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Sample records for drug screening assay

  1. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  2. A high content screening assay to predict human drug-induced liver injury during drug discovery.

    Science.gov (United States)

    Persson, Mikael; Løye, Anni F; Mow, Tomas; Hornberg, Jorrit J

    2013-01-01

    Adverse drug reactions are a major cause for failures of drug development programs, drug withdrawals and use restrictions. Early hazard identification and diligent risk avoidance strategies are therefore essential. For drug-induced liver injury (DILI), this is difficult using conventional safety testing. To reduce the risk for DILI, drug candidates with a high risk need to be identified and deselected. And, to produce drug candidates without that risk associated, risk factors need to be assessed early during drug discovery, such that lead series can be optimized on safety parameters. This requires methods that allow for medium-to-high throughput compound profiling and that generate quantitative results suitable to establish structure-activity-relationships during lead optimization programs. We present the validation of such a method, a novel high content screening assay based on six parameters (nuclei counts, nuclear area, plasma membrane integrity, lysosomal activity, mitochondrial membrane potential (MMP), and mitochondrial area) using ~100 drugs of which the clinical hepatotoxicity profile is known. We find that a 100-fold TI between the lowest toxic concentration and the therapeutic Cmax is optimal to classify compounds as hepatotoxic or non-hepatotoxic, based on the individual parameters. Most parameters have ~50% sensitivity and ~90% specificity. Drugs hitting ≥2 parameters at a concentration below 100-fold their Cmax are typically hepatotoxic, whereas non-hepatotoxic drugs typically hit based on nuclei count, MMP and human Cmax, we identified an area without a single false positive, while maintaining 45% sensitivity. Hierarchical clustering using the multi-parametric dataset roughly separates toxic from non-toxic compounds. We employ the assay in discovery projects to prioritize novel compound series during hit-to-lead, to steer away from a DILI risk during lead optimization, for risk assessment towards candidate selection and to provide guidance of safe

  3. Feasibility evaluation of 3 automated cellular drug screening assays on a robotic workstation.

    Science.gov (United States)

    Soikkeli, Anne; Sempio, Cristina; Kaukonen, Ann Marie; Urtti, Arto; Hirvonen, Jouni; Yliperttula, Marjo

    2010-01-01

    This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation-the Caspase-Glo 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol-and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 cells (Z' 0.8, S/B 2.4) but not with the Caco-2 cells (Z' -0.8, S/B 1.4). The automated bidirectional Caco-2 permeability experiments separated successfully low- and high-permeability compounds (Z' 0.8, S/B 84.2) and passive drug permeation from efflux-mediated transport (Z' 0.5, S/B 8.6). Of the assays, the homogeneous Caspase-Glo 3/7 assay benefits the most from automation, but also the heterogeneous SRB assay and Caco-2 permeability experiments gain advantages from automation.

  4. Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

    Directory of Open Access Journals (Sweden)

    Alexandra Ibáñez Escribano

    2012-08-01

    Full Text Available A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL. The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

  5. Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

    Directory of Open Access Journals (Sweden)

    Deepika Kanojia

    2017-11-01

    Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

  6. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

    Science.gov (United States)

    Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

    1988-02-01

    For the past 30 years strategies for the preclinical 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 cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

  7. Smart polymer platforms for in vitro drug screening assays based on drug-loaded nanoparticles

    DEFF Research Database (Denmark)

    Faralli, Adele

    -electrodes for co-localization of drug-loaded nanoparticles (liposomes) and cancer cells. PEGDA hydrogels are widely used in different fields including tissue engineering and in vivo drug delivery. A home-made setup for the fabrication of PEGDA hydrogels through visible-light photopolymerization is described...

  8. Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Cristiana Lalli

    2015-01-01

    Full Text Available 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.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.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.

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

    Science.gov (United States)

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

    2015-12-01

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

  10. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

    NARCIS (Netherlands)

    D'Alessandro, S.; Silvestrini, F.; Dechering, K.; Corbett, Y.; Parapini, S.; Timmerman, M.; Galastri, L.; Basilico, N.; Sauerwein, R.; Alano, P.; Taramelli, D.

    2013-01-01

    OBJECTIVES: Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the

  11. Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine

    International Nuclear Information System (INIS)

    Lim, Sue Ping; Callen, David F; Kumar, Raman; Akkamsetty, Yamini; Wang, Wen; Ho, Kristen; Neilsen, Paul M; Walther, Diego J; Suetani, Rachel J; Prestidge, Clive

    2013-01-01

    Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression

  12. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Science.gov (United States)

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  13. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

    Full Text Available A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10% were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

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

    Science.gov (United States)

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

    2015-01-01

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

  15. Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

    Science.gov (United States)

    Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

    2015-01-01

    Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

  16. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

    Directory of Open Access Journals (Sweden)

    Michael J Smout

    Full Text Available 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 IC(50 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.

  17. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

    OpenAIRE

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-thr...

  18. Urine drug screen

    Science.gov (United States)

    Drug screen - urine ... detect the presence of illegal and some prescription drugs in your urine. Their presence may indicate that you recently used these drugs. Some drugs may remain in your system for ...

  19. Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

    Science.gov (United States)

    Rotem, Asaf; Garraway, Levi; Su, Mei-Ju; Basu, Anindita; Regev, Aviv; Struhl, Kevin

    2017-02-01

    Three-dimensional growth conditions reflect the natural environment of cancer cells and are crucial to be performed at drug screens. We developed a 3D assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the 50-year old benchmark assay-soft agar. Using GILA, we performed high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. This phenotypic approach is complementary to our genetic approach that utilizes single-cell RNA-sequencing of a patient sample to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein. Currently, we are dealing with a big challenge in our field- the limited number of cells that might be extracted from a biopsy. Small patient-derived samples are hard to test in the traditional multiwell plate and it will be helpful to minimize the culture area and the experimental system. We managed to design a suitable microfluidic device for limited number of cells and perform the assay using image analysis. We aim to test drugs on tumor cells, outside of the patient body- and recommend on the ideal treatment that is tailored to the individual. This device will help to minimize biopsy-sampling volumes and minimize interventions in the patient's tumor.

  20. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

    Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

  1. An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

    Science.gov (United States)

    Loo, Jacky Fong-Chuen; Yang, Chengbin; Tsang, Hing Lun; Lau, Pui Man; Yong, Ken-Tye; Ho, Ho Pui; Kong, Siu Kai

    2017-10-07

    With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

  2. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

    Science.gov (United States)

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

    2014-10-03

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Screening of antifungal azole drugs and agrochemicals with an adapted alamarBlue-based assay demonstrates antibacterial activity of croconazole against Mycobacterium ulcerans.

    Science.gov (United States)

    Scherr, Nicole; Röltgen, Katharina; Witschel, Matthias; Pluschke, Gerd

    2012-12-01

    An alamarBlue-based growth inhibition assay has been adapted for the thermosensitive and slow-growing pathogen Mycobacterium ulcerans. The standardized test procedure enables medium-throughput screening of preselected compound libraries. Testing of a set of 48 azoles with known antifungal activity led to the identification of an imidazole antifungal displaying an inhibitory dose (ID) of 9 μM for M. ulcerans.

  4. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

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

    KAUST Repository

    Soufan, Othman; Ba Alawi, Wail; Afeef, Moataz A.; Essack, Magbubah; Kalnis, Panos; Bajic, Vladimir B.

    2016-01-01

    Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods

  6. Digital Drug Dosing: Dosing in Drug Assays by Light-Defined Volumes of Hydrogels with Embedded Drug-Loaded Nanoparticles

    DEFF Research Database (Denmark)

    Faralli, Adele; Melander, Fredrik; Larsen, Esben Kjær Unmack

    2014-01-01

    Polyethylene glycol (PEG)-based hydrogels are widely used for biomedical applications, including matrices for controlled drug release. We present a method for defining drug dosing in screening assays by light-activated cross-linking of PEG-diacrylate hydrogels with embedded drug-loaded liposome...

  7. Urine drug screening in the medical setting.

    Science.gov (United States)

    Hammett-Stabler, Catherine A; Pesce, Amadeo J; Cannon, Donald J

    2002-01-01

    The term drug screen is a misnomer since it implies screening for all drugs, which is not possible. Current practice is to limit the testing to the examination of serum for several drugs such as ethanol, acetaminophen, salicylate, and of urine for several specific drugs or classes of drugs. In the emergency setting the screen should be performed in less than one hour. Controversies continue to exist regarding the value of urine drug testing in the medical setting. The reasons for these include the drugs involved, the sample, the methods utilized to perform the tests, and the level of understanding of the physician using the data, all of which are closely related to the other. Current automated methods provide rapid results demanded in emergency situations, but are often designed for, or adapted from, workplace testing and are not necessarily optimized for clinical applications. Furthermore, the use of these methods without consideration of the frequency in which the drugs are found in a given area is not cost-effective. The laboratory must understand the limitations of the assays used and provide this information to the physician. Additionally, the laboratory and the physicians using the data must cooperate to determine which drugs are appropriate and necessary to measure for their institution and clinical setting. In doing so it should be remembered that for many drugs, the sample, urine, contains the end product(s) of drug metabolism, not the parent drug. Furthermore, it is necessary to understand the pharmacokinetic parameters of the drug of interest when interpreting data. Finally, while testing for some drugs may not appear cost-effective, the prevention or reduction of morbidity and mortality may offset any laboratory costs. While the literature is replete with studies concerning new methods and a few regarding physician understanding, there are none that we could find that thoroughly, objectively, and fully addressed the issues of utility and cost-effectiveness.

  8. Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming

    2015-01-01

    that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell...... and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology....

  9. Semiautomated radioimmunoassay for mass screening of drugs of abuse

    International Nuclear Information System (INIS)

    Sulkowski, T.S.; Lathrop, G.D.; Merritt, J.H.; Landez, J.H.; Noe, E.R.

    1975-01-01

    A rapid, semiautomated radioimmunoassay system for detection of morphine, barbiturates, and amphetamines is described. The assays are applicable to large drug abuse screening programs. The heart of the system is the automatic pipetting station which can accomplish 600 pipetting operations per hour. The method uses 15 to 30 μl for the amphetamine and combined morphine/barbiturate assays. A number of other drugs were tested for interference with the assays and the results are discussed

  10. Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

    Science.gov (United States)

    Winborn, Jessica; Kerrigan, Sarah

    2017-06-01

    Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Directory of Open Access Journals (Sweden)

    Michael A. Partridge

    2016-01-01

    Full Text Available Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.

  12. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

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

  13. Receptor-based screening assays for the detection of antibiotics residues - A review.

    Science.gov (United States)

    Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui

    2017-05-01

    Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    KAUST Repository

    Soufan, Othman

    2015-12-14

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

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

    KAUST Repository

    Soufan, Othman; Ba Alawi, Wail; Afeef, Moataz A.; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

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

  16. Nitric oxide production in mouse and rat macrophages: a rapid and efficient assay for screening of drugs immunostimulatory effects in human cells

    Czech Academy of Sciences Publication Activity Database

    Kmoníčková, Eva; Melkusová, Petra; Farghali, H.; Holý, Antonín; Zídek, Zdeněk

    2007-01-01

    Roč. 17, - (2007), s. 160-169 ISSN 1089-8603 R&D Projects: GA MŠk 1M0508; GA ČR GA305/07/0061 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z40550506 Keywords : Cytokines * Nitric oxide * Immunobiological screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.900, year: 2007

  17. Lack of cross-reactivity of Ambien (zolpidem) with drugs in standard urine drug screens.

    Science.gov (United States)

    Piergies, A A; Sainati, S; Roth-Schechter, B

    1997-04-01

    To determine in healthy volunteers (men and women; 18 to 40 years old) the potential cross-reactivity of Ambien (zolpidem) and/or its metabolites with drugs that are screened by the Syva EMIT II and the Abbott ADx urine drug screens assays. Open-label, fixed-treatment sequence of 1 night each of treatment with zolpidem (10 mg) and temazepam (15 mg). Clinical Pharmacology Unit within a teaching hospital. Over a 24-hour period, presence or absence of positive results on the Syva EMIT II or the Abbott ADx urine drug assay system, each performed at two different laboratory assay sites. Following ingestion of zolpidem, no subject had any positive response in either laboratory to the Syva EMIT II or the Abbott ADx urine drug screen assays at 0, 4, 8, 12, and 24 hours postdose. During the same time period, all subjects had measurable zolpidem plasma concentrations at 1.5 and 8 hours postdose, with mean concentrations of 115.2 ng/mL and 30.1 ng/mL, respectively (in agreement with its half-life of 2.5 hours). The positive response rate at 10 hours after ingestion of Restoril (temazepam) among the four laboratory/assay combinations ranged from 36.8% to 73.7%, a range that is within the reported response rates for these tests. These data indicate that zolpidem will not cross-react in standard urine drug screens with benzodiazepines, opiates, barbiturates, cocaine, cannabinoids, or amphetamines.

