) chosen for deriving new virtual screening approaches cannot fully represent all relevant chemical space for potential new compounds. In this study, we have taken a retrospective approach to evaluate virtual screening methods for the leukemia target kinase ABL1 and its drug-resistant mutant ABL1-T315I. ‘Dual active’ inhibitors against both targets were grouped together with inactive ligands chosen from different decoy sets and tested with virtual screening approaches with and without explicit use of target structures (docking). We show how various scoring functions and choice of inactive ligand sets influence overall and early enrichment of the libraries. Although ligand-based methods, for example principal component analyses of chemical properties, can distinguish some decoy sets from active compounds, the addition of target structural information via docking improves enrichment, and explicit consideration of multiple target conformations (i.e. types I and II) achieves best enrichment of active versus inactive ligands, even without assuming knowledge of the binding mode. We believe that this study can be extended to other therapeutically important kinases in prospective virtual screening studies. PMID:23746052

  4. Transgenic Plants as Low-Cost Platform for Chemotherapeutic Drugs Screening

    PubMed Central

    Vergara, Daniele; de Domenico, Stefania; Maffia, Michele; Piro, Gabriella; Di Sansebastiano, Gian-Pietro

    2015-01-01

    In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects. PMID:25608652

  5. Transgenic plants as low-cost platform for chemotherapeutic drugs screening.

    PubMed

    Vergara, Daniele; de Domenico, Stefania; Maffia, Michele; Piro, Gabriella; Di Sansebastiano, Gian-Pietro

    2015-01-01

    In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects. PMID:25608652

  6. Toxicity Screening of the ToxCast Chemical Library Using a Zebrafish Developmental Assay

    EPA Science Inventory

    As part of the chemical screening and prioritization research program of the U.S. Environmental Protection Agency, the toxicity of the 320 ToxCast? Phase I chemicals were assessed using a vertebrate screen of developmental toxicity. Zebrafish embryos/larvae (Danio rerio) were exp...

  7. Dose response screening of the Toxcast chemical library using a Zebrafish developmental assay

    EPA Science Inventory

    As part of the chemical screening and prioritization research program of the U.S. Environmental Protection Agency, the toxicity of the 320 ToxCaspM Phase I chemicals was assessed using a vertebrate screen of developmental toxicity. Zebrafish embryos/larvae (Danio rerio) were expo...

  8. NMR screening in fragment-based drug design: a practical guide.

    PubMed

    Kim, Hai-Young; Wyss, Daniel F

    2015-01-01

    Fragment-based drug design (FBDD) comprises both fragment-based screening (FBS) to find hits and elaboration of these hits to lead compounds. Typical fragment hits have lower molecular weight (<300-350 Da) and lower initial potency but higher ligand efficiency when compared to those from high-throughput screening. NMR spectroscopy has been widely used for FBDD since it identifies and localizes the binding site of weakly interacting hits on the target protein. Here we describe ligand-based NMR methods for hit identification from fragment libraries and for functional cross-validation of primary hits. PMID:25618347

  9. Structure-based virtual screening for drug discovery: a problem-centric review.

    PubMed

    Cheng, Tiejun; Li, Qingliang; Zhou, Zhigang; Wang, Yanli; Bryant, Stephen H

    2012-03-01

    Structure-based virtual screening (SBVS) has been widely applied in early-stage drug discovery. From a problem-centric perspective, we reviewed the recent advances and applications in SBVS with a special focus on docking-based virtual screening. We emphasized the researchers' practical efforts in real projects by understanding the ligand-target binding interactions as a premise. We also highlighted the recent progress in developing target-biased scoring functions by optimizing current generic scoring functions toward certain target classes, as well as in developing novel ones by means of machine learning techniques. PMID:22281989

  10. ASSESSMENT OF THE TLC/SALMONELLA ASSAY FOR SCREENING HAZARDOUS WASTES

    EPA Science Inventory

    Using a modified version of the TLC/Salmonella assay developed by Bjorseth et al. (1982), 10 complex hazardous wastes were tested for mutagenic activity. The method couples thin layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of m...

  11. An Improved Whole-Seed Assay for Screening Wheat Germplasm for Polyphenol Oxidase Activity

    Microsoft Academic Search

    James V. Anderson; Craig F. Morris

    2001-01-01

    portant for consumer acceptance (Moss, 1971; Miskelly, 1984, 1996), while low L* values indicate undesirable Polyphenol oxidase (PPO) causes darkening and discoloration of discoloration or darkening of noodles. wheat (Triticum aestivum L.) foods such as noodles. Consequently, a simple, nondestructive, quantitative assay for determining PPO on There is a need for a simple, quantitative assay for one to a few

  12. Validation of Screening Assays for Developmental Toxicity: An Exposure-Based Approach

    EPA Science Inventory

    There continue to be widespread efforts to develop assay methods for developmental toxicity that are shorter than the traditional Segment 2 study and use fewer or no animals. As with any alternative test method, novel developmental toxicity assays must be validated by evaluating ...

  13. A simple plate-assay for screening extracellular naringinase produced by streptomycetes.

    PubMed

    Caraveo, Leonela; Medina, Héctor; Rodríguez-Buenfil, Ingrid; Montalvo-Romero, Carlos; Evangelista-Martínez, Zahaed

    2014-07-01

    A simple plate-assay was developed with the purpose of detecting extracellular naringinase in streptomycetes. The naringin agar medium (NAM) was prepared by mixing carbon utilization medium (ISP9) and naringin. The clearing zones around colonies are correlated with the enzymatic activity. The assay validation was performed through the use of commercial naringinase (Penicillium decumbens). PMID:24742969

  14. Indeterminate Human Immunodeficiency Virus Western Blot Profiles in Ethiopians with Discordant Screening-Assay Results

    Microsoft Academic Search

    Hailu Meles; Dawit Wolday; Arnaud Fontanet; Aster Tsegaye; Tesfaye Tilahun; Mathias Aklilu; Eduard Sanders; Tobias F. Rinke De Wit

    2002-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB reactivity among Ethiopians are hardly known. Here, we describe the profiles of

  15. Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential

    Microsoft Academic Search

    M. K. Johnson

    1977-01-01

    The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent

  16. Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda

    PubMed Central

    2013-01-01

    Background The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. Methods The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. Results The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. Conclusions The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation. PMID:23782846

  17. The association of pseudoephedrine sales restrictions on emergency department urine drug screen results in Oklahoma.

    PubMed

    Brandenburg, M A; Brown, S J; Arneson, W L; Arneson, D L

    2007-11-01

    On June 15, 2004, Oklahoma became the first state to reclassify pseudoephedrine as a Schedule V drug. Arrests in Oklahoma for the manufacture of methamphetamines in clandestine laboratories precipitously declined. It was hypothesized that a decrease in methamphetamine use could be shown in the patient population in Oklahoma's largest emergency department. To test this hypothesis, all urine drug screen results in the Saint Francis Hospital Trauma Emergency Center from January 2003 through May 2005 were reviewed. There was a significant increase in the total tests performed and the percentage of positive test results for the amphetamine drug class (p = 0.0004, R2 = 0.3785) over time. These results suggest that methamphetamine usage has not decreased in the emergency department patient population. Possibly, methamphetamine usage in Oklahoma has not been impacted by the passage of HB 2176 due to an increase in drug trafficking of methamphetamine into the state. PMID:18183861

  18. Screening for adolescent alcohol and drug use in pediatric health-care settings: predictors and implications for practice and policy

    PubMed Central

    2012-01-01

    Objective This paper used data from a study of pediatric primary care provider (PCP) screening practices to examine barriers to and facilitators of adolescent alcohol and other drug (AOD) screening in pediatric primary care. Methods A web-based survey (N?=?437) was used to examine the influence of PCP factors (attitudes and knowledge, training, self-efficacy, comfort with alcohol and drug issues); patient characteristics (age, gender, ethnicity, comorbidities and risk factors); and organizational factors (screening barriers, staffing resources, confidentiality issues) on AOD screening practices. Self-reported and electronic medical record (EMR)-recorded screening rates were also assessed. Results More PCPs felt unprepared to diagnose alcohol abuse (42%) and other drug abuse (56%) than depression (29%) (p?screen boys than girls, and male PCPs were even more likely than female PCPs to screen boys (23% versus 6%, p?screen and review results were identified as potential screening facilitators. Self-reported screening rates were significantly higher than actual (EMR-recorded) rates for all substances. Feeling prepared to diagnose AOD problems predicted higher self-reported screening rates (OR?=?1.02, p <0.001), and identifying time constraints as a barrier to screening predicted lower self-reported screening rates (OR?=?0.91, p?screening rates (OR?=?1.11, p?screening may impact adolescent substance-abuse screening and intervention, but organizational approaches (e.g., EMR tools and workflow) may matter more than PCP or patient factors in determining screening. PMID:23186254

  19. Assay of residual organic solvents in topiramate drug substance by capillary gas chromatography.

    PubMed

    Micheel, A P; Ko, C Y; Evans, C R

    1993-01-01

    The analysis of residual organic solvents (methanol, ethanol and toluene) in topiramate drug substance was investigated. Topiramate is a potent anticonvulsant drug under clinical evaluation. The drug is recrystallized from ethanol denatured by either methanol or toluene, and each residual solvent is controlled at 0.1% (w/w) level. A capillary gas chromatography (GC) method described in this manuscript utilizes a DB-WAX, 1 micron thick, 30 m x 0.53 mm i.d., column. Since topiramate is a thermally labile compound, the selection of the proper injector temperature is critical to the success of the analysis. The injector temperature was set at 120 degrees C to prevent degradation. The initial oven temperature was set at 55 degrees C for 8 min and programmed at a rate of 30 degrees C min-1 to a final temperature of 160 degrees C for 11 min. Helium was used as a carrier gas. The sample solvent selected was dimethylformamide pretreated with molecular sieves to remove trace amounts of alcohols that may interfere with the assay. The method was validated to be specific, linear, precise, sensitive, rugged and showed excellent recovery. PMID:8123739

  20. Spectrofluorimetric methods of stability-indicating assay of certain drugs affecting the cardiovascular system

    NASA Astrophysics Data System (ADS)

    Moussa, B. A.; Mohamed, M. F.; Youssef, N. F.