  18. Rapid screening assay for calcium bioavailability studies

    International Nuclear Information System (INIS)

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

    1986-01-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium ( 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 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 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

  19. Bead-based screening in chemical biology and drug discovery

    DEFF Research Database (Denmark)

    Komnatnyy, Vitaly V.; Nielsen, Thomas Eiland; Qvortrup, Katrine

    2018-01-01

    libraries for early drug discovery. Among the various library forms, the one-bead-one-compound (OBOC) library, where each bead carries many copies of a single compound, holds the greatest potential for the rapid identification of novel hits against emerging drug targets. However, this potential has not yet...... been fully realized due to a number of technical obstacles. In this feature article, we review the progress that has been made towards bead-based library screening and applications to the discovery of bioactive compounds. We identify the key challenges of this approach and highlight key steps needed......High-throughput screening is an important component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds requires assays amanable to miniaturisation and automization. Combinatorial chemistry holds a unique promise to deliver structural diverse...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  1. Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

    Science.gov (United States)

    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 PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ 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 PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  2. Determination of antipsychotic drug in human serum by radioreceptor assay

    International Nuclear Information System (INIS)

    Wu Jinchang; Jiang Yimin

    1989-01-01

    Serum antipsychotic drug in 50 psychosis cases were measured by radioreceptor assay (RRA) and the values were compared in parallel with that by radioimmunoassay (RIA). The results showed that the RRA values were lower than the RIA values, but both assays gave significant correlation between the serum drug level and antipsychotic dose

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

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

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

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

    KAUST Repository

    Soufan, Othman

    2016-11-10

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

  5. Complementing in vitro screening assays with in silico ...

    Science.gov (United States)

    High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive under assay conditions but that can generate active metabolites under in vivo conditions. To address this potential issue, a case study will be presented to demonstrate the use of in silico tools to identify inactive parents with the ability to generate active metabolites. This case study used the results from an orthogonal assay designed to improve confidence in the identification of active chemicals tested across eighteen estrogen receptor (ER)-related in vitro assays by accounting for technological limitations inherent within each individual assay. From the 1,812 chemicals tested within the orthogonal assay, 1,398 were considered inactive. These inactive chemicals were analyzed using Chemaxon Metabolizer software to predict the first and second generation metabolites. From the nearly 1,400 inactive chemicals, over 2,200 first-generation (i.e., primary) metabolites and over 5,500 second-generation (i.e., secondary) metabolites were predicted. Nearly 70% of primary metabolites were immediately detoxified or converted to other metabolites, while over 70% of secondary metabolites remained stable. Among these predicted metabolites, those that are most likely to be produced and remain

  6. An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

    Science.gov (United States)

    Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

    2012-03-01

    The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

  7. Understanding drugs in breast cancer through drug sensitivity screening.

    Science.gov (United States)

    Uhr, Katharina; Prager-van der Smissen, Wendy J C; Heine, Anouk A J; Ozturk, Bahar; Smid, Marcel; Göhlmann, Hinrich W H; Jager, Agnes; Foekens, John A; Martens, John W M

    2015-01-01

    With substantial numbers of breast tumors showing or acquiring treatment resistance, it is of utmost importance to develop new agents for the treatment of the disease, to know their effectiveness against breast cancer and to understand their relationships with other drugs to best assign the right drug to the right patient. To achieve this goal drug screenings on breast cancer cell lines are a promising approach. In this study a large-scale drug screening of 37 compounds was performed on a panel of 42 breast cancer cell lines representing the main breast cancer subtypes. Clustering, correlation and pathway analyses were used for data analysis. We found that compounds with a related mechanism of action had correlated IC50 values and thus grouped together when the cell lines were hierarchically clustered based on IC50 values. In total we found six clusters of drugs of which five consisted of drugs with related mode of action and one cluster with two drugs not previously connected. In total, 25 correlated and four anti-correlated drug sensitivities were revealed of which only one drug, Sirolimus, showed significantly lower IC50 values in the luminal/ERBB2 breast cancer subtype. We found expected interactions but also discovered new relationships between drugs which might have implications for cancer treatment regimens.

  8. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    Science.gov (United States)

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

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

    International Nuclear Information System (INIS)

    Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

    2015-01-01

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

  11. Development in Assay Methods for in Vitro Antimalarial Drug Efficacy Testing: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Shweta Sinha

    2017-10-01

    Full Text Available The emergence and spread of drug resistance are the major challenges in malaria eradication mission. Besides various strategies laid down by World Health Organization, such as vector management, source reduction, early case detection, prompt treatment, and development of new diagnostics and vaccines, nevertheless the need for new and efficacious drugs against malaria has become a critical priority on the global malaria research agenda. At several screening stages, millions of compounds are screened (1,000–2,000,000 compounds per screening campaign, before pre-clinical trials to select optimum lead. Carrying out in vitro screening of antimalarials is very difficult as different assay methods are subject to numerous sources of variability across different laboratories around the globe. Despite this, in vitro screening is an essential part of antimalarial drug development as it enables to resource various confounding factors such as host immune response and drug–drug interaction. Therefore, in this article, we try to illustrate the basic necessity behind in vitro study and how new methods are developed and subsequently adopted for high-throughput antimalarial drug screening and its application in achieving the next level of in vitro screening based on the current approaches (such as stem cells.

  12. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Science.gov (United States)

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  13. Microfluidic Devices for Drug Delivery Systems and Drug Screening

    Science.gov (United States)

    Kompella, Uday B.; Damiati, Safa A.

    2018-01-01

    Microfluidic devices present unique advantages for the development of efficient drug carrier particles, cell-free protein synthesis systems, and rapid techniques for direct drug screening. Compared to bulk methods, by efficiently controlling the geometries of the fabricated chip and the flow rates of multiphase fluids, microfluidic technology enables the generation of highly stable, uniform, monodispersed particles with higher encapsulation efficiency. Since the existing preclinical models are inefficient drug screens for predicting clinical outcomes, microfluidic platforms might offer a more rapid and cost-effective alternative. Compared to 2D cell culture systems and in vivo animal models, microfluidic 3D platforms mimic the in vivo cell systems in a simple, inexpensive manner, which allows high throughput and multiplexed drug screening at the cell, organ, and whole-body levels. In this review, the generation of appropriate drug or gene carriers including different particle types using different configurations of microfluidic devices is highlighted. Additionally, this paper discusses the emergence of fabricated microfluidic cell-free protein synthesis systems for potential use at point of care as well as cell-, organ-, and human-on-a-chip models as smart, sensitive, and reproducible platforms, allowing the investigation of the effects of drugs under conditions imitating the biological system. PMID:29462948

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

    International Nuclear Information System (INIS)

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

    2004-01-01

    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

  15. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

    Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

    2011-09-01

    A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Assaying of drugs in body fluids

    International Nuclear Information System (INIS)

    Braestrup, C.; Squires, R.F.

    1981-01-01

    The invention provides, in general terms, a process for determining the concentration of a psychotropically active benzodiazepine drug in blood or other body fluid or urine, including bringing a sample of the fluid or urine into contact with brain tissue and with tritium labelled molecules of a benzodiazepine which can bind reversibly to receptors of the brain tissue to induce binding of molecules of the unlabelled drug and of the tritium labelled benzodiazepine to the receptors, and determining the radioactivity of the brain tissue, preferably by scintillation counting. (author)

  17. Drug Target Interference in Immunogenicity Assays: Recommendations and Mitigation Strategies.

    Science.gov (United States)

    Zhong, Zhandong Don; Clements-Egan, Adrienne; Gorovits, Boris; Maia, Mauricio; Sumner, Giane; Theobald, Valerie; Wu, Yuling; Rajadhyaksha, Manoj

    2017-11-01

    Sensitive and specific methodology is required for the detection and characterization of anti-drug antibodies (ADAs). High-quality ADA data enables the evaluation of potential impact of ADAs on the drug pharmacokinetic profile, patient safety, and efficacious response to the drug. Immunogenicity assessments are typically initiated at early stages in preclinical studies and continue throughout the drug development program. One of the potential bioanalytical challenges encountered with ADA testing is the need to identify and mitigate the interference mediated by the presence of soluble drug target. A drug target, when present at sufficiently high circulating concentrations, can potentially interfere with the performance of ADA and neutralizing antibody (NAb) assays, leading to either false-positive or, in some cases, false-negative ADA and NAb assay results. This publication describes various mechanisms of assay interference by soluble drug target, as well as strategies to recognize and mitigate such target interference. Pertinent examples are presented to illustrate the impact of target interference on ADA and NAb assays as well as several mitigation strategies, including the use of anti-target antibodies, soluble versions of the receptors, target-binding proteins, lectins, and solid-phase removal of targets. Furthermore, recommendations for detection and mitigation of such interference in different formats of ADA and NAb assays are provided.

  18. The loss-of-allele assay for ES cell screening and mouse genotyping.

    Science.gov (United States)

    Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

  19. Fluorescence-based assay as a new screening tool for toxic chemicals

    Science.gov (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

    2016-09-01

    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-01

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

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

    International Nuclear Information System (INIS)

    Morton, M.J.; Armstrong, D.; Abi Gerges, N.; Bridgland-Taylor, M.; Pollard, C.E.; Bowes, J.; Valentin, J.-P.

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Junaid Iqbal

    2013-01-01

    Full Text Available 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.

  3. Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

    Science.gov (United States)

    Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

    2018-05-17

    In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. G protein-coupled receptor internalization assays in the high-content screening format.

    Science.gov (United States)

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  5. Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

    Science.gov (United States)

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

    With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

  6. Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay.

    Science.gov (United States)

    Rotherham, D; Harbison, S A

    2011-04-15

    Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  7. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Directory of Open Access Journals (Sweden)

    Prerna Grover

    Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

  8. "Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

    Science.gov (United States)

    Görgens, Christian; Guddat, Sven; Orlovius, Anne-Katrin; Sigmund, Gerd; Thomas, Andreas; Thevis, Mario; Schänzer, Wilhelm

    2015-07-01

    In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT  0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

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

    International Nuclear Information System (INIS)

    Keating, Christopher; Martinez, Vicente; Ewart, Lorna; Gibbons, Stephen; Grundy, Luke; Valentin, Jean-Pierre; Grundy, David

    2010-01-01

    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.

  10. A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

    Science.gov (United States)

    Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

    2018-05-01

    The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

  11. Rectal swab screening assays of public health importance in molecular diagnostics: Sample adequacy control.

    Science.gov (United States)

    Glisovic, Sanja; Eintracht, Shaun; Longtin, Yves; Oughton, Matthew; Brukner, Ivan

    Rectal swabs are routinely used by public health authorities to screen for multi-drug resistant enteric bacteria including vancomycin-resistant enterococci (VRE) and carbapenem-resistant enterobacteriaceae (CRE). Screening sensitivity can be influenced by the quality of the swabbing, whether performed by the patient (self-swabbing) or a healthcare practitioner. One common exclusion criterion for rectal swabs is absence of "visible soiling" from fecal matter. In our institution, this criterion excludes almost 10% of rectal swabs received in the microbiology laboratory. Furthermore, over 30% of patients in whom rectal swabs are cancelled will not be re-screened within the next 48h, resulting in delays in removing infection prevention measures. We describe two quantitative polymerase chain reaction (qPCR)-based assays, human RNAse P and eubacterial 16S rDNA, which might serve as suitable controls for sampling adequacy. However, lower amounts of amplifiable human DNA make the 16s rDNA assay a better candidate for sample adequacy control. Copyright © 2017. Published by Elsevier Ltd.

  12. Open innovation for phenotypic drug discovery: The PD2 assay panel.

    Science.gov (United States)

    Lee, Jonathan A; Chu, Shaoyou; Willard, Francis S; Cox, Karen L; Sells Galvin, Rachelle J; Peery, Robert B; Oliver, Sarah E; Oler, Jennifer; Meredith, Tamika D; Heidler, Steven A; Gough, Wendy H; Husain, Saba; Palkowitz, Alan D; Moxham, Christopher M

    2011-07-01

    Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

  13. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    Science.gov (United States)

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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. (letter)

  15. Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

    Science.gov (United States)

    Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

    2004-12-01

    The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

  16. The development of high-content screening (HCS) technology and its importance to drug discovery.

    Science.gov (United States)

    Fraietta, Ivan; Gasparri, Fabio

    2016-01-01

    High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development. Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology. Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.

  17. A Mos1 transposase in vivo assay to screen new HIV-1 integrase inhibitors.

    Science.gov (United States)

    Cancian, Mariana; Loreto, Elgion L S

    2018-04-01

    The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.

  18. Development of fluorescence imaging-based assay for screening cardioprotective compounds from medicinal plants.

    Science.gov (United States)

    Wang, Yi; Zhao, Xiaoping; Gao, Xiumei; Nie, Xiaojing; Yang, Yingxin; Fan, Xiaohui

    2011-09-19

    Medicinal plants have been widely recognized as a renewable resource for the discovery of novel leads and drug. In this study, an approach for screening and identification compounds with cardioprotective activity from medicinal plant extracts by cellular-fluorescence imaging technique was developed. It is a cell-based assay for measuring mitochondrial membrane potential changes in H9c2 cardiac muscle cells exposed to H(2)O(2) by using a fluorescence automatic microscopy screening platform. Rhodamine 123 was used as the fluorescent dye to indicate the change of mitochondrial membrane potential. The sensitivity and linear range of the proposed approach were evaluated and validated using vitamin C, an antioxidative compound. The method was applied to screen active components with potent cardioprotective effects from a traditional Chinese formula. The potential cardioprotective components were identified by liquid chromatography coupled with mass spectrometry (LC/MS). Moreover, the utility of the proposed approach was further validated by three compounds (salvianolic acid B, protocatechuic aldehyde, and tanshinone II A) identified from the formula which showed cardioprotective effects in a dose-dependent manner. These applications suggested that the proposed rapid and sensitive screening approach offers an efficient way to discover active components or compounds from medicinal plants. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    KAUST Repository

    Soufan, Othman

    2016-01-01

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

  20. Drug discovery for Duchenne muscular dystrophy via utrophin promoter activation screening.

    Directory of Open Access Journals (Sweden)

    Catherine Moorwood

    Full Text Available Duchenne muscular dystrophy (DMD is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    , better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput...... the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range...... screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009: 173-180)...

  3. Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives.

    Science.gov (United States)

    Marugán, Carlos; Torres, Raquel; Lallena, María José

    2015-01-01

    Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

  4. Phenotypic screening approaches to develop Aurora kinase inhibitors: Drug Discovery perspectives

    Directory of Open Access Journals (Sweden)

    Carlos eMarugán

    2016-01-01

    Full Text Available Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins such as Aurora A and B. Current drugs which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules, have several side effects (neutropenia, alopecia, emesis. Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype.We will briefly describe two multiplexing technologies (high-content imaging, microarrays and flow cytometry and two key processes for drug discovery research (assay development and validation following our own published industry quality standards. We will further focus on high-content imaging as a useful tool for phenotypic screening and will provide a concrete example of high-content imaging assay to detect Aurora A or B selective inhibitors discriminating the off-target effects related to inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

  5. Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

    Science.gov (United States)

    Linares, María; Viera, Sara; Crespo, Benigno; Franco, Virginia; Gómez-Lorenzo, María G; Jiménez-Díaz, María Belén; Angulo-Barturen, Íñigo; Sanz, Laura María; Gamo, Francisco-Javier

    2015-11-05

    The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. The quantification of new infections to determine parasite viability offers important

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

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

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

  7. USER S GUIDE FOR THE RANDOM DRUG SCREENING SYSTEM

    Energy Technology Data Exchange (ETDEWEB)

    McNeany, Karen I [ORNL

    2013-12-01

    The Random Drug Screening System (RDSS) is a desktop computing application designed to assign nongameable drug testing dates to each member in a population of employees, within a specific time line. The program includes reporting capabilities, test form generation, unique test ID number assignment, and the ability to flag high-risk employees for a higher frequency of drug testing than the general population.