    2011-01-01

    Two stability-indicating spectrofluorimetric methods have been developed for the determination of ezetimibe and olmesartan medoxomil, drugs affecting the cardiovascular system, and validated in the presence of their degradation products. The first method, for ezetimibe, is based on an oxidative coupling reaction of ezetimibe with 3-methylbenzothiazolin-2-one hydrazone hydrochloride in the presence of cerium (IV) ammonium sulfate in an acidic medium. The quenching effect of ezetimibe on the fluorescence of excess cerous ions is measured at the emission wavelength, ?em, of 345 nm with the excitation wavelength, ?ex, of 296 nm. Factors affecting the reaction were carefully studied and optimized. The second method, for olmesartan medoxomil, is based on measuring the native fluorescence intensity of olmesartan medoxomil in methanol at ?em = 360 nm with ?ex = 286 nm. Regression plots revealed good linear relationships in the assay limits of 10-120 and 8-112 g/ml for ezetimibe and olmesartan medoxomil, respectively. The validity of the methods was assessed according to the United States Pharmacopeya guidelines. Statistical analysis of the results exposed good Student's t-test and F-ratio values. The introduced methods were successfully applied to the analysis of ezetimibe and olmesartan medoxomil in drug substances and drug products as well as in the presence of their degradation products.

  1. Characterization of a new small bowel adenocarcinoma cell line and screening of anti-cancer drug against small bowel adenocarcinoma.

    PubMed

    Suzuki, Hirobumi; Hirata, Yoshihiro; Suzuki, Nobumi; Ihara, Sozaburo; Sakitani, Kosuke; Kobayashi, Yuka; Kinoshita, Hiroto; Hayakawa, Yoku; Yamada, Atsuo; Watabe, Hirotsugu; Tateishi, Keisuke; Ikenoue, Tsuneo; Yamaji, Yutaka; Koike, Kazuhiko

    2015-02-01

    Small bowel adenocarcinoma (SBA) is a rare, aggressive malignancy with a poor prognosis, and the mechanisms of carcinogenesis in SBA remain unclear. Our aims were to investigate the molecular mechanisms underlying SBA and to identify treatments by establishing and characterizing an SBA cell line and performing anti-cancer drug screening. SIAC1 cells, established from jejunal SBA, showed epithelial characteristics and formed organoids in 3D culture. SIAC1 cells had a heterozygous ?-catenin deletion mutation, resulting in a stable ?-catenin protein with enhanced Wnt/?-catenin activity. SIAC1 cells lacked MLH1 and MSH6 expression, and target genes such as TGFBR2 and ACVR2 showed frameshift mutations. Among 10 clinical SBA samples, 2 (20%) had interstitial deletions in ?-catenin, expression of mismatch repair protein was aberrant in 4 (40%), and heterozygous frameshift mutations of three target genes were found in all 10 samples. On screening assay using 140 compounds, eribulin significantly inhibited SIAC1 cell growth both in vitro and in vivo by inhibition of the Wnt/?-catenin pathway via enhanced degradation of ?-catenin. In conclusion, we established an SBA cell line with molecular characteristics similar to those of clinical SBA samples, including ?-catenin deletion and mismatch repair protein deficiency, that will be useful for SBA research. Eribulin might be a candidate for SBA treatment due to its inhibitory effect on Wnt/?-catenin signaling. PMID:25478808

  2. Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

    E-print Network

    Paris-Sud XI, Université de

    ), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 2 Screening Technology & Pharmacology Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 3 Active Compound Space Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 4 Image Mining Group, Institut Pasteur Korea

  3. Performance Evaluation of three Liquid Chromatography Mass Spectrometry Methods for Broad Spectrum Drug Screening

    PubMed Central

    Lynch, Kara L.; Breaud, Autumn R.; Vandenberghe, Hilde; Wu, Alan H. B.; Clarke, William

    2010-01-01

    BACKGROUND Liquid chromatography-mass spectrometry (LC-MS) and tandem LC-MS (LC-MS/MS) are increasingly used in toxicology laboratories as a complementary method to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ultraviolet detection (LC-UV) for comprehensive drug screening (CDS). This study was designed to characterize the sensitivity and specificity of three LC-MS(/MS) vendor-supplied methods for targeted CDS and identify the current limitations associated with the use of these technologies. METHODS Five methods for broad spectrum CDS, including LC-UV (REMEDi), full scan GC-MS, LC-MS (ZQ™-Mass Detector with MassLynx™-software), LC-QTRAP-MS/MS (3200-QTRAP® with Cliquid®-software) and LC-LIT-MS/MS (LXQ™ Linear Ion Trap with ToxID™-software) were evaluated based on their ability to detect drugs in 48 patient urine samples. RESULTS The tandem MS methods identified 15% more drugs than the single stage MS or LC-UV methods. Use of two broad spectrum screening methods identified more drugs than any single system alone. False negatives and false positives generated by the LC-MS(/MS) software programs were identified upon manual review of the raw data. CONCLUSIONS The LC-MS/MS methods detected a broader menu of drugs; however, it is essential to establish manual data review criteria for all LC-MS(/MS) drug screening methods. Use of an EI-GC-MS and ESI-LC-MS/MS combination for targeted CDS may be optimal due to the complementary nature of the chromatographic and ionization techniques. PMID:20540936

  4. Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals.

    PubMed

    Iguchi, Taisen; Irie, Fumi; Urushitani, Hiroshi; Tooi, Osamu; Kawashima, Yukio; Roberts, Mike; Norrgren, Leif; Hutchinson, Thomas

    2006-01-01

    Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelope proteins and their mRNAs are produced in the liver of oviparous species by stimulation of endogenous estrogen and exogenous estrogenic chemicals. The VTG assay based on enzyme-linked immunosorbent assay (ELISA) has been widely used for many fish species to screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents using immature fish and/or male fish. In order to reduce the number of fish for screening of estrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can be used. In fact, primary cultured hepatocytes have been successfully used for the detection of estrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fish species, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream (Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish (Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these include frogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallus domesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantitative reverse transcription-polymerase chain reaction has also been successfully applied in the primary cultured hepatocytes of various species. PMID:16883298

  5. Nanotechnology in drug delivery: the need for more cell culture based studies in screening

    PubMed Central

    2014-01-01

    Advances in biomedical science are leading to upsurge synthesis of nanodelivery systems for drug delivery. The systems were characterized by controlled, targeted and sustained drug delivery ability. Humans are the target of these systems, hence, animals whose systems resembles humans were used to predict outcome. Thus, increasing costs in money and time, plus ethical concerns over animal usage. However, with consideration and planning in experimental conditions, in vitro pharmacological studies of the nanodelivery can mimic the in vivo system. This can function as a simple method to investigate the effect of such materials without endangering animals especially at screening phase. PMID:25057288

  6. Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

    PubMed Central

    Couturier, Cyril; Deprez, Benoit

    2012-01-01

    Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed “interactome.” Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET) technique was primarily developed to allow the dynamic monitoring of protein/protein interactions (PPI) in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of PPI and here is described why and how to set up and optimize a high throughput screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence of substrate concentration, number of cells and medium composition used on the Z? factor, and expected interferences from colored or fluorescent compounds. PMID:22973258

  7. Screening Helix-threading Peptides for RNA Binding Using a Thiazole Orange Displacement Assay

    PubMed Central

    Krishnamurthy, Malathy; Schirle, Nicole T.; Beal, Peter A.

    2008-01-01

    The fluorescent intercalator displacement assay using thiazole orange has been adapted to the study of RNA-binding helix-threading peptides (HTPs). This assay is highly sensitive with HTP-binding RNAs and provides binding affinity data in good agreement with quantitative ribonuclease footprinting without the need for radiolabeling or gel electrophoresis. The FID assay was used to define structure activity relationships for a small library of helix-threading peptides. Results of these studies indicate their RNA binding is dependent on peptide sequence, ?-amino acid stereochemistry, and cyclization (versus linear peptides), but independent of macrocyclic ring size for the penta-, tetra- and tri peptides analyzed. PMID:18789700

  8. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation

    PubMed Central

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A.; Liu, Jun O.; Parsons, Michael J.

    2011-01-01

    Pancreatic ?-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing ?-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of ?-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in ?-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis. PMID:22084084

  9. What is the cost utility of screening for hepatitis C virus (HCV) in intravenous drug users?

    Microsoft Academic Search

    Porsotam Leal; Ken Stein; William Rosenberg

    Objectives—To model the likely cost util- ity of the prevalence round of a screening programme for hepatitis C (HCV) in intravenous drug users (IVDUs) in con- tact with services in the South and West health region of the UK. Methods—Information on the prevalence of HCV, performance of diagnostic tests, and eVectiveness of interferon Æ (IFNÆ) for treatment of chronic hepatitis

  10. Computational chemistry, data mining, high-throughput synthesis and screening - informatics and integration in drug discovery

    PubMed Central

    Manly, Charles J.

    2001-01-01

    Drug discovery today includes considerable focus of laboratory automation and other resources on both combinatorial chemistry and high-throughput screening, and computational chemistry has been a part of pharmaceutical research for many years. The real benefit of these technologies is beyond the exploitation of each individually. Only recently have significant efforts focused on effectively integrating these and other discovery disciplines to realize their larger potential. This technical note will describe one example of these integration efforts. PMID:18924712

  11. Lysosomal storage disorder 4+1 multiplex assay for newborn screening using tandem mass spectrometry: Application to a small-scale population study for five

    E-print Network

    Gelb, Michael

    Lysosomal storage disorder 4+1 multiplex assay for newborn screening using tandem mass spectrometry,e , Michele Caggana a a New York State Dept. of Health, Wadsworth Center, Albany, NY 12201-0509, United States Keywords: Newborn screening Lysosomal storage disorder Multiplex Tandem mass spectrometry Background: We

  12. AN APPROACH FOR SCREENING CHOLINESTERASE INHIBITORS IN DRINKING WATER USING AN IMMOBILIZED ENZYME ASSAY

    EPA Science Inventory

    A simple, inexpensive and sensitive method for detecting organophosphate and carbamate insecticides is reported. Acetylcholinesterase was immobilized to PorexR Lateral-FloTM membrane material and remained active for several months at room temperature. The assay was sensitive ...

  13. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...

  14. PROTEOMICS OF MINUTE AMOUNTS OF TISSUE SAMPLE IN AQUATIC SPECIES: APPLICATION TO DEVELOPMENT OF NON-MAMMALIAN SCREENING ASSAYS FOR ENDOCRINE DISRUPTERS

    EPA Science Inventory

    When combined with other biochemical/toxicology assays, this type of information will contribute to the classification of chemicals based upon their effects at different levels of biological organization, and to the development of a cost-effective, non-mammalian screening assay f...

  15. VARIATION IN PROSTATE SPECIFIC ANTIGEN RESULTS FROM 2 DIFFERENT ASSAY PLATFORMS: CLINICAL IMPACT ON 2,304 PATIENTS UNDERGOING PROSTATE CANCER SCREENING

    Microsoft Academic Search

    RICHARD E. LINK; SHAHROKH F. SHARIAT; CUONG V. NGUYEN; ANITA FARR; ARMIN D. WEINBERG; RONALD A. MORTON; BARBARA RICHARDSON; DAVID BERNARD; KEVIN M. SLAWIN

    2004-01-01

    Purpose:Urologists must contend with fluctuating prostate specific antigen (PSA) results from different assay platforms when deciding whether prostate biopsy is indicated. The contribution of cross-platform variation to these fluctuations is not well understood. To address this question we performed a cross-sectional study comparing 2 popular PSA assays during prostate cancer screening.

  16. Development of a semi-automated colorimetric assay for screening anti-leishmanial agents

    Microsoft Academic Search

    Sudipto Ganguly; Samiran Bandyopadhyay; Arup Sarkar; Mitali Chatterjee

    2006-01-01

    MTS or {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium, inner salt) is converted into soluble formazan by mitochondrial dehydrogenase of viable cells, thus serving as an indicator of cell viability. Accordingly, a MTS-based assay was developed to evaluate anti-leishmanial activity in Leishmania promastigotes from strains responsible for visceral, cutaneous or mucocutaneous leishmaniasis. The assay was initially optimized for the appropriate wavelength (490 nm), culture medium (M-199),

  17. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    SciTech Connect

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  18. New-generation screening assays for the detection of anti-influenza compounds targeting viral and host functions

    PubMed Central

    Beyleveld, Grant; White, Kris M.; Ayllon, Juan; Shaw, Megan L.

    2013-01-01

    Current options for influenza antiviral therapy are limited to the neuraminidase inhibitors, and knowledge that high levels of oseltamivir resistance have been seen amongst previously circulating H1N1 viruses increases the urgency to find new influenza therapeutics. To feed this pipeline, assays that are appropriate for use in high-throughput screens are being developed and are discussed in this review. Particular emphasis is placed on cell-based assays that capture both inhibitors of viral functions as well as the host functions that facilitate optimal influenza virus replication. Success in this area has been fueled by a greater understanding of the genome structure of influenza viruses and the ability to generate replication-competent recombinant viruses that carry a reporter gene, allowing for easy monitoring of viral infection in a high-throughput setting. This article forms part of a symposium in Antiviral Research on “Treatment of influenza: targeting the virus or the host.” PMID:23933115

  19. A comparative study of ultrasonication, Fenton's oxidation and ferro-sonication treatment for degradation of carbamazepine from wastewater and toxicity test by Yeast Estrogen Screen (YES) assay.

    PubMed

    Mohapatra, D P; Brar, S K; Tyagi, R D; Picard, P; Surampalli, R Y

    2013-03-01

    A comparative study of ultrasonication (US), Fenton's oxidation (FO) and ferro-sonication (FS) (combination of ultrasonication and Fenton's oxidation) advanced oxidation processes (AOPs) for degradation of carbamazepine (CBZ) from wastewater (WW) is reported for the first time. CBZ is a worldwide used antiepileptic drug, found as a persistent emerging contaminant in many wastewater treatment plants (WWTPs) effluents and other aquatic environments. The oxidation treatments of WW caused an effective removal of the drug. Among the various US, FO and FS pre-treatments carried out, higher soluble chemical oxygen demand (SCOD) and soluble organic carbon (SOC) increment (63 to 86% and 21 to 34%, respectively) was observed during FO pre-treatment process, resulting in higher removal of CBZ (84 to 100%) from WW. Furthermore, analysis of by-products formed during US, FO and FS pre-treatment in WW was carried out by using laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) coupled to tandem mass spectrometry (MS/MS). LDTD-APCI-MS/MS analysis indicated formation of two by-products, such as epoxycarbamazepine and hydroxycarbamazepine due to the reaction of hydroxyl radicals (OH) with CBZ during the three types of pre-treatment processes. In addition, the estrogenic activity of US, FO and FS pre-treated sample with CBZ and its by-products was carried out by Yeast Estrogen Screen (YES) assay method. Based upon the YES test results, none of the pre-treated samples showed estrogenic activity. PMID:23410855

  20. Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

    PubMed

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L; Siminovitch, Katherine A; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K; Worthington, Jane; Gupta, Namrata; Clemons, Paul A; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M

    2013-05-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P?=?1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ?33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P?=?10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-?B transcription factor. Finally, we develop a high-throughput NF-?B luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-?B signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA. PMID:23696745

  1. Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

    PubMed Central

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J.; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L.; Siminovitch, Katherine A.; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K.; Worthington, Jane; Gupta, Namrata; Clemons, Paul A.; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M.

    2013-01-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P?=?1.4×10?9). Second, we demonstrate that subjects homozygous for the RA risk allele have ?33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P?=?10?9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-?B transcription factor. Finally, we develop a high-throughput NF-?B luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-?B signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA. PMID:23696745

  2. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

    PubMed Central

    Pantel, Jacques; Williams, Savannah Y.; Mi, Dehui; Sebag, Julien; Corbin, Jackie D.; Weaver, C. David; Cone, Roger D.

    2011-01-01

    The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: ?2AR agonists –the ?2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  3. Optimized methods for in vitro and in vivo anti-inflammatory assays and its applications in herbal and synthetic drug analysis.

    PubMed

    Nile, Shivraj Hariram; Park, Se Won

    2013-01-01

    Inflammatory diseases including, different types of rheumatic diseases are the major problems associated with the presently available non-steroidal anti-inflammatory agents. The numbers of plant derived drugs have been screened for their anti-inflammatory and anti-arthritic activity. Drug development in the recent times often relies on use of natural and synthetic drugs, which are promising candidates as therapeutic agents for prevention of diseases and disorders. These drugs possess different chemical structures, with wide range of therapeutic activities. The mechanism of Inflammation mainly involve in development of serious diseases, such as cancer, rheumatoid arthritis, sprains, bronchitis, muscle pains, chronic inflammatory bowel disease, persistent asthma, and liver fibrosis. Development of inflammatory events basically related to various chemicals, such as glucocorticoids (GCs) and mometasone furoate (MF); endogenous factors such as tumor necrosis factor alpha (TNF-?); enzymes and proteins such as copper and zinc-superoxide dismutase (SOD), proinflammatory peptide substance (PPS), RGD peptides, interleukin-4 (IL-4), IL-10, interferon-? (IFN-?), COX, LOX, cytokines such as interleukin-1 (IL-1); reactive oxygen species (ROS), nitric oxide (NO) and prostaglandin E2; as well as pro-inflammatory cells such as T and NK cells are well known to have an important role. Based on these correlations, numerous assays were used for inflammatory mechanism research, which was described in this paper. PMID:22876959

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

    A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption.