  8. Expression and Purification of PI3 Kinase {alpha} and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

    DEFF Research Database (Denmark)

    Boldyreff, Brigitte; Rasmussen, Tine L; Jensen, Hans H

    2008-01-01

    Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal....... In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. (Journal of Biomolecular Screening XXXX:xx-xx)....

  9. Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

    Science.gov (United States)

    Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

    2017-12-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

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

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

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

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

    Science.gov (United States)

    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 μg /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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

  13. A polychromatic turbidity microplate assay to distinguish discovery stage drug molecules with beneficial precipitation properties.

    Science.gov (United States)

    Morrison, John; Nophsker, Michelle; Elzinga, Paul; Donoso, Maria; Park, Hyunsoo; Haskell, Roy

    2017-10-05

    A material sparing microplate screening assay was developed to evaluate and compare the precipitation of discovery stage drug molecules as a function of time, concentration and media composition. Polychromatic turbidity time course profiles were collected for cinnarizine, probucol, dipyridamole as well as BMS-932481, and compared with turbidity profiles of monodisperse particle size standards. Precipitation for select sample conditions were further characterized at several time points by size, morphology, amount and form via laser diffraction, microscopy, size based particle counting and X-ray diffraction respectively. Wavelength dependent turbidity was found indicative of nanoprecipitate, while wavelength independent turbidity was consistent with larger microprecipitate formation. A transition from wavelength dependent to wavelength independent turbidity occurred for nanoparticle to microparticle growth, and a decrease in wavelength independent turbidity correlated with continued growth in size of microparticles. Other sudden changes in turbidity signal over time such as rapid fluctuation, a decrease in slope or a sharp inversion were correlated with very large or aggregated macro-precipitates exceeding 100μm in diameter, a change in the rate of precipitate formation or an amorphous to crystalline form conversion respectively. The assay provides an effective method to efficiently monitor and screen the precipitation fates of drug molecules, even during the early stages of discovery with limited amounts of available material. This capability highlights molecules with beneficial precipitation properties that are able to generate and maintain solubility enabling amorphous or nanoparticle precipitates. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Microfractionation revisited: a 1536 well high resolution screening assay

    NARCIS (Netherlands)

    Giera, M.A.; Heus, F.; Janssen, L.; Kool, J.; Lingeman, H.; Irth, H.

    2009-01-01

    The aim of the here presented study was to combine high performance liquid chromatography with plate reader technology in order to overcome certain drawbacks of integrated online systems as well as offline plate reader approaches. The described method combines an "at-line" enzyme assay for the

  15. A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

    Science.gov (United States)

    Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

    2011-08-15

    A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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 β-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.

  17. Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

    Science.gov (United States)

    The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

  18. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection

    OpenAIRE

    Kremastinou, J.; Polymerou, V.; Lavranos, D.; Aranda Arrufat, A.; Harwood, J.; Mart?nez Lorenzo, M. J.; Ng, K. P.; Queiros, L.; Vereb, I.; Cusini, M.

    2016-01-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum. The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient popul...

  19. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  20. A High-Throughput Screening Assay to Detect Thyroperoxidase Inhibitors (Teratology Society)

    Science.gov (United States)

    In support of the Endocrine Disruption Screening Program (EDSP21), the US EPA ToxCast program is developing assays to enable screening for chemicals that may disrupt thyroid hormone synthesis. Thyroperoxidase (TPO) is critical for TH synthesis and is a known target of thyroid-dis...

  1. Preformulation compatibility screening of dika fat-drug mixtures ...

    African Journals Online (AJOL)

    Differential scanning calorimetry (DSC) was used as screening technique for assessing compatibility between dika fat and drug substances. Dika fat was found to be compatible with aspirin, ascorbic acid, paracetamol, sulphanilamide, phenylpropanolamine hydrochloride, bromopheniramine maleate, chlorpheniramire ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Vanparys, Caroline, E-mail: caroline.vanparys@ua.ac.be [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)

    2010-09-15

    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 to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

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

    International Nuclear Information System (INIS)

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

    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 to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC 50 value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R 2 = 0.98), the estrogen receptor (ER) binding (R 2 = 0.84) and the ER transcription activation assay (R 2 = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of

  4. A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

    Science.gov (United States)

    Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

    2015-12-01

    Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

  5. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

    Science.gov (United States)

    Li, Guannan; Huang, Ke; Nikolic, Dejan; van Breemen, Richard B

    2015-11-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan [Department of Medicine, Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Durmus, Naside Gozde, E-mail: udemirci@rics.bwh.harvard.edu [School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI (United States)

    2011-09-15

    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.

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

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    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.

  9. Thermodynamic Studies for Drug Design and Screening

    Science.gov (United States)

    Garbett, Nichola C.; Chaires, Jonathan B.

    2012-01-01

    Introduction A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions. Areas covered This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 – 2011 using the Science Citation Index and PUBMED and the keywords listed below. Expert opinion The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically-driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development towards an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design. PMID:22458502

  10. Thermodynamic studies for drug design and screening.

    Science.gov (United States)

    Garbett, Nichola C; Chaires, Jonathan B

    2012-04-01

    A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions. This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 - 2011 using the Science Citation Index and PUBMED and the keywords listed below. The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development toward an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in the design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design. © 2012 Informa UK, Ltd.

  11. High-throughput screening using pseudotyped lentiviral particles: a strategy for the identification of HIV-1 inhibitors in a cell-based assay.

    Science.gov (United States)

    Garcia, Jean-Michel; Gao, Anhui; He, Pei-Lan; Choi, Joyce; Tang, Wei; Bruzzone, Roberto; Schwartz, Olivier; Naya, Hugo; Nan, Fa-Jun; Li, Jia; Altmeyer, Ralf; Zuo, Jian-Ping

    2009-03-01

    Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

  12. Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence "transmission blocking" assay.

    Directory of Open Access Journals (Sweden)

    Joël Lelièvre

    Full Text Available 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Recommended Immunological Assays to Screen for Ricin-Containing Samples

    Directory of Open Access Journals (Sweden)

    Stéphanie Simon

    2015-11-01

    Full Text Available Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120. Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests. Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

  15. Alcohol and drug screening of occupational drivers for preventing injury

    NARCIS (Netherlands)

    Cashman, Clodagh M.; Ruotsalainen, Jani H.; Greiner, Birgit A.; Beirne, Paul V.; Verbeek, Jos H.

    2009-01-01

    BACKGROUND: Workforce alcohol and drug testing is commonplace but its effect in reducing occupational injuries remains unclear. OBJECTIVES: To assess the effects of alcohol and drug screening of occupational drivers (operating a motorised vehicle) in preventing injury or work-related effects such as

  16. Microreactor for electrochemical conversion: in drug screening and proteomics

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus

    2016-01-01

    The majority of marketed drugs are metabolized through oxidation by enzymes of the cytochrome P450 family, thereby producing phase I metabolites. For pharmaceutical companies it is essential to thoroughly screen candidate drugs for potentially toxic metabolites, in order to avoid high costs

  17. A multi-infrastructure gateway for virtual drug screening

    NARCIS (Netherlands)

    Jaghoori, Mohammad Mahdi; van Altena, Allard J.; Bleijlevens, Boris; Ramezani, Sara; Font, Juan Luis; Olabarriaga, Silvia D.

    2015-01-01

    In computer-aided drug design, software tools are used to narrow down possible drug candidates, thereby reducing the amount of expensive in vitro research, by a process called virtual screening. This process includes large computations that require advanced computing infrastructure; however, using

  18. Hierarchical virtual screening approaches in small molecule drug discovery.

    Science.gov (United States)

    Kumar, Ashutosh; Zhang, Kam Y J

    2015-01-01

    Virtual screening has played a significant role in the discovery of small molecule inhibitors of therapeutic targets in last two decades. Various ligand and structure-based virtual screening approaches are employed to identify small molecule ligands for proteins of interest. These approaches are often combined in either hierarchical or parallel manner to take advantage of the strength and avoid the limitations associated with individual methods. Hierarchical combination of ligand and structure-based virtual screening approaches has received noteworthy success in numerous drug discovery campaigns. In hierarchical virtual screening, several filters using ligand and structure-based approaches are sequentially applied to reduce a large screening library to a number small enough for experimental testing. In this review, we focus on different hierarchical virtual screening strategies and their application in the discovery of small molecule modulators of important drug targets. Several virtual screening studies are discussed to demonstrate the successful application of hierarchical virtual screening in small molecule drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-01-01

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  20. Optimisation of the microplate resazurin assay for screening and bioassay-guided fractionation of phytochemical extracts against Mycobacterium tuberculosis.

    Science.gov (United States)

    O'Neill, Taryn E; Li, Haoxin; Colquhoun, Caitlyn D; Johnson, John A; Webster, Duncan; Gray, Christopher A

    2014-01-01

    Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate resazurin assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the assay can be problematic and unreliable when screening methanolic phytochemical extracts. To optimise the MRA for the screening and bioassay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. The effects of varying assay duration, resazurin solution composition, solvent (dimethyl sulphoxide - DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioassay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL resazurin in 5% aqueous Tween 80 over 96 h incubation. The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The assay procedure has been used successfully for the screening and bioassay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.

  1. Enzymatic assays for detecting lactose and sucrose in urine to reveal intravenous drug abuse with emphasis on buprenorphine.

    Science.gov (United States)

    Keltanen, T; Mariottini, C; Walta, A M; Rahikainen, A L; Ojanperä, I

    2017-06-01

    Buprenorphine and methadone are commonly used medications for opioid maintenance treatment (OMT), using sublingual and oral administration, respectively. Although beneficial for OMT, these drugs can also be abused by intravenous administration. In intravenous abuse cases, the adjuvants lactose and sucrose are excreted in urine without hydrolysis to monosaccharides, since there are no disaccharidases in the blood. We validated enzymatic methods for the analysis of lactose and sucrose in urine. The analytical performance of both assays was considered appropriate for detecting intravenous drug abuse. The principle was proven by analyzing 93 postmortem (PM) urine samples for lactose, following comprehensive toxicological drug screening. In addition, 32 clinical urine samples from potential drug abusers were analyzed to assess the effect of PM changes on the assay. The mean level of lactose was low in clinical samples and relatively low in PM samples in which no drugs were found. Markedly elevated levels were seen in many of the buprenorphine positive samples, suggesting intravenous administration. Enzymatic methods could provide a simple and cost effective way to assess the intravenous administration of OMT drugs or drugs of abuse. Very high levels of glucose in urine may interfere with the assays. Furthermore, other causes for elevated urine disaccharides, such as hypolactasia and increased intestinal permeability, need to be considered in the interpretation of the results. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    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.

  3. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

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

    Directory of Open Access Journals (Sweden)

    Seri Jeong

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

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

    Science.gov (United States)

    Jeong, Seri; Yang, Heeyoung; Hwang, Hyunyong

    2017-01-01

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

  6. Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

    Science.gov (United States)

    2013-01-01

    Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome

  7. The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

    Science.gov (United States)

    Bosserman, Linda; Prendergast, Franklyn; Herbst, Roy; Fleisher, Martin; Salom, Emery; Strickland, Steven; Raptis, Anastasios; Hallquist, Allan; Perree, Mathieu; Rajurkar, Swapnil; Karimi, Misagh; Rogers, Karl; Davidson, Dirk; Willis, Carl; Penalver, Manuel; Homesley, Howard; Burrell, Matthew; Garrett, Audrey; Rutledge, James; Chernick, Michael; Presant, Cary A

    2012-08-15

    A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development. ©2012 AACR.

  8. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis

    OpenAIRE

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-01-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive sample...

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

    OpenAIRE

    Wittbrodt, Jonas N.; Liebel, Urban; Gehrig, Jochen

    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. ...

  10. Understanding drugs in breast cancer through drug sensitivity screening

    NARCIS (Netherlands)

    K. Uhr (Katharina); W.J.C. Prager-van der Smissen (Wendy); A.A.J. Heine (Anouk); B. Ozturk (Bahar); M. Smid (Marcel); H.W.H. Göhlmann (Hinrich W. H.); A. Jager (Agnes); J.A. Foekens (John); J.W.M. Martens (John)

    2015-01-01

    textabstractWith substantial numbers of breast tumors showing or acquiring treatment resistance, it is of utmost importance to develop new agents for the treatment of the disease, to know their effectiveness against breast cancer and to understand their relationships with other drugs to best assign

  11. Assay method for organic calcium antagonist drugs and a kit for such an assay

    International Nuclear Information System (INIS)

    Snyder, S. H.; Gould, R. J.

    1985-01-01

    A method for measuring the level of organic calcium antagonist drug in a body fluid comprises preparing a mixture of a radioactive calcium antagonist drug, a body fluid containing a calcium antagonist drug and a calcium antagonist receptor material, measuring the radioactivity of the radioactive calcium antagonist drug bound to said calcium antagonist receptor material and deriving the concentration of the calcium antagonist drug in the body fluid from a standard curve indicating the concentration of calcium antagonist drug versus inhibition of binding of said radioactive calcium antagonist drug to said receptor sites caused by the calcium antagonist drug in said body fluid. A kit for measuring the level of an organic calcium drug comprises a receptacle containing a radioactive calcium antagonist drug, a calcium antagonist receptor material and a standard amount of a nonradioactive calcium antagonist drug

  12. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines.

    Science.gov (United States)

    Volpe, Donna A

    2011-12-01

    The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.

  13. An FDA-Drug Library Screen for Compounds with Bioactivities against Meticillin-Resistant Staphylococcus aureus (MRSA

    Directory of Open Access Journals (Sweden)

    Qiu Ying Lau

    2015-10-01

    Full Text Available The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA, a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA for bioactivities against MRSA in a 10 μM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections.

  14. Performance Evaluation of a Novel Chemiluminescence Assay Detecting Treponema Pallidum Antibody as a Syphilis Screening Method.