  5. OCTN cation transporters in health and disease: role as drug targets and assay development.

    PubMed

    Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Indiveri, Cesare

    2013-09-01

    The three members of the organic cation transporter novel subfamily are known to be involved in interactions with xenobiotic compounds. These proteins are characterized by 12 transmembrane segments connected by nine short loops and two large hydrophilic loops. It has been recently pointed out that acetylcholine is a physiological substrate of OCTN1. Its transport could be involved in nonneuronal cholinergic functions. OCTN2 maintains the carnitine homeostasis, resulting from intestinal absorption, distribution to tissues, and renal excretion/reabsorption. OCTN3, identified only in mouse, mediates also carnitine transport. OCTN1 and OCTN2 are associated with several pathologies, such as inflammatory bowel disease, primary carnitine deficiency, diabetes, neurological disorders, and cancer, thus representing useful pharmacological targets. The function and interaction with drugs of OCTNs have been studied in intact cell systems and in proteoliposomes. The latter experimental model enables reduced interference from other transporters or enzyme pathways. Using proteoliposomes, the molecular bases of toxicity of some drugs have recently been revealed. Therefore, proteoliposomes represent a promising experimental tool suitable for large-scale molecular screening of interactions of OCTNs with chemicals regarding human health. PMID:23771822

  6. Sediment toxicity screening with cost-effective microbiotests and conventional assays: A comparative study

    SciTech Connect

    Vanciheluwe, M.L.; Janssen, C.R.; Persoone, G. [Univ. of Ghent (Belgium). Lab. for Biological Research in Aquatic Pollution

    1995-12-31

    A large monitoring study of freshwater sediments, using the TRIAD approach, was conducted in Flanders (Belgium). This paper reports on the results of the toxicity assessment of 80 sediment samples evaluated with a battery of microbiotests and conventional assays. Sediment pore waters, extracted by squeezing, were tested with the Microtox{reg_sign} (Vibrio fischerii) and Thamnotoxkit{trademark} F (Thamnocephalus platyurus) microbiotests and the conventional (acute) assays with algae (Selenastrum capricornutum) and daphnids (Daphnia magna). A newly developed 5 day ELS test with the catfish Clarias gariepinus was also applied to the pore waters. Solid-phase testing was performed with the Microtox Sp{reg_sign} assay and the 10 day tests with Chironomus riparius and Hyalella azteca. Uni- and multivariate statistical techniques were applied to the data matrix to select a minimal test battery from the water phase and solid phase assays and from all tests combined. The influence of sediment associated confounding factors on the validity of the test results obtained with the various assays will be discussed. Finally a comparison of the predictive power of the selected battery of signal tests and that of the complete battery will be made and the potential use of the minimal battery for the initial hazard assessment of contaminated sediments will be reviewed.

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

    NASA Astrophysics Data System (ADS)

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

    2002-06-01

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

  8. Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.

    PubMed

    Bichara, Darío; Calcaterra, Nora B; Arranz, Silvia; Armas, Pablo; Simonetta, Sergio H

    2014-02-01

    Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large-scale assays, as they demand either a time-consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC??), showing a strong linear correlation (R² ?=?0.91) with the LC?? values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4 ?>?2,4-dinitrophenol?>?3,4-dichloroaniline?>?SDS?>?sodium benzoate?>?EDTA?>?K?CrO4 ; regarding solvents, EtOH???DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale-up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole-organism high-throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds. PMID:23401233

  9. [Assay of screening and prevention issues for high risk populations with prodromes of severe mental illness].

    PubMed

    Ma, Wei-Fen; Yang, Tzu-Ching; Chen, Yu-Hsia

    2013-06-01

    Before the appearance of specific psychotic symptoms, most individuals experience a period of prodromal symptoms associated with severe mental illness. Early intervention during this pre-psychotic phase may improve treatment outcomes, alter the natural course of disease, and prevent or delay mental disease onset. This paper aimed to synthesize four screening strategies (genetic high-risk populations selected by family history with mental illness, finding ultra-high-risk population, the close-in strategy, and the pre-psychotic phase by assessing basic symptoms); two intervention dimensions (psychosocial interventions and antipsychotic medicine); discussions of ethnic issues; and three Taiwanese nurses' roles (the role of assessment for screening, the role of development for screening tools, and the role of preventive intervention providers) to attain early diagnoses and prevention of mental illness. This article provides more information to advanced Taiwanese mental health nurses responsible to promote /s enhance the health of patients with prodromes of severe mental illness. PMID:23729347

  10. Assessment of the TLC/Salmonella assay for screening hazardous wastes

    SciTech Connect

    Houk, V.S.; Claxton, L.D.

    1987-09-01

    Using a modified version of the TLC/Salmonella assay developed by Bjorseth et al. (1982), 10 complex hazardous wastes were tested for mutagenic activity. The method couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude hazardous wastes and selected hazardous-waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed by applying a single overlay of minimal growth agar containing a tester strain of Salmonella and the optional metabolic activation system directly onto the developed chromatogram. Seven of 10 hazardous wastes demonstrated mutagenic activity when tested by the method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Eleven of the 14 mutagens were positive in the test system.

  11. Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

    PubMed

    Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

    2013-06-15

    The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. PMID:23497869

  12. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    PubMed

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function. PMID:15090141

  13. Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells

    PubMed Central

    Ketola, Kirsi; Kallioniemi, Olli; Iljin, Kristiina

    2012-01-01

    Background Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. Methods and Findings In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. Conclusions Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell growth. Disulfiram-sunitinib combination was identified as one of the potent synergistic approaches. Since sunitinib alone has been reported to lack efficacy in prostate cancer clinical trials, our results provide a rationale for novel combinatorial approach to target prostate cancer more efficiently. PMID:23251544

  14. Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening**

    EPA Science Inventory

    The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. As such, we are exploring a behavioral testing paradigm, which can assess the effect of sublethal and subteratogenic concentrations of de...

  15. Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

    EPA Science Inventory

    Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...

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

    EPA Science Inventory

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

  17. Using pathway modules as targets for assay development in xenobiotic screening

    EPA Science Inventory

    Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

  18. Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening

    EPA Science Inventory

    The U.S. Environmental Protection Agency is developing and evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. Towards this goal, we are exploring methods to detect developmental neurotoxicants in very young larval zebrafish. We have...

  19. Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

    PubMed Central

    Hu, Zongyi; Lan, Keng-Hsin; He, Shanshan; Swaroop, Manju; Hu, Xin; Southall, Noel; Zheng, Wei

    2014-01-01

    Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 ?M were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 ?M from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries. PMID:24277038

  20. SCREENING FOR TOXIC INDUSTRIAL CHEMICALS USING SEMIPERMEABLE MEMBRANE DEVICES WITH RAPID TOXICITY ASSAYS

    EPA Science Inventory

    A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...

  1. In house validation of recombinant yeast estrogen and androgen receptor agonist and antagonist screening assays

    Microsoft Academic Search

    S. N. Kolle; H. G. Kamp; H.-A. Huener; J. Knickel; A. Verlohner; C. Woitkowiak; R. Landsiedel; B. van Ravenzwaay

    2010-01-01

    Besides other modes of action, endocrine disruptors may interact with hormone receptors thereby modifying the physiological function of endogenous hormones. In the present study, we report the results obtained with yeast based assays to detect the (anti-)estrogenic potential (YES) and the (anti-)androgenic potential (YAS) of 105 substances. The results show very high reproducibility and good concordance with literature data of

  2. Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening

    PubMed Central

    Huang, Ruili; Southall, Noel; Cho, Ming-Hsuang; Xia, Menghang; Inglese, James; Austin, Christopher P.

    2009-01-01

    Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms. PMID:18281954

  3. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. ?Toxicity Testin...

  4. VAPOR SAMPLING DEVICE FOR INTERFACE WITH MICROTOX ASSAY FOR SCREENING TOXIC INDUSTRIAL CHEMICALS

    EPA Science Inventory

    A time-integrated sampling system interfaced with a toxicity-based assay is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethyl sulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...

  5. Poly(thymine)-templated fluorescent copper nanoparticles for ultrasensitive label-free nuclease assay and its inhibitors screening.

    PubMed

    Qing, Zhihe; He, Xiaoxiao; Qing, Taiping; Wang, Kemin; Shi, Hui; He, Dinggeng; Zou, Zhen; Yan, Lvan; Xu, Fengzhou; Ye, Xiaosheng; Mao, Zhengui

    2013-12-17

    Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10(-7) units ?L(-1) S1 nuclease, which was about 1-4 orders of magnitude more sensitive than the developed approaches. PMID:24236868

  6. Screening diabetic cats for hypersomatotropism: performance of an enzyme-linked immunosorbent assay for insulin-like growth factor 1.

    PubMed

    Rosca, Madalina; Forcada, Yaiza; Solcan, Gheorghe; Church, David B; Niessen, Stijn J M

    2014-02-01

    Screening diabetic cats for feline hypersomatotropism (HS) is currently dependent on using a radioimmunoassay (RIA) for measurement of growth hormone or insulin-like growth factor 1 (IGF-1), both of which require radioactivity, are costly and have limited availability. Performance of an enzyme-linked immunosorbent assay (ELISA) using anti-human IGF-1 antibodies was assessed. Total IGF-1 was determined in diabetic cat samples across a wide range of IGF-concentrations using a previously validated RIA (serum: 92 cats; plasma: 31 cats). Repeat IGF-1 measurement was then performed using the ELISA-system. Mean IGF-1 recovery after serial dilution proved satisfactory with a correlation coefficient of 0.96 (serum) and 0.97 (plasma). Appropriate precision was established [intra-assay coefficient of variation (CV) 9.5 ± 2% (serum) and 13.6 ± 7% (plasma); inter-assay CV 11.4 ± 4% (serum) and 7.6 ± 6% (plasma)] and significant effect of hyperlipidaemia, haemoglobinaemia, bilirubinaemia and storage was excluded, with the exception of an increase in serum IGF-1 when left at room temperature for more than 24 h. ELISA concentrations correlated significantly with RIA concentrations (serum Pearson r(2): 0.75; plasma: 0.83, P <0.001). Receiver operating characteristics analysis showed an area under the curve of 0.99 (serum) and 0.96 (plasma), and indicated high diagnostic accuracy for categorising a diabetic cat correctly as suspicious for HS at a serum IGF-1 cut-off of 997 ng/ml (sensitivity, 100%; specificity, 88.1%). The current study is the first to validate an easy-to-use and economical IGF-1 ELISA for the screening for HS among diabetic cats, which is important given the suspected significant prevalence of HS-induced diabetes mellitus. PMID:23828811

  7. Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.

    PubMed

    Mejia, Monica; Heghinian, Mari D; Busch, Alexandra; Marí, Frank; Godenschwege, Tanja A

    2012-01-01

    Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D?7 nicotinic acetylcholine receptors (nAChRs). Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles. Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster" from Augustin et al, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique. PMID:22525737

  8. Real-time cytotoxicity assays.

    PubMed

    Wlodkowic, Donald; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M

    2011-01-01

    Validation of new therapeutic targets calls for the advance in innovative assays that probe both spatial and temporal relationships in signaling networks. Cell death assays have already found a widespread use in pharmacological profiling of anticancer drugs. Such assays are, however, predominantly restricted to end point DEAD/LIVE parameter that provides only a snapshot of inherently stochastic process such as tumor cell death. Development of new methods that can offer kinetic real-time analysis would be highly advantageous for the pharmacological screening and predictive toxicology. In the present work we outline innovative protocols for the real-time analysis of tumor cell death, based on propidium iodide (PI) and SYTOX Green probes. These can be readily adapted to both flow cytometry and time-lapse fluorescence imaging. Considering vast time savings and kinetic data acquisition such assays have the potential to be applied in a number of areas including accelerated anticancer drug discovery and high-throughput screening routines. PMID:21516415

  9. A high-throughput screening campaign for detection of ca(2+)-activated k(+) channel activators and inhibitors using a fluorometric imaging plate reader-based tl(+)-influx assay.

    PubMed

    Jørgensen, Susanne; Dyhring, Tino; Brown, David T; Strøbæk, Dorte; Christophersen, Palle; Demnitz, Joachim

    2013-04-01

    The intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) has been proposed to play many physiological roles, and modulators of KCa3.1 activity are potentially interesting as new drugs. In order to identify new chemical scaffolds, high-throughput screening (HTS) assays are needed. In the current study, we present an HTS assay that has been optimized for the detection of inhibitors as well as activators of KCa3.1 in a combined assay. We used HEK293 cells heterologously expressing KCa3.1 in a fluorescence-based Tl(+) influx assay, where the permeability of potassium channels to Tl(+) is taken advantage of. We found the combined activator-and-inhibitor assay to be robust and insensitive to dimethyl sulfoxide (up to 1%), and conducted an HTS campaign of 217,119 small molecules. In total, 224 confirmed activators and 312 confirmed inhibitors were found, which corresponded to a hit rate of 0.10% and 0.14%, respectively. The confirmed hits were further characterized in a fluorometric imaging plate reader-based concentration-response assay, and selected compounds were subjected to secondary testing in an assay for endogenous KCa3.1 activity using human erythrocytes (red blood cell assay). Although the estimated potencies were slightly higher in the RBC assay, there was an overall good correlation across all clusters. The campaign led to the identification of several chemical series of KCa3.1 activators and inhibitors, comprising already known pharmacophores and new chemical series. One of these were the benzothiazinones that constitute a new class of highly potent KCa3.1 inhibitors, exemplified by 4-{[3-(trifluoromethyl)phenyl]methyl}-2H-1,4-benzothiazin-3(4H)-one (NS6180). PMID:23198866

  10. Overcoming the toxicity effects of municipal wastewater sludge and biosolid extracts in the Yeast Estrogen Screen (YES) assay.

    PubMed

    Citulski, Joel; Farahbakhsh, Khosrow

    2012-04-01

    For nearly two decades, the Yeast Estrogen Screen (YES) has been used as a valuable tool for determining the total estrogenic potency of various environmental samples, including influent and effluent streams at municipal wastewater plants. However, applying the YES assay to wastewater sludges and stabilized biosolids has been problematic. This is due to co-extracted compounds from the solids either proving toxic to the yeast or masking the presence of estrogenic substances. The present research describes the development and validation of sample preparation steps that mitigate the toxicity effects of municipal wastewater sludge and biosolid samples in the YES assay, while allowing for reliable dose-dependent expression of estrogenic activity. A copper work-up for sulfur removal and chromatographic cleanup with silica and alumina were required in addition to solid-phase extraction to adequately remove interfering compounds. Sample stabilization methods such as autoclaving, lyophilization and formaldehyde treatment were found to be detrimental to the assay. Hence, heat-drying is recommended to prevent cytotoxicity and the degradation of estrogenic substances. PMID:22277884

  11. Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.

    PubMed

    Jiang, Wenxiao; Zhang, Huiyan; Li, Xiangmei; Liu, Xinxin; Zhang, Suxia; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2013-11-20

    To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 ?g/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 ?g/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process. PMID:24147865

  12. Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay.

    PubMed

    Sharma, Pooja; Jindal, Swati; Bala, Kiran; Kumari, Kirti; Niharika, Neha; Kaur, Jasvinder; Pandey, Gunjan; Pandey, Rinku; Russell, Robyn J; Oakeshott, John G; Lal, Rup

    2014-04-01

    Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified. PMID:23740574

  13. In Silico Assay Development for Screening of Tetracyclic Triterpenoids as Anticancer Agents against Human Breast Cancer Cell Line MCF7

    PubMed Central

    Prakash, Om; Ahmad, Ateeque; Tripathi, Vinay Kumar; Tandon, Sudeep; Pant, Aditya Bhusan; Khan, Feroz

    2014-01-01

    Experimental activity of a compound on cancer cell line/target is mostly analyzed in the form of percentage inhibition at different concentration gradient and time of incubation. In this study a statistical model has been developed referred as in silico assay using support vector regression model, which can act with change in concentration gradient and time of incubation. This model is a function of concentration gradient, treatment hour and independent components; which calculate the percentage inhibition in combination of above three components. This model is designed to screen tetracyclic triterpenoids active against human breast cancer cell line MCF7. The model has been statistically validated, checked for applicability domain and predicted results were reconfirmed by MTT assay, for example Oenotheranstrol derivatives, OenA & B. Computational SAR, target and docking studies were performed to understand the cytotoxic mechanism of action of Oenotheranstrol compounds. The proposed in silico assay model will work for specific chemical family for which it will be optimized. This model can be used to analyze growth kinetics pattern on different human cancer cell lines for designed compounds. PMID:25365399

  14. Retinal and Ocular Toxicity in Ocular Application of Drugs and Chemicals – Part I: Animal Models and Toxicity Assays

    Microsoft Academic Search

    Fernando Marcondes Penha; Eduardo B. Rodrigues; Maurício Maia; Eduardo Dib; Elaine Fiod Costa; Bruno A. Furlani; Milton Nunes Moraes Filho; Juliana L. Dreyfuss; Juliana Bottós; Michel E. Farah

    2010-01-01

    Aims: Experimental retinal research has gained great importance due to the ophthalmic pharmacotherapy era. An increasing number of drugs are constantly released into the market for the treatment of retinal diseases. In this review, animal species, animal models and toxicity assays in retinal research are discussed. Methods: An extensive search of the literature was performed to review various aspects of

  15. Serial Pediatric Symptom Checklist Screening in Children with Prenatal Drug Exposure

    PubMed Central

    Whitaker, Toni M.; Bada, Henrietta S.; Bann, Carla M.; Shankaran, Seetha; LaGasse, Linda; Lester, Barry M.; Bauer, Charles R.; Hammond, Jane; Higgins, Rosemary