    Science.gov (United States)

    Chen, Qixia; An, Jingna; Rao, Chenli; Wang, Tingting; Li, Dongdong; Feng, Shu; Tao, Chuanmin

    2016-01-01

    Syphilis is a major concern to global public health with increasing incidence. So its screening test should have sufficient sensitivity and specificity. We evaluated the performance of the Lumipulse G TP-N assay detection for syphilis screening and compared it with the InTec ELISA test kit for TP, which is widely used. Samples of several patient groups including 133 clinical and serologically characterized syphilitic sera, 175 samples containing potentially interfering agents, and 2290 unselected samples submitted for routine screening were detected by both the Lumipulse G TP-N assay and the InTec ELISA test kit for TP. Inconsistent samples were confirmed by RecomLine Treponema IgG, IgM immunoblot. Coefficient of variations of the Lumipulseo G TP-N assay at both levels were below 5% and of the InTec ELISA test kit for TP both over 5%. The sensitivity of the Lumipulse G TP-N assay and the InTec ELISA test kit for TP were 100% for all stages of syphilis. The two methods had consistent analytical specificity of 100% (95% CI: 97.21 - 100.00), while the clinical specificity was 100% (95% CI: 99.79 - 100.00) and 99.82% (95% CI: 99.51 - 99.94), respectively. Between them, Spearman's correlation coefficient was 0.455 and kappa value was 0.986. The overall sensitivity and specificity of the Lumipulse G TP-N assay was higher than the InTec ELISA test kit for TP (sensitivity: 100.0 versus 99.5, specificity: 100.0 versus 99.8). The automated Lumipulse G TP-N assay demonstrated excellent diagnostic sensitivity and specificity when evaluated as a screening test for syphilis. Thus, it can be an alternative to the treponemal screening test.

  15. A new in vitro screening system for anticancer drugs for the treatment of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Hanauske, U.; Hanauske, A.R.; Clark, G.M.; Tsen, D.; Buchok, J.; Hoff, D.D. von

    1989-01-01

    We have evaluated a semiautomated radiometric assay (BACTEC 460 system) for screening of activity of anticancer drugs against human non-small cell lung cancer cell lines. Cells from seven cell lines were exposed to standard antineoplastic agents at four different concentrations using a 1-h incubation. Alpha 2-interferon was tested using a continuous incubation. In vitro drug activity was analyzed as a function of the clinically achievable serum concentration. Our results indicate that two cell lines (CALU-3, SK-MES-1) exhibit in vitro drug sensitivity patterns closest to those observed in clinical studies. These two cell lines might therefore be most useful for screening new anticancer compounds for activity against non-small cell lung cancer. The radiometric assay is a semiautomated system which has advantages over other, more time-consuming screening systems

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

    Science.gov (United States)

    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

  17. Reliability, Validity and Factor Structure of Drug Abuse Screening Test

    OpenAIRE

    Sayed Hadi Sayed Alitabar; Mojtaba Habibi; Maryam Falahatpisheh; Musa Arvin

    2016-01-01

    Background and Objective: According to the increasing of substance use in the country, more researches about this phenomenon are necessary. This Study Investigates the Validity, Reliability and Confirmatory Factor Structure of the Drug Abuse Screening test (DAST). Materials and Methods: The Sample Consisted of 381 Patients (143 Women and 238 Men) with a Multi-Stage Cluster Sampling of Areas 2, 6 and 12 of Tehran Were Selected from Each Region, 6 Randomly Selected Drug Rehabilitation Center. T...

  18. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    Science.gov (United States)

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. Copyright © 2016 Kremastinou et al.

  19. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection

    Science.gov (United States)

    Kremastinou, J.; Polymerou, V.; Lavranos, D.; Aranda Arrufat, A.; Harwood, J.; Martínez Lorenzo, M. J.; Ng, K. P.; Queiros, L.; Vereb, I.

    2016-01-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum. The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  20. Development of the screening assay for identification of compounds targeting influenza A

    Czech Academy of Sciences Publication Activity Database

    Karlukova, Elena; Bachmannová, Christina; Machara, Aleš; Berenguer Albiñana, Carlos; Konvalinka, Jan; Kožíšek, Milan

    2017-01-01

    Roč. 15, č. 1 (2017), s. 13 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : influenza virus A * screening assay Subject RIV: CE - Biochemistry

  1. Concordant testing results between various Human Papillomavirus assays in primary cervical cancer screening

    DEFF Research Database (Denmark)

    de Thurah, Lena; Bonde, Jesper; Hoa Lam, Janni Uyen

    2018-01-01

    OBJECTIVES: Human Papillomavirus (HPV) assays are increasingly used for primary cervical screening and HPV vaccination effect monitoring. We undertook a systematic literature review to determine the concordance in positive test results (i.e., detection of HPV infections) between Hybrid Capture 2 ...

  2. Performance of the Xpert HPV assay in women attending for cervical screening

    Directory of Open Access Journals (Sweden)

    Jack Cuzick

    2015-12-01

    Full Text Available Objectives: This study evaluated the Xpert HPV Assay in women attending screening in general practice by comparing Xpert with two established HPV tests, cytology and histology. Methods: A prospective study in women aged 20–60 years attending screening in Bristol, Edinburgh and London using residual Preservcyt cytology samples. Sample order was randomised between Roche cobas4800 and Cepheid Xpert assays with Qiagen hc2 third. Results: 3408 cases were included in the primary analysis. Positivity for Xpert was 19.6%, cobas 19.2% and hc2 19.9% with high concordance (kappa=86.8% vs cobas, 81.55 vs hc2. Xpert, cobas and hc2 showed similar sensitivity (98.7%, 97.5%, 98.7% for CIN2+. All pairwise comparisons had high concordance (Kappa ≥0.78 with any abnormal cytology. Xpert and hc2 were positive for all cases of ≥moderate dyskaryosis (N=63, cobas was negative in two. Histology was available for 172 participants. 79 reported CIN2+, 47 CIN3+. All CIN3+ was positive on Xpert and hc2 and one case negative for cobas. One case of CIN2 was negative for all assays. Conclusions: The performance of Xpert HPV Assay in a general screening population is comparable to established HPV tests. It offers simplicity of testing, flexibility with non-batching of individual samples and rapid turnaround time. Keywords: Human papillomavirus, Xpert, Cervical screening, HPV testing

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

    Science.gov (United States)

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

  4. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Science.gov (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

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

    Directory of Open Access Journals (Sweden)

    Guangchao Yu

    2014-01-01

    Full Text Available 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 µL, 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.

  6. Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle.

    Science.gov (United States)

    Willi, Barbara; Meli, Marina L; Lüthy, Ruedi; Honegger, Hanspeter; Wengi, Nicole; Hoelzle, Ludwig E; Reusch, Claudia E; Lutz, Hans; Hofmann-Lehmann, Regina

    2009-12-01

    Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.

  7. Saliva Polymerase-Chain-Reaction Assay for Cytomegalovirus Screening in Newborns

    Science.gov (United States)

    Boppana, Suresh B.; Ross, Shannon A.; Shimamura, Masako; Palmer, April L.; Ahmed, Amina; Michaels, Marian G.; Sánchez, Pablo J.; Bernstein, David I.; Tolan, Robert W.; Novak, Zdenek; Chowdhury, Nazma; Britt, William J.; Fowler, Karen B.

    2011-01-01

    BACKGROUND Congenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives — real-time polymerase-chain-reaction (PCR)–based testing of a liquid-saliva or dried-saliva specimen obtained at birth — have been developed. METHODS In our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth. RESULTS A total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively. CONCLUSIONS Real-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be

  8. A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

    2015-03-01

    The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    Science.gov (United States)

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. © 2015 Society for Laboratory Automation and Screening.

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

    Directory of Open Access Journals (Sweden)

    Hampus Sunner

    2015-09-01

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

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

    Science.gov (United States)

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

    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.

  12. Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

    Directory of Open Access Journals (Sweden)

    Mary T. Bausch-Jurken

    2017-06-01

    Full Text Available Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay. The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay.

  13. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library.

    Science.gov (United States)

    Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G; Zhang, Ying

    2016-01-01

    Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

  14. High-throughput assay of 9 lysosomal enzymes for newborn screening.

    Science.gov (United States)

    Spacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C Ronald; Turecek, Frantisek; Gelb, Michael H

    2013-03-01

    There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

  15. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-induced Round Bodies of Borrelia burgdorferi Persisters from an FDA Drug Library

    Directory of Open Access Journals (Sweden)

    Jie eFeng

    2016-05-01

    Full Text Available Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that are not killed by current Lyme antibiotics. To identify more effective drugs that are active against the round bodies of B. burgdorferi, we established a round body persister model induced by amoxicillin and screened the Food and Drug Administration (FDA drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide (PI viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven of these scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. While some drug candidates such as daptomycin and clofazimine overlapped with a previous screen against stationary phase B. burgdorferi persisters, additional drug candidates active against round bodies we identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even if pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

  16. poolHiTS: A Shifted Transversal Design based pooling strategy for high-throughput drug screening

    Directory of Open Access Journals (Sweden)

    Woolf Peter J

    2008-05-01

    Full Text Available Abstract Background A key goal of drug discovery is to increase the throughput of small molecule screens without sacrificing screening accuracy. High-throughput screening (HTS in drug discovery involves testing a large number of compounds in a biological assay to identify active compounds. Normally, molecules from a large compound library are tested individually to identify the activity of each molecule. Usually a small number of compounds are found to be active, however the presence of false positive and negative testing errors suggests that this one-drug one-assay screening strategy can be significantly improved. Pooling designs are testing schemes that test mixtures of compounds in each assay, thereby generating a screen of the whole compound library in fewer tests. By repeatedly testing compounds in different combinations, pooling designs also allow for error-correction. These pooled designs, for specific experiment parameters, can be simply and efficiently created using the Shifted Transversal Design (STD pooling algorithm. However, drug screening contains a number of key constraints that require specific modifications if this pooling approach is to be useful for practical screen designs. Results In this paper, we introduce a pooling strategy called poolHiTS (Pooled High-Throughput Screening which is based on the STD algorithm. In poolHiTS, we implement a limit on the number of compounds that can be mixed in a single assay. In addition, we show that the STD-based pooling strategy is limited in the error-correction that it can achieve. Due to the mixing constraint, we show that it is more efficient to split a large library into smaller blocks of compounds, which are then tested using an optimized strategy repeated for each block. We package the optimal block selection algorithm into poolHiTS. The MATLAB codes for the poolHiTS algorithm and the corresponding decoding strategy are also provided. Conclusion We have produced a practical version

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

    Directory of Open Access Journals (Sweden)

    Windy A Boyd

    2007-12-01

    Full Text Available 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.Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants, and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50 of 2 microM.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

  18. Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

    Science.gov (United States)

    Tang, Huaping; Shen, Ding Ren; Han, Yong-Hae; Kong, Yan; Balimane, Praveen; Marino, Anthony; Gao, Mian; Wu, Sophie; Xie, Dianlin; Soars, Matthew G; O'Connell, Jonathan C; Rodrigues, A David; Zhang, Litao; Cvijic, Mary Ellen

    2013-10-01

    Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

  19. DNA Comet Assay. A simple screening technique for identification of some irradiated foods

    International Nuclear Information System (INIS)

    Khan, A.A.; Khan, H.M.

    2008-01-01

    DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test. (author)

  20. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis.

    Science.gov (United States)

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-07-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive samples, 704 fresh syphilis-negative samples, 48 preselected potentially interfering samples, and 30 "borderline" samples and was compared head to head with the commercially available Lumipulse G TP-N. In this study, the HISCL anti-TP assay was in perfect agreement with the applied testing algorithms with an overall agreement of 100%, comparable to that of Lumipulse G TP-N (99.63%). The sensitivity and specificity of the HISCL anti-TP assay were 100% (95% confidence interval [CI], 98.42% to 100%) and 100% (95% CI, 99.37% to 100%), respectively. Considering the excellent ease of use and automation, high throughput, and its favorable sensitivity and specificity, the HISCL anti-TP assay may represent a new choice for syphilis screening in high-volume laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

    Directory of Open Access Journals (Sweden)

    Jonathan A Lee

    Full Text Available Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS pharmaceutical collection (NPC is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/ (AID 1117321. Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

  2. Evaluation of e-liquid toxicity using an open-source high-throughput screening assay

    Science.gov (United States)

    Keating, James E.; Zorn, Bryan T.; Kochar, Tavleen K.; Wolfgang, Matthew C.; Glish, Gary L.; Tarran, Robert

    2018-01-01

    The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography–mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition. PMID:29584716

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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. (paper)

  4. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  5. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2015-08-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

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

    Science.gov (United States)

    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)

  7. Towards novel therapeutics for HIV through fragment-based screening and drug design.

    Science.gov (United States)

    Tiefendbrunn, Theresa; Stout, C David

    2014-01-01

    Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

  8. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

    In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30-65 years in Copenhagen, Denmark....... Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected...... more ≥CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results...