    2011-01-01

    Objective To examine screening results obtained by serial annual behavioral assessment of children with prenatal drug exposure. Method The Maternal Lifestyle Study enrolled children with prenatal cocaine exposure (PCE) at birth for longitudinal assessments of developmental, behavioral, and health outcomes. At 8, 9, 10, 11, and 12 years of age, caregivers rated participants on the Pediatric Symptom Checklist (PSC). Serial PSC results were compared to an established broad-based behavioral measure at 9, 11, and 13 years. PSC results were analyzed for 1,081 children who had at least 2 annual screens during the 5-year time span. Most subjects (87%) had 4 or more annual screens rated by the same caregiver (80%). PSC scores (and Positive screens) over time were compared at different time points for those with and without PCE. Covariates, including demographic factors and exposures to certain other substances, were controlled. Results Children with PCE had significantly higher scores overall, with more Positive screens for behavior problems than children without PCE. Children with PCE had more externalizing behavior problems. Children exposed to tobacco pre- and post-natally also showed higher PSC scores. Over time, PSC scores differed slightly from the 8-year scores, without clear directional trend. Earlier PSC results predicted later behavioral outcomes. Conclusion Findings of increased total PSC scores and Positive PSC screens for behavioral concerns in this group of children with prenatal substance exposure support the growing body of evidence that additional attention to identification of mental health problems may be warranted in this high-risk group. PMID:21200328

  16. Identification and elimination of target-related matrix interference in a neutralizing anti-drug antibody assay.

    PubMed

    Schwickart, Martin; Mehrzai, Freshta; Pearson, Jennifer; Shaghasi, Nabila; Chavez, Carlos; Schneider, Amy; Wu, Spencer; Roskos, Lorin; Liang, Meina

    2014-01-31

    Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2? antagonistic antibody against PDGFR-?. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-?. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-? present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay. PMID:24287421

  17. Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet Syndrome treatment

    PubMed Central

    Baraban, Scott C.; Dinday, Matthew T.; Hortopan, Gabriela A.

    2013-01-01

    Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterise zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviors. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviors and electrographic seizures. This approach represents a new direction in modeling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder. PMID:24002024

  18. Screening and personalizing nootropic drugs and cognitive modulator regimens in silico.

    PubMed

    Jellen, Leslie C; Aliper, Alexander; Buzdin, Anton; Zhavoronkov, Alex

    2015-01-01

    The go-to cognitive enhancers of today are those that are widely available rather than optimal for the user, including drugs typically prescribed for treatment of ADHD (e.g., methylphenidate) and sleep disturbances such as narcolepsy (modafinil). While highly effective in their intended therapeutic role, performance gains in healthy populations are modest at best and profoundly inconsistent across subgroups and individuals. We propose a method for in silico screening of possible novel cognitive enhancers followed by high-throughput in vivo and in vitro validation. The proposed method uses gene expression data to evaluate the the collection of activated or suppressed signaling pathways in tissues or neurons of the cognitively enhanced brain. An algorithm maps expression data onto signaling pathways and quantifies their individual activation strength. The collective pathways and their activation form what we term the signaling pathway cloud, a biological fingerprint of cognitive enhancement (or any other condition of interest). Drugs can then be screened and ranked based on their ability to minimize, mimic, or exaggerate pathway activation or suppression within that cloud. Using this approach, one may predict the efficacy of many drugs that may enhance various aspects of cognition before costly preclinical studies and clinical trials are undertaken. PMID:25705179

  19. Screening and personalizing nootropic drugs and cognitive modulator regimens in silico

    PubMed Central

    Jellen, Leslie C.; Aliper, Alexander; Buzdin, Anton; Zhavoronkov, Alex

    2015-01-01

    The go-to cognitive enhancers of today are those that are widely available rather than optimal for the user, including drugs typically prescribed for treatment of ADHD (e.g., methylphenidate) and sleep disturbances such as narcolepsy (modafinil). While highly effective in their intended therapeutic role, performance gains in healthy populations are modest at best and profoundly inconsistent across subgroups and individuals. We propose a method for in silico screening of possible novel cognitive enhancers followed by high-throughput in vivo and in vitro validation. The proposed method uses gene expression data to evaluate the the collection of activated or suppressed signaling pathways in tissues or neurons of the cognitively enhanced brain. An algorithm maps expression data onto signaling pathways and quantifies their individual activation strength. The collective pathways and their activation form what we term the signaling pathway cloud, a biological fingerprint of cognitive enhancement (or any other condition of interest). Drugs can then be screened and ranked based on their ability to minimize, mimic, or exaggerate pathway activation or suppression within that cloud. Using this approach, one may predict the efficacy of many drugs that may enhance various aspects of cognition before costly preclinical studies and clinical trials are undertaken. PMID:25705179

  20. Flow screen-printed amperometric detection of p -nitrophenol in alkaline phosphatase-based assays

    Microsoft Academic Search

    Pablo Fanjul-Bolado; María Begoña González-García; Agustín Costa-García

    2006-01-01

    p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble\\u000a product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare\\u000a screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system

  1. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    Microsoft Academic Search

    Serge Leimanis; Sandrine Hamels; Florence Naze ´; Guillaume Mbongolo Mbella; Myriam Sneyers; Rupert Hochegger; Hermann Broll; Lillian Roth; Klára Dallmann; Adrienn Micsinai; Maria Pla; Claudia Brünen-Nieweler; Nina Papazova; Isabel Taverniers; Norbert Hess; Britta Kirschneit; Yves Bertheau; Colette Audeon; Valérie Laval; Ulrich Busch; Sven Pecoraro; Katrin Neumann; Sibylle Rösel; Jeroen van Dijk; Esther Kok; Gianni Bellocchi; Nicoletta Foti; Marco Mazzara; William Moens; José Remacle; Guy Van Den Eede

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray,\\u000a has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target\\u000a elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip® GMO, Eppendorf, Germany).

  2. An analytical evaluation of eight on-site oral fluid drug screening devices using laboratory confirmation results from oral fluid

    Microsoft Academic Search

    Tom Blencowe; Anna Pehrsson; Pirjo Lillsunde; Kari Vimpari; Sjoerd Houwing; Beitske Smink; René Mathijssen; Trudy Van der Linden; Sara-Ann Legrand; Kristof Pil; Alain Verstraete

    2011-01-01

    The performance of eight on-site oral fluid drug screening devices was studied in Belgium, Finland and the Netherlands as a part of the EU-project DRUID. The main objective of the study was to evaluate the reliability of the devices for testing drivers suspected of driving under the influence of drugs (DUID). The performance of the devices was assessed by their

  3. Active screening of multi-drug resistant bacteria effectively prevent and control the potential infections.

    PubMed

    Ren, Yuguo; Ma, Guoliang; Peng, Lin; Ren, Yufeng; Zhang, Fengmei

    2015-03-01

    Our objective is to determine if actively screen the multi-drug resistant bacteria (MDRB) infection in intensive care unit (ICU) to prevent, control, and decrease the infection rate and transmission of MDRB. The patients admitted in ICU of one hospital in 2013 were analyzed. The throat swab, blood, defecation, and urine of patients were actively collected for bacteria cultures to screen Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii in patients. All patients received screening of MDRB infection and colonization within 2 days and after 2 days of admission, the results showed that there were 418 infectious bacterial strains in total and P. aeruginosa was the main bacterium. The asymptomatic infection rates of P. aeruginosa, K. pneumonia, E. coli, S. aureus, and A. baumannii were 39.02, 24.74, 44.00, 29.17, and 33.33 %, respectively; the symptomatic infection rates were 60.98, 75.26, 56.00, 70.83, and 66.67 %. 59.70 % patients received antibiotics treatment, 27.45 % patients received trachea cannula, 32.95 % patients received mechanism ventilation, 2.27 % patients received arterial cannula or venous cannula and 4.00 % patients received indwelling urinary catheters. The main MDRB in ICU is P. aeruginosa. The active screening of MDRB infection and colonization can provide the opportunity to take the life-saving measure against MDRB and treat patients. This can decrease the infection risk and the nosocomial transmission of MDRB. PMID:25416582

  4. A nonradioactive 96-well plate assay for screening of trans-sialidase activity

    Microsoft Academic Search

    Silke Schrader; Evelin Tiralongo; Teruo Yoshino; Roland Schauer

    2003-01-01

    Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably ?2,3-linked sialic acid to another glycan or glycoconjugate, forming a new ?2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-?-d-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using

  5. Screening for toxic industrial chemicals using semipermeable membrane devices with rapid toxicity assays

    Microsoft Academic Search

    K. R. Rogers; S. L. Harper; G. Robertson

    2005-01-01

    A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor phase. Uptake kinetics experiments for one of these compounds (acrolein) indicated that it was significantly concentrated (i.e., 10% of the 24h maximum) in

  6. Application and challenges in using LC-MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery.

    PubMed

    Zheng, Joanna; Mehl, John; Zhu, Yongxin; Xin, Baomin; Olah, Timothy

    2014-03-01

    As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery. PMID:24702115

  7. Data-independent Proteomic Screen Identifies Novel Tamoxifen Agonist that Mediates Drug Resistance

    PubMed Central

    Hengel, Shawna Mae; Murray, Euan; Langdon, Simon; Hayward, Larry; O’Donoghue, Jean; Panchaud, Alexandre; Hupp, Ted; Goodlett, David R.

    2011-01-01

    A label-free quantitative variation of the recently developed data-independent shotgun proteomic method Precursor Acquisition Independent From Ion Count (PAcIFIC) was used to identify novel proteins implicated in cancer progression and resistance. Specifically, this screen identified the pro-metastatic protein anterior gradient 2 (AGR2) as significantly up-regulated in tamoxifen treated cells. Highlighting the need for direct proteome profiling methods like PAcIFIC, neither data-dependent gas-phase fractionation nor a transcriptomic screen detected AGR2 protein/transcript at significantly up-regulated levels. Further cell-based experiments using human cancer cell lines and in vivo xenografts confirmed the PAcIFIC hypothesis that AGR2 is up-regulated in MCF-7 cells post tamoxifen treatment, and that it is implicated in drug resistance mediation. PMID:21936522

  8. Monitoring ribosomal frameshifting as a platform to screen anti-riboswitch drug candidates.

    PubMed

    Yu, Chien-Hung; Olsthoorn, René C L

    2015-01-01

    Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target. PMID:25605396

  9. Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

    PubMed Central

    Ginouves, Marine; Carme, Bernard; Couppie, Pierre

    2014-01-01

    In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

  10. Performance evaluation of the DrugWipe ® 5\\/5 + on-site oral fluid screening device

    Microsoft Academic Search

    Anna Pehrsson; Tom Blencowe; Kari Vimpari; Antti Impinen; Teemu Gunnar; Pirjo Lillsunde

    This study presents a retrospective performance evaluation of an on-site oral fluid drug screening device DrugWipe® 5\\/5+ (Securetec). The results obtained by the device were compared with gas chromatography–mass spectrometry confirmation analysis\\u000a results in whole blood. Data used in the comparison were based on 1,807 real cases in which the Finnish police had conducted\\u000a an on-site drug test on persons

  11. INTERVAL ESTIMATION OF QUANTILE RATIOS APPLIED TO ANTI-CANCER DRUG SCREENING BY XENOGRAFT EXPERIMENTS

    PubMed Central

    Cheng, Cheng; Wu, Jianrong

    2012-01-01

    The current practice in analyzing data from anti-cancer drug screening by xenograft experiments lacks statistical consideration to account for experimental noise, and a sound inference procedure is necessary. A novel confidence bound and interval procedure for estimating quantile ratios developed in this paper fills the void. Justified by rigorous large-sample theory and a simulation study of small-sample performance, the proposed method performs well in a wide range of scenarios involving right-skewed distributions. By providing rigorous inference and much more interpretable statistics that account for experimental noise, the proposed method improves the current practice of analyzing drug activity data in xenograft experiments. The proposed method is fully nonparametric, simple to compute, performs equally well or better than known nonparametric methods, and is applicable to any statistical inference of a “fold change” that can be formulated as a quantile ratio. PMID:20799257

  12. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    PubMed Central

    Choi, Yeonim; Wang, Hye-Young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae

    2013-01-01

    Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections. PMID:23447637

  13. Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

    PubMed Central

    Lewis, Russell E.; Liao, Guangling; Young, Katherine; Douglas, Cameron

    2014-01-01

    Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-?B and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the ?-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-?B/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z. PMID:24395226

  14. Three Dimensional Microfluidic Cell Arrays for ex Vivo Drug Screening with Mimicked Vascular Flow

    PubMed Central

    2015-01-01

    Currently, there are no reliable ex vivo models that predict anticancer drug responses in human tumors accurately. A comprehensive method of mimicking a 3D microenvironment to study effects of anticancer drugs on specific cancer types is essential. Here, we report the development of a three-dimensional microfluidic cell array (3D ?FCA), which reconstructs a 3D tumor microenvironment with cancer cells and microvascular endothelial cells. To mimic the in vivo spatial relationship between microvessels and nonendothelial cells embedded in extracellular matrix, three polydimethylsiloxane (PDMS) layers were built into this array. The multilayer property of the device enabled the imitation of the drug delivery in a microtissue array with simulated blood circulation. This 3D ?FCA system may provide better predictions of drug responses and identification of a suitable treatment for a specific patient if biopsy samples are used. To the pharmaceutical industry, the scaling-up of our 3D ?FCA system may offer a novel high throughput screening tool. PMID:24568664

  15. Three dimensional microfluidic cell arrays for ex vivo drug screening with mimicked vascular flow.

    PubMed

    Dereli-Korkut, Zeynep; Akaydin, H Dogus; Ahmed, A H Rezwanuddin; Jiang, Xuejun; Wang, Sihong

    2014-03-18

    Currently, there are no reliable ex vivo models that predict anticancer drug responses in human tumors accurately. A comprehensive method of mimicking a 3D microenvironment to study effects of anticancer drugs on specific cancer types is essential. Here, we report the development of a three-dimensional microfluidic cell array (3D ?FCA), which reconstructs a 3D tumor microenvironment with cancer cells and microvascular endothelial cells. To mimic the in vivo spatial relationship between microvessels and nonendothelial cells embedded in extracellular matrix, three polydimethylsiloxane (PDMS) layers were built into this array. The multilayer property of the device enabled the imitation of the drug delivery in a microtissue array with simulated blood circulation. This 3D ?FCA system may provide better predictions of drug responses and identification of a suitable treatment for a specific patient if biopsy samples are used. To the pharmaceutical industry, the scaling-up of our 3D ?FCA system may offer a novel high throughput screening tool. PMID:24568664

  16. A nonradioactive 96-well plate assay for screening of trans-sialidase activity.

    PubMed

    Schrader, Silke; Tiralongo, Evelin; Paris, Gastón; Yoshino, Teruo; Schauer, Roland

    2003-11-15

    Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems. PMID:14596820

  17. A cell-based, high-throughput homogeneous time-resolved fluorescence assay for the screening of potential ?-opioid receptor agonists

    PubMed Central

    Wang, Yue; Yan, Ming; Zheng, Guang-yao; He, Ling; Yang, Huan

    2014-01-01

    Aim: The aim of this study was to identify ?-opioid receptor (KOR) agonists from a library of 80 000 small-molecule compounds and provide the experimental basis for the development of new analgesic candidates. Methods: The cell-based, high-throughput screen for human KOR agonists was based on the LANCE™ cAMP assay. Preliminary structure-activity relationship (SAR) analysis was applied according to the compounds' structures. An acetic acid twisting experiment was used to verify the pharmacodynamics. Results: In total, 31 compounds were identified as KOR agonists after preliminary and secondary screening. Of these compounds, five demonstrated significant KOR-stimulating activity that was comparable to U-50,488, a selective KOR agonist. The EC50 values for I-7, I-8, I-10, II-5, and II-8 were 13.34±1.65, 14.01±1.84, 9.57±0.19, 14.94±0.64, and 8.74±0.72 nmol/L, respectively. Based on SAR studies, the stimulating activity of compounds with 5-phenyl-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo [1, 5-a] pyrimidine (group I) and 3,4-dimethoxy-N-(2-oxoethyl)-N-p-tolylbenzenesulfonamide (group II) parent structures were higher than the compound with a 5-hydroxy-2-methylbenzofuran-3-carboxylic acid (group III) parent structure. Pharmacodynamic experiments indicated that 20–40 ?g/kg ip of compounds I-10 and II-8 significantly decreased the number of writhes induced by acetic acid; this finding is consistent with the SAR studies. Furthermore, the analgesic effects of compounds I-10 and II-8 were significantly antagonized in the presence of the selective KOR antagonist nor-BNI. Conclusion: These findings collectively indicate that compounds I-10 and II-8 exhibit significant analgesic activities, providing evidence, at least in part, for their clinical application as new analgesic drugs. PMID:24930486

  18. Evaluation of on-site oral fluid screening using Drugwipe-5(+), RapidSTAT and Drug Test 5000 for the detection of drugs of abuse in drivers.

    PubMed

    Wille, Sarah M R; Samyn, Nele; Ramírez-Fernández, Maria del Mar; De Boeck, Gert

    2010-05-20

    Driving under the influence of drugs is a major problem worldwide. At the moment, several countries have adopted a 'per se' legislation to address this problem. One of the key elements in the enforcement process is the possibility of rapid on-site screening tests to take immediate administrative measures. In this study, the reliability of three oral fluid screening devices (Mavand RapidSTAT, Securetec Drugwipe-5(+), and Dräger DrugTest 5000) was assessed by comparing their on-site results with confirmatory GC-MS plasma analysis. Our results demonstrate that for amphetamine screening, the oral fluid on-site devices on the market today are certainly sensitive enough. RapidSTAT, Drugwipe-5(+), and DrugTest 5000 demonstrated respectively a sensitivity of 93%, 100% and 92% for amphetamine/MDMA. For cocaine screening, sensitivities of 75%, 78% and 67% were obtained for the RapidSTAT, Drugwipe-5(+), and DrugTest 5000 devices, respectively. The studied devices were able to detect about 70% of all cannabis users in a roadside setting. However, a newer version of the DrugTest 5000 test cassette demonstrated a sensitivity of 93%, indicating an increased detection of Delta(9)-tetrahydrocannabinol using 'new generation' oral fluid screening tests with lowered cut-offs. Due to these promising results police officers and judicial experts are keen to use oral fluid screening devices. They believe that their ease of use and diminished amount of false positive results in comparison with urine screening will lead to more roadside tests and more appropriate juridical measures. PMID:19913376

  19. A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases.

    PubMed

    Kaiser, Annette; Khomutov, Alex R; Simonian, Alina; Agostinelli, Enzo

    2012-05-01

    Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/?l. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2'-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker. PMID:21360085

  20. High-Throughput Microsomal Stability Assay for Screening New Chemical Entities in Drug Discovery

    Microsoft Academic Search

    Massimiliano Fonsi; Maria V. Orsale; Edith Monteagudo

    2008-01-01

    In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC\\/MS\\/MS) analytical methods were also developed, and a rapid

  1. In vitro testing of chemotherapeutic drug combinations in acute myelocytic leukaemia using the fluorometric microculture cytotoxicity assay (FMCA).