  9. Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

    Science.gov (United States)

    Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

    2013-06-28

    Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

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

    Science.gov (United States)

    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

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Orbitrap technology for comprehensive metabolite-based liquid chromatographic–high resolution-tandem mass spectrometric urine drug screening – Exemplified for cardiovascular drugs

    International Nuclear Information System (INIS)

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

    2015-01-01

    LC–high resolution (HR)-MS well established in proteomics has become more and more important in bioanalysis of small molecules over the last few years. Its high selectivity and specificity provide best prerequisites for its use in broad screening approaches. Therefore, Orbitrap technology was tested for developing a general metabolite-based LC–HR-MS/MS screening approach for urinalysis of drugs necessary in clinical and forensic toxicology. After simple urine precipitation, the drugs and their metabolites were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan with positive/negative switching, and subsequent data dependent acquisition (DDA) mode. Identification criteria were the presence of accurate precursor ions, isotopic patterns, five most intense fragment ions, and comparison with full HR-MS/MS library spectra. The current library contains over 1900 parent drugs and 1200 metabolites. The method was validated for typical drug representatives and metabolites concerning recovery, matrix effects, process efficiency, and limits showed acceptable results. The applicability was tested first for cardiovascular drugs, which should be screened for in poisoning cases and for medication adherence of hypertension patients. The novel LC–HR-MS/MS method allowed fast, simple, and robust urine screening, particularly for cardiovascular drugs showing the usefulness of Orbitrap technology for drug testing. - Highlights: • First study on the application of Orbitrap technology for comprehensive drug screening in clinical and forensic toxicology. • Simple workup, sufficient separation, and powerful screening and identification using modern high resolution MS. • Validation of the assay according to guidelines for qualitative approaches. • Elucidation of the power of new data evaluation software in combination with a new reference drug and metabolite library. • Great relevance for science and practice in clinical and forensic

  12. Orbitrap technology for comprehensive metabolite-based liquid chromatographic–high resolution-tandem mass spectrometric urine drug screening – Exemplified for cardiovascular drugs

    Energy Technology Data Exchange (ETDEWEB)

    Helfer, Andreas G.; Michely, Julian A.; Weber, Armin A.; Meyer, Markus R.; Maurer, Hans H., E-mail: hans.maurer@uks.eu

    2015-09-03

    LC–high resolution (HR)-MS well established in proteomics has become more and more important in bioanalysis of small molecules over the last few years. Its high selectivity and specificity provide best prerequisites for its use in broad screening approaches. Therefore, Orbitrap technology was tested for developing a general metabolite-based LC–HR-MS/MS screening approach for urinalysis of drugs necessary in clinical and forensic toxicology. After simple urine precipitation, the drugs and their metabolites were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan with positive/negative switching, and subsequent data dependent acquisition (DDA) mode. Identification criteria were the presence of accurate precursor ions, isotopic patterns, five most intense fragment ions, and comparison with full HR-MS/MS library spectra. The current library contains over 1900 parent drugs and 1200 metabolites. The method was validated for typical drug representatives and metabolites concerning recovery, matrix effects, process efficiency, and limits showed acceptable results. The applicability was tested first for cardiovascular drugs, which should be screened for in poisoning cases and for medication adherence of hypertension patients. The novel LC–HR-MS/MS method allowed fast, simple, and robust urine screening, particularly for cardiovascular drugs showing the usefulness of Orbitrap technology for drug testing. - Highlights: • First study on the application of Orbitrap technology for comprehensive drug screening in clinical and forensic toxicology. • Simple workup, sufficient separation, and powerful screening and identification using modern high resolution MS. • Validation of the assay according to guidelines for qualitative approaches. • Elucidation of the power of new data evaluation software in combination with a new reference drug and metabolite library. • Great relevance for science and practice in clinical and forensic

  13. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    OpenAIRE

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

  14. A new in vivo screening paradigm to accelerate antimalarial drug discovery.

    Directory of Open Access Journals (Sweden)

    María Belén Jiménez-Díaz

    Full Text Available The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR, which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0 of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1 or induce parasite clearance (PRR >1 with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a

  15. A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery

    Science.gov (United States)

    Jiménez-Díaz, María Belén; Viera, Sara; Ibáñez, Javier; Mulet, Teresa; Magán-Marchal, Noemí; Garuti, Helen; Gómez, Vanessa; Cortés-Gil, Lorena; Martínez, Antonio; Ferrer, Santiago; Fraile, María Teresa; Calderón, Félix; Fernández, Esther; Shultz, Leonard D.; Leroy, Didier; Wilson, David M.; García-Bustos, José Francisco; Gamo, Francisco Javier; Angulo-Barturen, Iñigo

    2013-01-01

    The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task

  16. Sample preparation composite and replicate strategy for assay of solid oral drug products.

    Science.gov (United States)

    Harrington, Brent; Nickerson, Beverly; Guo, Michele Xuemei; Barber, Marc; Giamalva, David; Lee, Carlos; Scrivens, Garry

    2014-12-16

    In pharmaceutical analysis, the results of drug product assay testing are used to make decisions regarding the quality, efficacy, and stability of the drug product. In order to make sound risk-based decisions concerning drug product potency, an understanding of the uncertainty of the reportable assay value is required. Utilizing the most restrictive criteria in current regulatory documentation, a maximum variability attributed to method repeatability is defined for a drug product potency assay. A sampling strategy that reduces the repeatability component of the assay variability below this predefined maximum is demonstrated. The sampling strategy consists of determining the number of dosage units (k) to be prepared in a composite sample of which there may be a number of equivalent replicate (r) sample preparations. The variability, as measured by the standard error (SE), of a potency assay consists of several sources such as sample preparation and dosage unit variability. A sampling scheme that increases the number of sample preparations (r) and/or number of dosage units (k) per sample preparation will reduce the assay variability and thus decrease the uncertainty around decisions made concerning the potency of the drug product. A maximum allowable repeatability component of the standard error (SE) for the potency assay is derived using material in current regulatory documents. A table of solutions for the number of dosage units per sample preparation (r) and number of replicate sample preparations (k) is presented for any ratio of sample preparation and dosage unit variability.

  17. Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

    International Nuclear Information System (INIS)

    Ravanshad, M.; Sabahi, F.; Mahboudi, F.; Sabahi, F.

    2006-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2). However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four (12.8%) patients tested for HIV were positive for HIV-1 antibody. Nine (1.49%) have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention (CDC) criteria. Most (66.7%) of these indeterminate WB results were due to p24 reactivity. However, 2(22.2%) display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization (WHO) criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples 8(88.8%) became negative when retested subsequently while one (11.1%) remained indeterminate for more than a year and were thus considered negative. In addition all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there were was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting. (author)

  18. Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

    Science.gov (United States)

    Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

    2015-03-01

    The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

  19. Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Tolosa, Laia [Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); Gómez-Lechón, M. José [Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); CIBERehd, FIS, Barcelona 08036 (Spain); Jiménez, Nuria [Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); Hervás, David [Biostatistics Unit, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); Jover, Ramiro [Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); CIBERehd, FIS, Barcelona 08036 (Spain); Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia 46010 (Spain); Donato, M. Teresa, E-mail: donato_mte@gva.es [Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia 46026 (Spain); CIBERehd, FIS, Barcelona 08036 (Spain); Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia 46010 (Spain)

    2016-07-01

    Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis. - Highlights: • HepaRG cells were explored as an in vitro model to detect steatogenic potential. • Multiple toxicity-related endpoints were analysed by HCS. • HepaRG showed a greater sensitivity to drug-induced steatosis than HepG2 cells. • Changes in the expression of genes related to lipid metabolism were revealed. • HepaRG allow mechanistic understanding of liver damage induced by steatogenic drugs.

  20. Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis

    International Nuclear Information System (INIS)

    Tolosa, Laia; Gómez-Lechón, M. José; Jiménez, Nuria; Hervás, David; Jover, Ramiro; Donato, M. Teresa

    2016-01-01

    Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis. - Highlights: • HepaRG cells were explored as an in vitro model to detect steatogenic potential. • Multiple toxicity-related endpoints were analysed by HCS. • HepaRG showed a greater sensitivity to drug-induced steatosis than HepG2 cells. • Changes in the expression of genes related to lipid metabolism were revealed. • HepaRG allow mechanistic understanding of liver damage induced by steatogenic drugs.

  1. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Science.gov (United States)

    van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

    2015-01-01

    The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

  2. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Directory of Open Access Journals (Sweden)

    René van der Ploeg

    2015-07-01

    Full Text Available The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl isothiourea (A22 or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.

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

    Science.gov (United States)

    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.

  4. Drug abuse in the workplace: employee screening techniques

    International Nuclear Information System (INIS)

    Buzzeo, R.W.

    1984-01-01

    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

  5. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Johnson

    Full Text Available BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

  6. Miniature short hairpin RNA screens to characterize antiproliferative drugs.

    Science.gov (United States)

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

    2013-08-07

    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

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

    International Nuclear Information System (INIS)

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

    2011-01-01

    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.

  8. Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

    2017-08-12

    Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

  9. Sample preparation composite and replicate strategy case studies for assay of solid oral drug products.

    Science.gov (United States)

    Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei

    2017-11-30

    Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Systematic comparison of drug-tolerant assays for anti-drug antibodies in a cohort of adalimumab-treated rheumatoid arthritis patients

    NARCIS (Netherlands)

    Bloem, Karien; van Leeuwen, Astrid; Verbeek, Gerrit; Nurmohamed, Michael T.; Wolbink, Gerrit Jan; van der Kleij, Desiree; Rispens, Theo

    2015-01-01

    Drug interference complicates assessment of immunogenicity of biologicals and results in an underestimation of anti-drug antibody (ADA) formation. Drug-tolerant assays have the potential to overcome such limitations. However, to which extent drug-tolerant assays provide an unbiased picture of the

  11. A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

    Science.gov (United States)

    Röhm, Martina; Carle, Stefan; Maigler, Frank; Flamm, Johannes; Kramer, Viktoria; Mavoungou, Chrystelle; Schmid, Otmar; Schindowski, Katharina

    2017-10-30

    Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  12. A Novel Complementation Assay for Quick and Specific Screen of Genes Encoding Glycerol-3-Phosphate Acyltransferases

    Directory of Open Access Journals (Sweden)

    Jie Lei

    2018-03-01

    Full Text Available The initial step in glycerolipid biosynthesis, especially in diverse allopolyploid crop species, is poorly understood, mainly due to the lack of an effective and convenient method for functional characterization of genes encoding glycerol-3-phosphate acyltransferases (GPATs catalyzing this reaction. Here we present a novel complementation assay for quick and specific characterization of GPAT-encoding genes. Its key design involves rational construction of yeast conditional lethal gat1Δgat2Δ double mutant bearing the heterologous Arabidopsis AtGPAT1 gene whose leaky expression under repressed conditions does not support any non-specific growth, thereby circumventing the false positive problem encountered with the system based on the gat1Δgat2Δ mutant harboring the native episomal GAT1 gene whose leaky expression appears to be sufficient for generating enough GPAT activities for the non-specific restoration of the mutant growth. A complementation assay developed based on this novel mutant enables quick phenotypic screen of GPAT sequences. A high degree of specificity of our assay was exemplified by its ability to differentiate effectively GPAT-encoding genes from those of other fatty acyltransferases and lipid-related sequences. Using this assay, we show that Arabidopsis AtGPAT1, AtGPAT5, and AtGPAT7 can complement the phosphatidate biosynthetic defect in the double mutants. Collectively, our assay provides a powerful tool for rapid screening, validation and optimization of GPAT sequences, aiding future engineering of the initial step of the triacylglycerol biosynthesis in oilseeds.

  13. Detection of systemic hypersensitivity to drugs using standard guinea pig assays.

    Science.gov (United States)

    Weaver, James L; Staten, David; Swann, Joslyn; Armstrong, George; Bates, Melissa; Hastings, Kenneth L

    2003-12-01

    The most commonly used assays designed to detect either skin or systemic immune-based hypersensitivity reactions are those using guinea pigs (GP). We obtained data from various FDA records to evaluate the correlation between GP assay results and reported post-marketing systemic hypersensitivity reactions. We examined the new drug application (NDA) reviews of approved drugs for the results of GP assays. Post-marketing human data were extracted from the FDA adverse event reporting system (AERS). Drug usage data were obtained from a commercial database maintained by IMS Health Inc. We found 83 (21%) of 396 drugs approved between 1978 and 1998 had reported GP test results. Among these 83 drugs, 14 (17%) were found to have positive results in at least one GP assay. Simple reporting index (RI) values for systemic hypersensitivity reactions were calculated from AERS data and usage to produce the index of adverse event reports per million shipping units of drug. A variety of definitions of positive human response were examined. A statistically significant association was seen for rash between post-marketing and clinical trials adverse event reports. No statistically significant associations between human data and GP test results were observed. These data suggest that standard GP assays have limited ability to predict human systemic hypersensitivity potential for pharmaceuticals.

  14. Congestion game scheduling for virtual drug screening optimization

    Science.gov (United States)

    Nikitina, Natalia; Ivashko, Evgeny; Tchernykh, Andrei

    2018-02-01

    In virtual drug screening, the chemical diversity of hits is an important factor, along with their predicted activity. Moreover, interim results are of interest for directing the further research, and their diversity is also desirable. In this paper, we consider a problem of obtaining a diverse set of virtual screening hits in a short time. To this end, we propose a mathematical model of task scheduling for virtual drug screening in high-performance computational systems as a congestion game between computational nodes to find the equilibrium solutions for best balancing the number of interim hits with their chemical diversity. The model considers the heterogeneous environment with workload uncertainty, processing time uncertainty, and limited knowledge about the input dataset structure. We perform computational experiments and evaluate the performance of the developed approach considering organic molecules database GDB-9. The used set of molecules is rich enough to demonstrate the feasibility and practicability of proposed solutions. We compare the algorithm with two known heuristics used in practice and observe that game-based scheduling outperforms them by the hit discovery rate and chemical diversity at earlier steps. Based on these results, we use a social utility metric for assessing the efficiency of our equilibrium solutions and show that they reach greatest values.

  15. Comparison of RAW264.7, human whole blood and PBMC assays to screen for immunomodulators.

    Science.gov (United States)

    Elisia, Ingrid; Pae, Han Bee; Lam, Vivian; Cederberg, Rachel; Hofs, Elyse; Krystal, Gerald

    2018-01-01

    The RAW264.7 mouse macrophage cell line is used extensively to carry out in vitro screens for immunomodulators. Compounds that are effective at reducing the expression of pro-inflammatory cytokines or nitric oxide (NO) from lipopolysaccharide (LPS)-stimulated RAW264.7 cells are often considered candidate anti-inflammatory agents for humans. There is, however, very little data on the reliability of this screen to identify bona fide human immunomodulators. We compared the efficacy of 37 purported immunomodulators to modulate LPS or E. coli-induced inflammatory responses in RAW264.7 cell, whole human blood and human peripheral blood mononuclear cell (PBMC) assays. Interestingly, there was no significant correlation (R=0.315) between the responses obtained with RAW264.7 cells and the whole blood assay (WBA), suggesting that compounds demonstrating efficacy in RAW264.7 cells may be ineffective in humans, and, more importantly, compounds that are effective in humans may be missed with a RAW264.7 screen. Interestingly, there was also no significant correlation between the WBA and human PBMCs when the latter were cultured with 10% FCS, but a moderate correlation was seen when the PBMCs were cultured with 25% autologous plasma. The presence of plasma thus contributes to the overall inflammatory response observed in the WBA. We then asked if RAW264.7 cells, given that they are mouse macrophage-like cells, respond in a manner similar to primary murine derived macrophages. Intriguingly, there was no significant correlation (R=0.012) with the 37 putative immunomodulators, pointing to distinct inflammatory response mechanisms in the two model systems. We conclude that the use of a WBA to confirm potential immunomodulators obtained from high throughput screening with RAW264.7 cells is advisable and that future screens be carried out using a WBA. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    Science.gov (United States)

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

  17. A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

    Science.gov (United States)

    Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

    2017-07-01

    Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.