    PubMed Central

    Larsson, R.; Fridborg, H.; Kristensen, J.; Sundström, C.; Nygren, P.

    1993-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) was employed for analysing the effect of different chemotherapeutic drug combinations and their single constituents in 44 cases of acute myelocytic leukaemia (AML). A large heterogeneity with respect to cell kill was observed for all combinations tested, the interactions ranging from antagonistic to synergistic in terms of the multiplicative concept for drug interactions. However, an 'additive' model provided a significantly better fit of the data compared to the effect of the most active single agent of the combination (Dmax) for several common antileukaemic drug combinations. When the two interaction models were related to treatment outcome 38% of the non-responders showed preference for the additive model whereas the corresponding figure for responders was 80%. Overall, in 248 of 290 (85%) tests performed with drug combinations, there was an agreement between the effect of the combination and that of the most active single component. Direct comparison of Dmax and the combination for correlation with clinical outcome demonstrated only minor differences in the ability to predict drug resistance. The results show that FMCA appear to report drug interactions in samples from patients with AML in accordance with clinical experience. Furthermore, testing single agents as a substitute for drug combinations may be adequate for detection of clinical drug resistance to combination therapy in AML. PMID:8494730

  2. Unbiased compound screening identifies unexpected drug sensitivities and novel treatment options for gastrointestinal stromal tumors.

    PubMed

    Boichuk, Sergei; Lee, Derek J; Mehalek, Keith R; Makielski, Kathleen R; Wozniak, Agnieszka; Seneviratne, Danushka S; Korzeniewski, Nina; Cuevas, Rolando; Parry, Joshua A; Brown, Matthew F; Zewe, James; Taguchi, Takahiro; Kuan, Shin-Fan; Schöffski, Patrick; Debiec-Rychter, Maria; Duensing, Anette

    2014-02-15

    Most gastrointestinal stromal tumors (GIST) are caused by oncogenic KIT or platelet-derived growth factor receptor activation, and the small molecule kinase inhibitor imatinib mesylate is an effective first-line therapy for metastatic or unresectable GIST. However, complete remissions are rare and most patients ultimately develop resistance, mostly because of secondary mutations in the driver oncogenic kinase. Hence, there is a need for novel treatment options to delay failure of primary treatment and restore tumor control in patients who progress under therapy with targeted agents. Historic data suggest that GISTs do not respond to classical chemotherapy, but systematic unbiased screening has not been performed. In screening a compound library enriched for U.S. Food and Drug Administration (FDA)-approved chemotherapeutic agents (NCI Approved Oncology Drugs Set II), we discovered that GIST cells display high sensitivity to transcriptional inhibitors and topoisomerase II inhibitors. Mechanistically, these compounds exploited the cells' dependency on continuous KIT expression and/or intrinsic DNA damage response defects, explaining their activity in GIST. Mithramycin A, an indirect inhibitor of the SP1 transcription factor, and mitoxantrone, a topoisomerase II inhibitor, exerted significant antitumor effects in mouse xenograft models of human GIST. Moreover, these compounds were active in patient-derived imatinib-resistant primary GIST cells, achieving efficacy at clinically relevant concentrations. Taken together, our findings reveal that GIST cells have an unexpectedly high and specific sensitivity to certain types of FDA-approved chemotherapeutic agents, with immediate implications for encouraging their clinical exploration. PMID:24385214

  3. [ALS disease modeling and drug screening using patient-specific iPS cells].

    PubMed

    Egawa, Naohiro; Inoue, Haruhisa

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which motor neuron (MN) loss in the spinal cord leads to progressive paralysis and death. Cytosolic aggregations in ALS MNs are composed of Tar DNA-binding protein-43 (TDP-43). Genetic analysis has identified more than twenty mutations of TDP-43 in ALS cases. Although accumulating evidence provides several hypotheses of disease mechanism, it is still needed to discover effective cure for ALS. We aimed to reveal cellular phenotypes in ALS MNs for identifying a drug-screening target for ALS using patient-specific induced pluripotent stem cells (iPSCs). To generate patient-specific iPSCs, dermal fibroblasts were obtained by biopsy from ALS patients carrying mutant TDP-43. The fibroblasts were reprogrammed by retrovirus or episomal vectors. Disease-specific iPSCs were differentiated into MNs expressing HB9 and SMI-32. Despite short culture period, ALS MNs recapitulated several disease phenotypes including detergent-insoluble TDP-43, shortened neurites and cellular vulnerability that observed in patient and animal models. Anacardic acid treatment reverted those phenotypes. Disease-specific iPSCs might provide a first step for drug-screening platform for ALS using patient-specific iPSCs. PMID:24291866

  4. Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

    PubMed

    Schneider, Adele; Nakagawa, Sachiko; Keep, Rosanne; Dorsainville, Darnelle; Charrow, Joel; Aleck, Kirk; Hoffman, Jodi; Minkoff, Sherman; Finegold, David; Sun, Wei; Spencer, Andrew; Lebow, Johannah; Zhan, Jie; Apfelroth, Stephen; Schreiber-Agus, Nicole; Gross, Susan

    2009-11-01

    Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice. PMID:19876898

  5. Screening of genetically modified organisms and specific detection of Bt176 maize in flours and starches by PCR-enzyme linked immunosorbent assay

    Microsoft Academic Search

    Laetitia Petit; Fabienne Baraige; Anne-Marie Balois; Yves Bertheau; Patrick Fach

    2003-01-01

    Polymerase chain reaction (PCR)-enzyme linked immunosorbent assays (ELISAs) targeting either the 35S promoter or the Bt176 specific junction sequence were developed to screen for the presence of genetically modified organisms (GMOs) and specifically detect Bt176 maize in flours and starches. Two additional PCR-ELISA assays were developed to validate the results: one, based on the detection of the maize alcohol dehydrogenase

  6. An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of ?-substituted alcohols.

    PubMed

    Wan, Nan-Wei; Liu, Zhi-Qiang; Xue, Feng; Huang, Kai; Tang, Ling-Jiao; Zheng, Yu-Guo

    2015-05-01

    Halohydrin dehalogenases (HHDHs) are an important class of enzymes for preparing optically active haloalcohols, epoxides, and ?-substituted alcohols. However, natural HHDHs rarely meet the requirements of industrial applications. Here, a novel high-throughput screening (HTS) methodology for directed evolution of HHDH was developed based on the colorimetric determination of azide. In this method, azide was involved in the HHDH-catalyzed ring-opening process and the decrease of azide was used to quantitatively evaluate HHDH activity. The HTS methodology was simple and sensitive (?460?=?1.2173?×?10(4) L mol(-1) cm(-1)) and could be performed in a microplate format using whole cells. To verify the efficiency of the HTS methodology, it was adopted to engineer a HHDH (HHDH-PL) from Parvibaculum lavamentivorans DS-1, which was applied in the process for ethyl (R)-4-cyano-3-hydroxybutanoate (HN) by the conversion of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)). A random mutant library containing 2500 colonies was screened using the HTS methodology, and three beneficial mutants F176M, A187R, and A187S were obtained. By combining the beneficial mutated residues, the variant F176M/A187R was identified with 2.8-fold higher catalytic efficiency for preparation of HN. The high-throughput colorimetric assay would be very useful for directed evolution of HHDH for preparing ?-substituted alcohols. PMID:25805343

  7. Reporter Protein Complementation Imaging Assay to Screen and Study Nrf2 Activators in Cells and Living Animals

    PubMed Central

    Ramkumar, Kunka Mohanram; Sekar, Thillai Veerapazham; Foygel, Kira; Elango, Bhakkiyalakshmi; Paulmurugan, Ramasamy

    2013-01-01

    NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferase reporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents’ for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators pre-clinically in small animal models by non-invasive molecular imaging approach. PMID:23826874

  8. Reporter protein complementation imaging assay to screen and study Nrf2 activators in cells and living animals.

    PubMed

    Ramkumar, Kunka Mohanram; Sekar, Thillai Veerapazham; Foygel, Kira; Elango, Bhakkiyalakshmi; Paulmurugan, Ramasamy

    2013-08-01

    NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferase reporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents' for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators preclinically in small animal models by noninvasive molecular imaging approach. PMID:23826874

  9. Validation of a multiplex RT-PCR assay for screening significant oncogene fusion transcripts in children with acute lymphoblastic leukaemia.

    PubMed

    Ariffin, Hany; Chen, S P; Wong, H-L; Yeoh, A

    2003-10-01

    In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL. PMID:15024455

  10. Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

    PubMed Central

    2011-01-01

    Background Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. Results Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. Conclusion In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints. PMID:21345205

  11. Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity

    E-print Network

    Xu, Jinghai J.

    Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration ...

  12. An Image-Based Biosensor Assay Strategy to Screen for Modulators of the microRNA 21 Biogenesis Pathway

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Radu, Constantin; Calder, Paul; Ramirez, Christina N.; Djaballah, Hakim

    2013-01-