    Science.gov (United States)

    Ishida, Akihiko; Yamada, Yasuko; Kamidate, Tamio

    2008-11-01

    In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

  19. The trade-off between accuracy and accessibility of syphilis screening assays.

    Directory of Open Access Journals (Sweden)

    Pieter W Smit

    Full Text Available The availability of rapid and sensitive methods to diagnose syphilis facilitates screening of pregnant women, which is one of the most cost-effective health interventions available. We have evaluated two screening methods in Tanzania: an enzyme immunoassay (EIA, and a point-of-care test (POCT. We evaluated the performance of each test against the Treponema pallidum particle agglutination assay (TPPA as the reference method, and the accessibility of testing in a rural district of Tanzania. The POCT was performed in the clinic on whole blood, while the other assays were performed on plasma in the laboratory. Samples were also tested by the rapid plasma Reagin (RPR test. With TPPA as reference assay, the sensitivity and specificity of EIA were 95.3% and 97.8%, and of the POCT were 59.6% and 99.4% respectively. The sensitivity of the POCT and EIA for active syphilis cases (TPPA positive and RPR titer ≥ 1/8 were 82% and 100% respectively. Only 15% of antenatal clinic attenders in this district visited a health facility with a laboratory capable of performing the EIA. Although it is less sensitive than EIA, its greater accessibility, and the fact that treatment can be given on the same day, means that the use of POCT would result in a higher proportion of women with syphilis receiving treatment than with the EIA in this district of Tanzania.

  20. Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

    Science.gov (United States)

    Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

    2013-05-13

    Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

  1. Reliability, Validity and Factor Structure of Drug Abuse Screening Test

    Directory of Open Access Journals (Sweden)

    Sayed Hadi Sayed Alitabar

    2016-05-01

    Full Text Available Background and Objective: According to the increasing of substance use in the country, more researches about this phenomenon are necessary. This Study Investigates the Validity, Reliability and Confirmatory Factor Structure of the Drug Abuse Screening test (DAST. Materials and Methods: The Sample Consisted of 381 Patients (143 Women and 238 Men with a Multi-Stage Cluster Sampling of Areas 2, 6 and 12 of Tehran Were Selected from Each Region, 6 Randomly Selected Drug Rehabilitation Center. The DAST Was Used as Instrument. Divergent & Convergent Validity of this Scale Was Assessed with Problems Assessment for Substance Using Psychiatric Patients (PASUPP and Relapse Prediction Scale (RPS.Results: The DAST after the First Time Factor Structure of Using Confirmatory Factor Analysis Was Confirmed. The DAST Had a Good Internal Consistency (Cranach’s Alpha, and the Reliability of the Test Within a Week, 0.9, 0.8. Also this Scale Had a Positive Correlation with Problems Assessment for Substance Using Psychiatric Patients and Relapse Prediction Scale (P<0.01.Conclusion: The Overall Results Showed that the Drug Abuse Screening Test in Iranian Society Is Valid. It Can Be Said that Self-Report Scale Tool Is Useful for Research Purposes and Addiction.

  2. Screening Drug, Alcohol and Substance Abuse the Psychometric Measures

    Directory of Open Access Journals (Sweden)

    Othman Mohamad Hashim

    2018-01-01

    Full Text Available Urinalysis was used in previous studies among higher institution students (n=16252 in Malaysia to answer the question of whether university students are involved in drug abuse. However, the use of urinalysis had faced some problems. The problems were related to human rights issues and the cost to perform the urinalysis was expensive and quite impossible to be implemented to a large population of university students. To overcome this problem, this study was conducted to examine the effectiveness of psychometric measures in screening drug, alcohol and substance abuse. The Substance Abuse Subtle Screening Inventory A2 (SASSI-A2 was used for this purpose. SASSI-A2 is a brief screening tool designed to identify individuals who have a high probability of having a substance use disorder, including both substance abuse and substance dependence. SASSI-A2 comprises of 72 items that are rated on a two point scale with response; true and false. SASSI-A2 was translated into Malay language and it was refined through a back-translation technique and focus group approach. Psychometric testing was undertaken on a sample of 750 university students from five public universities in Malaysia. All participants were aged between 19 and 20 years. Internal consistency coefficients were calculated for the total scale and its subscales. Chronbach's alpha obtained for SASSI-A2 was 0.72. This relatively high level of Chronbach's alpha showed relatively high level of reliability. The results demonstrated that the whole SASSI-A2 meets the fundamental measurement properties and can discriminate groups of higher institution students from high to low on the substance dependency variable. The accuracy of the test has been found to be unaffected by gender, ethnicity, age and years of education. Although more rigorous validation studies are needed, it is recommended that SASSI-A2 be considered for usage to higher institution students populations when a brief, objective, and

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

    Directory of Open Access Journals (Sweden)

    Landry Losi

    2010-08-01

    Full Text Available Abstract 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 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.

  4. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Ting Xiao

    2016-10-01

    Full Text Available Eukaryotic elongation factor 2 kinase (eEF2K inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

  6. Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities

    DEFF Research Database (Denmark)

    Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B.

    2017-01-01

    Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any...... associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents...... including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong...

  7. Yeast Estrogen Screen Assay as a Tool for Detecting Estrogenic Activity in Water Bodies

    Directory of Open Access Journals (Sweden)

    Mirjana Bistan

    2012-01-01

    Full Text Available The presence of endocrine-disrupting compounds in wastewater, surface water, groundwater and even drinking water has become a major concern worldwide, since they negatively affect wildlife and humans. Therefore, these substances should be effectively removed from effluents before they are discharged into surface water to prevent pollution of groundwater, which can be a source of drinking water. Furthermore, an efficient control of endocrine-disrupting compounds in wastewater based on biological and analytical techniques is required. In this study, a yeast estrogen screen (YES bioassay has been introduced and optimized with the aim to assess potential estrogenic activity of waters. First, assay duration, concentration of added substrate to the assay medium and wavelength used to measure the absorbance of the substrate were estimated. Several compounds, such as 17-β-estradiol, 17-α-ethinylestradiol, bisphenol A, nonylphenol, genisteine, hydrocortisone, dieldrin, atrazine, methoxychlor, testosterone and progesterone were used to verify its specificity and sensitivity. The optimized YES assay was sensitive and responded specifically to the selected estrogenic and nonestrogenic compounds in aqueous samples. Potential estrogenicity of influent and effluent samples of two wastewater treatment plants was assessed after the samples had been concentrated by solid-phase extraction (SPE procedure using Oasis® HLB cartridges and methanol as eluting solvent. Up to 90 % of relative estrogenic activity was detected in concentrated samples of influents to wastewater treatment plants and estrogenic activity was still present in the concentrated effluent samples. We found that the introduced YES assay is a suitable screening tool for monitoring the potential estrogenicity of effluents that are discharged into surface water.

  8. The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

    Science.gov (United States)

    Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

    2016-02-01

    Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, PLumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Comparative analysis of three drug-drug interaction screening systems against probable clinically relevant drug-drug interactions: a prospective cohort study.

    Science.gov (United States)

    Muhič, Neža; Mrhar, Ales; Brvar, Miran

    2017-07-01

    Drug-drug interaction (DDI) screening systems report potential DDIs. This study aimed to find the prevalence of probable DDI-related adverse drug reactions (ADRs) and compare the clinical usefulness of different DDI screening systems to prevent or warn against these ADRs. A prospective cohort study was conducted in patients urgently admitted to medical departments. Potential DDIs were checked using Complete Drug Interaction®, Lexicomp® Online™, and Drug Interaction Checker®. The study team identified the patients with probable clinically relevant DDI-related ADRs on admission, the causality of which was assessed using the Drug Interaction Probability Scale (DIPS). Sensitivity, specificity, and positive and negative predictive values of screening systems to prevent or warn against probable DDI-related ADRs were evaluated. Overall, 50 probable clinically relevant DDI-related ADRs were found in 37 out of 795 included patients taking at least two drugs, most common of them were bleeding, hyperkalemia, digitalis toxicity, and hypotension. Complete Drug Interaction showed the best sensitivity (0.76) for actual DDI-related ADRs, followed by Lexicomp Online (0.50), and Drug Interaction Checker (0.40). Complete Drug Interaction and Drug Interaction Checker had positive predictive values of 0.07; Lexicomp Online had 0.04. We found no difference in specificity and negative predictive values among these systems. DDI screening systems differ significantly in their ability to detect probable clinically relevant DDI-related ADRs in terms of sensitivity and positive predictive value.

  10. Focused use of drug screening in overdose patients increases impact on management.

    OpenAIRE

    Erdmann, A.; Werner, D.; Hugli, O.; Yersin, B.

    2015-01-01

    UNLABELLED: Drug poisoning is a common cause for attendance in the emergency department. Several toxicology centres suggest performing urinary drug screens, even though they rarely influence patient management. STUDY OBJECTIVES: Measuring the impact on patient management, in a University Emergency Department with approximately 40 000 admissions annually, of a rapid urinary drug screening test using specifically focused indications. Drug screening was restricted to patients having a first p...

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

    Science.gov (United States)

    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

  12. A systematic screen of FDA-approved drugs for inhibitors of biological threat agents.

    Directory of Open Access Journals (Sweden)

    Peter B Madrid

    Full Text Available 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.

  13. Assessment of tobramycin RIA for drug monitoring and dosage regimen. Comparison with other assay technics

    International Nuclear Information System (INIS)

    Takahashi, S.; Shinozaki, K.; Tsujino, D.; Ohhara, H.; Tanaka, Y.; Arai, S.; Someya, K.; Sasaki, Y.

    1983-01-01

    Because of wide range of inter-individual difference of pharmacokinetic parameter, importance of monitoring blood concentration of aminoglycoside antibiotics in each patient has been recognized. With the purpose to use for monitoring of serum tobramycin (TOB) levels and for adequate dosage regimen RIA of TOB was evaluated in comparison with other assay technics. Gamma Coat TOB RIA kits (Clinical Assay-Travenol Japan) were used for RIA of TOB. The TOB concentrations in the same samples were also measured by two kinds of enzyme immunoassay (EIA) (EMIT EIA and SLFIA EIA), High Performance Liquid Chromatography (HPLC) and bioassay (BA). RIA of TOB is a useful assay method with high sensitivity and reasonably good precision to be used for drug monitoring and adequate dosage regimen. Modification of the method for rapid assay of a small number of samples will increase the clinical usefulness in individualized drug monitoring

  14. Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme

    NARCIS (Netherlands)

    Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.

    2005-01-01

    A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional

  15. Complementing in vitro screening assays with in silico molecular chemistry tools to examine potential in vivo metabolite-mediated effects

    Science.gov (United States)

    High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive un...

  16. Screening applications in drug discovery based on microfluidic technology.

    Science.gov (United States)

    Eribol, P; Uguz, A K; Ulgen, K O

    2016-01-01

    Microfluidics has been the focus of interest for the last two decades for all the advantages such as low chemical consumption, reduced analysis time, high throughput, better control of mass and heat transfer, downsizing a bench-top laboratory to a chip, i.e., lab-on-a-chip, and many others it has offered. Microfluidic technology quickly found applications in the pharmaceutical industry, which demands working with leading edge scientific and technological breakthroughs, as drug screening and commercialization are very long and expensive processes and require many tests due to unpredictable results. This review paper is on drug candidate screening methods with microfluidic technology and focuses specifically on fabrication techniques and materials for the microchip, types of flow such as continuous or discrete and their advantages, determination of kinetic parameters and their comparison with conventional systems, assessment of toxicities and cytotoxicities, concentration generations for high throughput, and the computational methods that were employed. An important conclusion of this review is that even though microfluidic technology has been in this field for around 20 years there is still room for research and development, as this cutting edge technology requires ingenuity to design and find solutions for each individual case. Recent extensions of these microsystems are microengineered organs-on-chips and organ arrays.

  17. Screening applications in drug discovery based on microfluidic technology

    Science.gov (United States)

    Eribol, P.; Uguz, A. K.; Ulgen, K. O.

    2016-01-01

    Microfluidics has been the focus of interest for the last two decades for all the advantages such as low chemical consumption, reduced analysis time, high throughput, better control of mass and heat transfer, downsizing a bench-top laboratory to a chip, i.e., lab-on-a-chip, and many others it has offered. Microfluidic technology quickly found applications in the pharmaceutical industry, which demands working with leading edge scientific and technological breakthroughs, as drug screening and commercialization are very long and expensive processes and require many tests due to unpredictable results. This review paper is on drug candidate screening methods with microfluidic technology and focuses specifically on fabrication techniques and materials for the microchip, types of flow such as continuous or discrete and their advantages, determination of kinetic parameters and their comparison with conventional systems, assessment of toxicities and cytotoxicities, concentration generations for high throughput, and the computational methods that were employed. An important conclusion of this review is that even though microfluidic technology has been in this field for around 20 years there is still room for research and development, as this cutting edge technology requires ingenuity to design and find solutions for each individual case. Recent extensions of these microsystems are microengineered organs-on-chips and organ arrays. PMID:26865904

  18. Newborn Congenital Cytomegalovirus Screening Based on Clinical Manifestations and Evaluation of DNA-based Assays for In Vitro Diagnostics.

    Science.gov (United States)

    Fujii, Tomoyuki; Oka, Akira; Morioka, Ichiro; Moriuchi, Hiroyuki; Koyano, Shin; Yamada, Hideto; Saito, Shigeru; Sameshima, Hiroshi; Nagamatsu, Takeshi; Tsuchida, Shinya; Inoue, Naoki

    2017-10-01

    To establish a strategy for congenital cytomegalovirus (cCMV) screening and to establish confirmatory assays approved as in vitro diagnostics by the regulatory authorities, we evaluated the clinical risks and performance of diagnostic assays developed by commercial companies, since cCMV infection has significant clinical consequences. Newborns with clinical manifestations considered to be consequences of cCMV infection (n = 575) were screened for the presence of cytomegalovirus (CMV) DNA in urine specimens collected onto filter paper placed in their diapers using the polymerase chain reaction-based assay reported previously. Liquid urine specimens were obtained from all of 20 CMV-positive newborns and 107 of the CMV-negative newborns identified in the screening. We used these 127 specimens, as well as 12 from cCMV cases identified in a previous study and 41 from healthy newborns, to compare the performance of 2 commercial assays and 1 in-house assay. The risk-based screening allowed the identification of cCMV cases at least 10-fold more efficiently than our previous universal screening, although there appears to be a limit to the identification of asymptomatically infected newborns. Although CMV-specific IgM during pregnancy was found frequently in mothers of cCMV newborns, CMV-IgM alone is not an effective diagnostic marker. The urine-filter-based assay and the 3 diagnostic assays yielded identical results. Although risk-based and universal newborn screening strategies for cCMV infection each have their respective advantages and disadvantages, urine-filter-based assay followed by confirmatory in vitro diagnostics assays is able to identify cCMV cases efficiently.

  19. Design of a micro colorimetric enzyme assay for screening of radioprotectors using the oriental fruit fly Bactrocera Philippine's Model

    International Nuclear Information System (INIS)

    Deocaris, Custer C.; Nato, Jr. Alejandro Q.; Dacanay, Elena T.; Marcelo, Samantha C.; Buenaventura, Dyan M.

    1998-01-01

    Loss of function and expression of a 109kDa protein is observed in the oriental fruit fly, Bactrocera philippinensis, upon exposure to a γ-radiation dose of 100 Gy. Found to possess tyrosinase activity, this marker enzyme is particularly important during quarantine treatment of export fruits. A semi-automated radioprotector screening assay for anti-cancer drug development at PNRI has been developed and optimized. Larvae of B.philippinensis are subjected to relatively high and low doses of standard radioprotectors (L-glutathione (GSH), tert-butylated hydroxyanisole (BHA), garlic bulb extracts), temperature treatments (37 degrees centegrade and 42 degrees centegrade) and relatively high and low radiation doses (10 and 40 Gy) following a 2-factorial design. Using mushroom tyrosinase as standard and 605 nm as reference wavelength, optimum precision, sensitivity and curve linearity are achieved at the 405 nm window within 60-minute reaction time with 2-methyl DOPA yielding dopachrome. Significant radioprotection and tyrosinase activity are observed. Results showed that GSH exhibited the best radioprotection with an emergence rate of 100% (GSHη 42 degrees10). Consequently, GSHη exhibited a high dopachrome level next to garlicη. Garlic approximates the performance of GSH and BHA, but the fact that dopachrome levels or garlich are exceeding high could be correlated with the relatively lower emergence rates observed. Dopachrome level of 0.45-005μg/ml exhibits the optimal radioprotection.Other radioprotectors will be screened in the future using this assay in search of potent and less toxic radioprotectors that could decrease radiation-induced morbidities and improve therapeutic gains in patients undergoing therapy. (Author)

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

    Directory of Open Access Journals (Sweden)

    Thomas J.J. Gintjee

    2014-11-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2014-11-14

    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.

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

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Asther Marcel

    2010-07-01

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

  4. Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

    Science.gov (United States)

    Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

    2017-06-01

    Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

  5. Characterization analysis for leaves of Leucaena leucocephala by using phytochemical screening assay

    Science.gov (United States)

    Zarina, Z.; Ghazali, C. M. R.; Sam, S. T.

    2017-09-01

    Leucaena Leucocephala (Lam.) de Wit (Petai Belalang) is a medium plant which belong in group of tropical breed that can survived in hot, dried and warm environment. In Malaysia, the plant is available abundantly. As there are still no commercial used, and no serious intention in finding the benefits of L. Leucocephala, this work come out with the idea to analyze the antioxidants contains in leaves of the plant by undergoes different extraction and chemical testing method. The phytochemical screening assay involved in this study are antioxidant activity by using free radical diphenylpicrylhydrazyl (DPPH) method, total phenolic content by using Folin-Ciocalteu method, total flavonoid content by using colorimetric assay with ascorbic acid and quercetin were used as reference standards while for phosphorus analysis, a molybdenum blue method or also known as ascorbic acid method was used. For antioxidant activity by using free radical diphenylpicrylhydrazyl (DPPH) method, higher concentration was recorded by extraction using methanol (dried sample) which is 8247.0 mg/L, for total phenolic content higher concentration was recorded by extraction using deionized water (dried sample) which is 4276.0 mg/L, for total flavonoid content by using colorimetric assay higher concentration was recorded by extraction using methanol (dried sample) which is 4439.0 mg/L, and for for phosphorus analysis higher concentration was recorded by extraction using methanol (dried sample) which is 71.057 mg/L.

  6. Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

    Science.gov (United States)

    Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B; Papamarkou, Theodore; Huber, Kilian V M; Mutz, Cornelia; Toretsky, Jeffrey A; Bennett, Keiryn L; Olsen, Jesper V; Brunak, Søren; Kovar, Heinrich; Superti-Furga, Giulio

    2017-01-01

    Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR. ©2016 American Association for Cancer Research.

  7. Outreach screening of drug users for cirrhosis with transient elastography

    DEFF Research Database (Denmark)

    Moessner, Belinda K; Jørgensen, Tina R; Skamling, Merete

    2011-01-01

    Aims  Transient elastography (TE) is a non-invasive sensitive tool for diagnosing cirrhosis in hospital-based cohorts. This study aimed to evaluate TE as a screening tool for cirrhosis among drug users. Design  Cross-sectional study. Setting  All treatment centres in the county of Funen, Denmark....... Participants  Drug users attending treatment centres during the presence of the study team. Measurements  Liver stiffness measurements (LSM) by transient elastography using the Fibroscan device; blood tests for viral hepatitis, HIV infection and hyaluronic acid (HA) levels; and routine liver tests. Individuals...... with LSM ≥ 8 kPa were referred to the hospital for treatment evaluation. Individuals with LSM ≥ 12 kPa were recommended a liver biopsy. Findings  Among 175 drug users negative for hepatitis C, 13% had LSM = 8-11.9 kPa and 4% had LSM ≥ 12 kPa; elevated LSM was associated with a body mass index (BMI) > 30...

  8. Evaluation of the microscopic observational drug susceptibility assay for rapid and efficient diagnosis of multi-drug resistant tuberculosis

    Directory of Open Access Journals (Sweden)

    R P Lazarus

    2012-01-01

    Full Text Available Purpose: Tuberculosis (TB is endemic in India and the burden of multi-drug-resistant tuberculosis (MDR-TB is high. Early detection of MDR-TB is of primary importance in controlling the spread of TB. The microscopic observational drug susceptibility (MODS assay has been described as a cost-effective and rapid method by which mycobacterial culture and the drug susceptibility test (DST can be done at the same time. Materials and Methods: A total of 302 consecutive sputum samples that were received in an accredited mycobacteriology laboratory for conventional culture and DST were evaluated by the MODS assay. Results: In comparison with conventional culture on Lowenstein Jensen (LJ media, the MODS assay showed a sensitivity of 94.12% and a specificity of 89.39% and its concordance with the DST by the proportion method on LJ media to isoniazid and rifampicin was 90.8% and 91.5%, respectively. The turnaround time for results by MODS was 9 days compared to 21 days by culture on LJ media and an additional 42 days for DST by the 1% proportion method. The cost of performing a single MODS assay was Rs. 250/-, compared to Rs. 950/- for culture and 1st line DST on LJ. Conclusion: MODS was found to be a sensitive and rapid alternative method for performing culture and DST to identify MDR-TB in resource poor settings.

  9. A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials

    Science.gov (United States)

    Eleuteri, C.; Olla, S.; Veroni, C.; Umeton, R.; Mechelli, R.; Romano, S.; Buscarinu, Mc.; Ferrari, F.; Calò, G.; Ristori, G.; Salvetti, M.; Agresti, C.

    2017-04-01

    There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds’ activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

  10. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  11. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  12. Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Zor, Kinga; Montini, Lucia

    2015-01-01

    In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, develo...

  13. Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-05-01

    Occupational exposure to antineoplastic drugs is associated with genotoxic effects, although comet assay analyzed parameters were higher in exposed comparing with controls, were not significant. Also the study of the susceptibility biomarkers did not show statistical significant differences, the small size of our sample hampered the finding of a possible association, let alone a causality relationship.

  14. Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

    Science.gov (United States)

    Pulido, Sergio A; Muñoz, Diana L; Restrepo, Adriana M; Mesa, Carol V; Alzate, Juan F; Vélez, Iván D; Robledo, Sara M

    2012-04-01

    Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity

  15. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Directory of Open Access Journals (Sweden)

    Kathleen Ingenhoven

    2017-07-01

    Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

  16. Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

    Science.gov (United States)

    Santillo, Michael F; Mapa, Mapa S T

    2018-02-28

    Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC 50 , 0.4-4.0 ng mL -1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g -1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

  17. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

    Science.gov (United States)

    Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

    2016-01-01

    Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

  18. A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device

    Directory of Open Access Journals (Sweden)

    Jonas Christoffersson

    2018-05-01

    Full Text Available Three-dimensional (3D models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

  19. Cell-patterned glass spray for direct drug assay using mass spectrometry

    International Nuclear Information System (INIS)

    Wu, Jing; Wang, Shiqi; Chen, Qiushui; Jiang, Hao; Liang, Shuping; Lin, Jin-Ming

    2015-01-01

    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R"2 = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  20. Cathepsin D immobilized capillary reactors for on-flow screening assays.

    Science.gov (United States)

    Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

    2018-03-20

    The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Sager J Gosai

    2010-11-01

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

  2. Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches.

    Science.gov (United States)

    Chalasani, Anita; Ji, Kyungmin; Sameni, Mansoureh; Mazumder, Samia H; Xu, Yong; Moin, Kamiar; Sloane, Bonnie F

    2017-01-01

    Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor microenvironment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

  3. Antibody screening by enzyme-linked immunosorbent assay using pooled soluble HLA in renal transplant candidates.

    Science.gov (United States)

    Zaer, F; Metz, S; Scornik, J C

    1997-01-15

    The enzyme-linked immunosorbent assay (ELISA) using HLA class I molecules purified from pooled platelets has the potential to detect HLA antibodies with increased efficiency without sacrificing sensitivity or specificity. This test, which was originally developed in our institution, has been independently validated by recent studies and is now commercially available. We now present evidence of its usefulness as a routine HLA antibody screening test for renal transplant patients. A total of 515 patients were tested monthly by ELISA (13.9 tests/patient) and by antiglobulin-enhanced panel reactivity (6.3 tests/patient). In patients found to be unsensitized, the incidence of false-positive results was less for ELISA than for the panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. Because 66% of our patients were not sensitized, the ELISA was effective in reducing the number of more involved tests aimed at characterizing the antibodies. These results provide a foundation to use the pooled platelet HLA ELISA on a routine basis for HLA antibody screening.

  4. The DNA 'comet assay' as a rapid screening technique to control irradiated food

    International Nuclear Information System (INIS)

    Cerda, H.; Delincee, H.; Haine, H.; Rupp, H.

    1997-01-01

    The exposure of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests, and to extend shelf-life, thereby contributing to a safer and more plentiful food supply. To ensure free consumer choice, irradiated food will be labelled as such, and to enforce labelling, analytical methods to detect the irradiation treatment in the food product itself are desirable. In particular, there is a need for simple and rapid screening methods for the control of irradiated food. The DNA comet assay offers great potential as a rapid tool to detect whether a wide variety of foodstuffs have been radiation processed. In order to simplify the test, the agarose single-layer set-up has been chosen, using a neutral protocol. Interlaboratory blind trials have been successfully carried out with a number of food products, both of animal and plant origin. This paper presents an overview of the hitherto obtained results and in addition the results of an intercomparison test with seeds, dried fruits and spices are described. In this intercomparison, an identification rate of 95% was achieved. Thus, using this novel technique, an effective screening of radiation-induced DNA fragmentation is obtained. Since other food treatments also may cause DNA fragmentation, samples with fragmented DNA suspected to have been irradiated should be analyzed by other validated methods for irradiated food, if such treatments which damage DNA cannot be excluded

  5. A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

    Science.gov (United States)

    Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

    2004-12-01

    With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

  6. Evaluation of a fluorescence-based method for antibabesial drug screening.

    Science.gov (United States)

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

    2014-08-01

    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 (r(2)) 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 IC(50)s determined by the fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) 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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. High-Throughput Screening Using Mass Spectrometry within Drug Discovery.

    Science.gov (United States)

    Rohman, Mattias; Wingfield, Jonathan

    2016-01-01

    In order to detect a biochemical analyte with a mass spectrometer (MS) it is necessary to ionize the analyte of interest. The analyte can be ionized by a number of different mechanisms, however, one common method is electrospray ionization (ESI). Droplets of analyte are sprayed through a highly charged field, the droplets pick up charge, and this is transferred to the analyte. High levels of salt in the assay buffer will potentially steal charge from the analyte and suppress the MS signal. In order to avoid this suppression of signal, salt is often removed from the sample prior to injection into the MS. Traditional ESI MS relies on liquid chromatography (LC) to remove the salt and reduce matrix effects, however, this is a lengthy process. Here we describe the use of RapidFire™ coupled to a triple-quadrupole MS for high-throughput screening. This system uses solid-phase extraction to de-salt samples prior to injection, reducing processing time such that a sample is injected into the MS ~every 10 s.

  8. In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay.

    Science.gov (United States)

    Sankar, Manimuthu Mani; Gopinath, Krishnamoorthy; Singla, Roopak; Singh, Sarman

    2008-07-11

    Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

  9. Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

    Science.gov (United States)

    Larson, Brad; Banks, Peter; Sherman, Hilary; Rothenberg, Mark

    2012-06-01

    Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.

  10. Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

    Science.gov (United States)

    Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

    2018-06-26

    Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

  11. Timing of specimen collection is crucial in urine screening of drug dependent mothers and newborns.

    Science.gov (United States)

    Halstead, A C; Godolphin, W; Lockitch, G; Segal, S

    1988-01-01

    We compared results of urine drug analysis with clinical data and history to test the usefulness of peripartum drug screening and to establish guidelines for optimal testing. Urine from 28 mothers and 52 babies was analysed. Drugs not suspected by history were found in 10 mothers and six babies. Results assisted in the management of neonatal withdrawal in three babies. Drugs suspected by history were not found in 11/22 mothers and 23/35 babies. About half of these results were associated with delayed urine collection. In 12/28 mothers, drugs administered in hospital could have confused interpretation of screen results. We conclude that urine drug screening without strict protocols for specimen collection is of limited usefulness for management of drug abuse in pregnancy and neonatal drug withdrawal. We favour testing of maternal urine obtained before drugs are administered in hospital. Neonatal urine, if used, should be collected in the first day of life.

  12. Bioassay Screening of the Essential Oil and Various Extracts of Nigella sativa L. Seeds Using Brine Shrimp Toxicity Assay

    Directory of Open Access Journals (Sweden)

    Fariba Sharififar

    2017-03-01

    Full Text Available Background and Aim: Since cytotoxicity screening is the first step necessary for any new drug development, this study was designed to find out and compare the cytotoxicity effects of the essential oil and various extracts of Nigella sativa L. seeds using Brine Shrimp Lethality (BSL assay. Materials and Methods: Essential oils and various extracts of N. sativa were assessed by two methods of disk and solution of BSL. Data analysis was carried out using SPSS statistical package version 17.0 (SPSS Inc., Chicago, IL, 250 USA. Data were processed in probit-analysis program to estimate LC50 values. Results: All of the tested fractions demonstrated more cytotoxicity in the solution method. Petroleum ether and chloroform extract of N. sativa showed the most cytotoxicity with LC50 values 7 and 21 μg/ml respectively; while aqueous and ethanolic had no significant cytotoxicity. Moreover, the GC/MS analysis of the essential oil of N. sativa showed the p-cymene (48.1%, α-thujone (14.38% and dihydro carveol (9.11% as the main compounds. Conclusion: These results suggest some limitation for using this spice in diet. Furthermore, this plant could be considered as a source of cytotoxic compounds which should be studied in details.

  13. A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards

    OpenAIRE

    Mordwinkin, Nicholas M.; Burridge, Paul W.; Wu, Joseph C.

    2012-01-01

    Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and ...

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

    Directory of Open Access Journals (Sweden)

    Cécile Voisset

    2014-04-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2014-04-01

    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.

  16. Enhancing Reproducibility in Cancer Drug Screening: How Do We Move Forward?

    DEFF Research Database (Denmark)

    Shi, Leming; Haibe-Kains, Benjamin; Birkbak, Nicolai Juul

    2014-01-01

    Large-scale pharmacogenomic high-throughput screening (HTS) studies hold great potential for generating robust genomic predictors of drug response. Two recent large-scale HTS studies have reported results of such screens, revealing several known and novel drug sensitivities and biomarkers...

  17. A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

    Science.gov (United States)

    Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

    2013-02-01

    Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

  18. Portable non-destructive assay methods for screening and segregation of radioactive waste

    International Nuclear Information System (INIS)

    Simpson, Alan; Jones, Stephanie; Clapham, Martin; Lucero, Randy

    2011-01-01

    Significant cost-savings and operational efficiency may be realised by performing rapid non-destructive classification of radioactive waste at or near its point of retrieval or generation. There is often a need to quickly categorize and segregate bulk containers (drums, crates etc.) into waste streams defined at various boundary levels (based on its radioactive hazard) in order to meet disposal regulations and consignor waste acceptance criteria. Recent improvements in gamma spectroscopy technologies have provided the capability to perform rapid in-situ analysis using portable and hand-held devices such as battery-operated medium and high resolution detectors including lanthanum halide and high purity germanium (HPGe). Instruments and technologies that were previously the domain of complex lab systems are now widely available as touch-screen 'off-the-shelf' units. Despite such advances, the task of waste stream screening and segregation remains a complex exercise requiring a detailed understanding of programmatic requirements and, in particular, the capability to ensure data quality when operating in the field. This is particularly so when surveying historical waste drums and crates containing heterogeneous debris of unknown composition. The most widely used portable assay method is based upon far-field High Resolution Gamma Spectroscopy (HRGS) assay using HPGe detectors together with a well engineered deployment cart (such as the PSC TechniCART TM technology). Hand-held Sodium Iodide (NaI) detectors are often also deployed and may also be used to supplement the HPGe measurements in locating hot spots. Portable neutron slab monitors may also be utilised in cases where gamma measurements alone are not suitable. Several case histories are discussed at various sites where this equipment has been used for in-situ characterization of debris waste, sludge, soil, high activity waste, depleted and enriched uranium, heat source and weapons grade plutonium, fission products

  19. Whole blood interferon-gamma assay for baseline tuberculosis screening among Japanese healthcare students.

    Directory of Open Access Journals (Sweden)

    Katsuyuki Hotta

    Full Text Available BACKGROUND: The whole blood interferon-gamma assay (QuantiFERON-TB-2G; QFT has not been fully evaluated as a baseline tuberculosis screening test in Japanese healthcare students commencing clinical contact. The aim of this study was to compare the results from the QFT with those from the tuberculin skin test (TST in a population deemed to be at a low risk for infection with Mycobacterium tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Healthcare students recruited at Okayama University received both the TST and the QFT to assess the level of agreement between these two tests. The interleukin-10 levels before and after exposure to M tuberculosis-specific antigens (early-secreted antigenic target 6-kDa protein [ESAT-6] and culture filtrate protein 10 [CFP-10] were also measured. Of the 536 healthcare students, most of whom had been vaccinated with bacillus-Calmette-Guérin (BCG, 207 (56% were enrolled in this study. The agreement between the QFT and the TST results was poor, with positive result rates of 1.4% vs. 27.5%, respectively. A multivariate analysis also revealed that the induration diameter of the TST was not affected by the interferon-gamma concentration after exposure to either of the antigens but was influenced by the number of BCG needle scars (p = 0.046. The whole blood interleukin-10 assay revealed that after antigen exposure, the median increases in interleukin-10 concentration was higher in the subgroup with the small increase in interferon-gamma concentration than in the subgroup with the large increase in interferon-gamma concentration (0.3 vs. 0 pg/mL; p = 0.004. CONCLUSIONS/SIGNIFICANCE: As a baseline screening test for low-risk Japanese healthcare students at their course entry, QFT yielded quite discordant results, compared with the TST, probably because of the low specificity of the TST results in the BCG-vaccinated population. We also found, for the first time, that the change in the interleukin-10 level after exposure to

  20. Screening for primary aldosteronism using the newly developed IDS-iSYS® automated assay system

    Directory of Open Access Journals (Sweden)

    P.M. O’Shea

    2017-04-01

    Full Text Available Background: The recommended approach to screening for primary aldosteronism (PA in at-risk populations is to determine the ratio of aldosterone concentration (serum (SAC/plasma (PAC to renin measured in plasma as activity (PRA or concentration (DRC. However, lack of assay standardisation mandates the need for method-specific decision thresholds and clinical validation in the local population. Aim: The study objective was to establish method-specific aldosterone: renin ratio (ARR cut-offs for PA in men and women using the IDS-iSYS® assay system (IDS plc. Methods: A prospective cohort study design was used. PAC and DRC were measured immunochemically in ethylenediamine-tetraacetic acid (EDTA plasma on the IDS-iSYS® instrument. Results: A total of 437 subjects (218 men, 219 women were recruited including: healthy normotensive volunteers (n=266 and women taking the oral contraceptive pill (OCP; n=15; patients with essential hypertension (EH; n=128; confirmed PA (n=16; adrenal cortical carcinoma (ACC; n=3; Addison's disease (AD; n=4 and phaeochromocytoma/paraganglioma (PPGL; n=5. In this population, an ARR cut-off at >37.4 pmol/mIU provided 100% diagnostic sensitivity, 96% specificity and positive likelihood ratio for PA of 23:1. When the ARR decision threshold was stratified according to gender, a cut-off of >26.1 pmol/mIU in men and >113.6 pmol/mIU in women resulted in diagnostic sensitivity and specificity of 100%. Conclusion: This study demonstrates that decision thresholds for PA should not only be method-specific but also gender-specific. However, given the small number of PA patients (n=16, particularly women (n=4, further validation through a prospective study with a larger PA cohort is required before the thresholds presented here could be recommended for routine clinical use. Keywords: Primary aldosteronism, Renin, Aldosterone, Aldosterone: renin ratio (ARR, Sensitivity, Specificity

  1. Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

    Science.gov (United States)

    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

  2. Evaluation of three rapid oral fluid test devices on the screening of multiple drugs of abuse including ketamine.

    Science.gov (United States)

    Tang, Magdalene H Y; Ching, C K; Poon, Simon; Chan, Suzanne S S; Ng, W Y; Lam, M; Wong, C K; Pao, Ronnie; Lau, Angus; Mak, Tony W L

    2018-05-01

    Rapid oral fluid testing (ROFT) devices have been extensively evaluated for their ability to detect common drugs of abuse; however, the performance of such devices on simultaneous screening for ketamine has been scarcely investigated. The present study evaluated three ROFT devices (DrugWipe ® 6S, Ora-Check ® and SalivaScreen ® ) on the detection of ketamine, opiates, methamphetamine, cannabis, cocaine and MDMA. A liquid chromatography tandem mass spectrometry (LCMS) assay was firstly established and validated for confirmation analysis of the six types of drugs and/or their metabolites. In the field test, the three ROFT devices were tested on subjects recruited from substance abuse clinics/rehabilitation centre. Oral fluid was also collected using Quantisal ® for confirmation analysis. A total of 549 samples were collected in the study. LCMS analysis on 491 samples revealed the following drugs: codeine (55%), morphine (49%), heroin (40%), methamphetamine (35%), THC (8%), ketamine (4%) and cocaine (2%). No MDMA-positive cases were observed. Results showed that the overall specificity and accuracy were satisfactory and met the DRUID standard of >80% for all 3 devices. Ora-Check ® had poor sensitivities (ketamine 36%, methamphetamine 63%, opiates 53%, cocaine 60%, THC 0%). DrugWipe ® 6S showed good sensitivities in the methamphetamine (83%) and opiates (93%) tests but performed relatively poorly for ketamine (41%), cocaine (43%) and THC (22%). SalivaScreen ® also demonstrated good sensitivities in the methamphetamine (83%) and opiates (100%) tests, and had the highest sensitivity for ketamine (76%) and cocaine (71%); however, it failed to detect any of the 28 THC-positive cases. The test completion rate (proportion of tests completed with quality control passed) were: 52% (Ora-Check ® ), 78% (SalivaScreen ® ) and 99% (DrugWipe ® 6S). Copyright © 2018 Elsevier B.V. All rights reserved.

  3. The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening: Special focus on IK1.

    Science.gov (United States)

    Goversen, Birgit; van der Heyden, Marcel A G; van Veen, Toon A B; de Boer, Teun P

    2018-03-01

    Preclinical drug screens are not based on human physiology, possibly complicating predictions on cardiotoxicity. Drug screening can be humanised with in vitro assays using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, in contrast to adult ventricular cardiomyocytes, iPSC-CMs beat spontaneously due to presence of the pacemaking current I f and reduced densities of the hyperpolarising current I K1 . In adult cardiomyocytes, I K1 finalises repolarisation by stabilising the resting membrane potential while also maintaining excitability. The reduced I K1 density contributes to proarrhythmic traits in iPSC-CMs, which leads to an electrophysiological phenotype that might bias drug responses. The proarrhythmic traits can be suppressed by increasing I K1 in a balanced manner. We systematically evaluated all studies that report strategies to mature iPSC-CMs and found that only few studies report I K1 current densities. Furthermore, these studies did not succeed in establishing sufficient I K1 levels as they either added too little or too much I K1 . We conclude that reduced densities of I K1 remain a major flaw in iPSC-CMs, which hampers their use for in vitro drug screening. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. A single-question screening test for drug use in primary care.

    Science.gov (United States)

    Smith, Peter C; Schmidt, Susan M; Allensworth-Davies, Donald; Saitz, Richard

    2010-07-12

    Drug use (illicit drug use and nonmedical use of prescription drugs) is common but underrecognized in primary care settings. We validated a single-question screening test for drug use and drug use disorders in primary care. Adult patients recruited from primary care waiting rooms were asked the single screening question, "How many times in the past year have you used an illegal drug or used a prescription medication for nonmedical reasons?" A response of at least 1 time was considered positive for drug use. They were also asked the 10-item Drug Abuse Screening Test (DAST-10). The reference standard was the presence or absence of current (past year) drug use or a drug use disorder (abuse or dependence) as determined by a standardized diagnostic interview. Drug use was also determined by oral fluid testing for common drugs of abuse. Of 394 eligible primary care patients, 286 (73%) completed the interview. The single screening question was 100% sensitive (95% confidence interval [CI], 90.6%-100%) and 73.5% specific (95% CI, 67.7%-78.6%) for the detection of a drug use disorder. It was less sensitive for the detection of self-reported current drug use (92.9%; 95% CI, 86.1%-96.5%) and drug use detected by oral fluid testing or self-report (81.8%; 95% CI, 72.5%-88.5%). Test characteristics were similar to those of the DAST-10 and were affected very little by participant demographic characteristics. The single screening question accurately identified drug use in this sample of primary care patients, supporting the usefulness of this brief screen in primary care.

  5. High-throughput micro-plate HCL-vanillin assay for screening tannin content in sorghum grain

    Science.gov (United States)

    Sorghum contains tannin which is a phenolic compound that offers health promoting antioxidant capacity. The HCl-vanillin assay is a common and time consuming method for determining tannin content, but is not efficient for screening large sample sets as seen in association mapping panels or breeding ...

  6. Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

    Science.gov (United States)

    AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

  7. High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts

    NARCIS (Netherlands)

    de Jong, C.F.; Derks, R.J.E.; Bruyneel, B.; Niessen, W.M.A.; Irth, H.

    2006-01-01

    The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the

  8. Unique Nanoparticle Optical Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Screening and Ranking

    Science.gov (United States)

    Nanoparticles (NPs) are novel materials having at least one dimension less than 100 nm and display unique physicochemical properties due to their nanoscale size. An emphasis has been placed on developing high throughput screening (HTS) assays to characterize and rank the toxiciti...

  9. Validation of an optical surface plasmon resonance biosensor assay for screening (fluoro)quinolones in egg, fish and poultry

    NARCIS (Netherlands)

    Huet, A.C.; Charlier, C.; Weigel, S.; Benrejeb Godefroy, S.; Delahaut, P.

    2009-01-01

    A surface plasmon resonance biosensor immunoassay has been developed for multi-residue determination of 13 (fluoro)quinolone antibiotics in poultry meat, eggs and fish. The following performance characteristics were determined according to the guidelines laid down for screening assay validation in

  10. A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Dyhring, Tino

    2011-01-01

    PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ...

  11. Off-target effects of psychoactive drugs revealed by genome-wide assays in yeast.

    Directory of Open Access Journals (Sweden)

    Elke Ericson

    2008-08-01

    Full Text Available To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac interfered with establishment of cell polarity, cyproheptadine (Periactin targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol and pimozide (Orap. Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.

  12. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

    Directory of Open Access Journals (Sweden)

    Tom A. Ewing

    2018-01-01

    Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

  13. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

    Science.gov (United States)

    Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

    2018-01-14

    Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

  14. Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

    Science.gov (United States)

    Jacobs, K R; Guillemin, G J; Lovejoy, D B

    2018-02-01

    Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

  15. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening

    Directory of Open Access Journals (Sweden)

    Pothineni VR

    2016-04-01

    Full Text Available Venkata Raveendra Pothineni,1 Dhananjay Wagh,1 Mustafeez Mujtaba Babar,1 Mohammed Inayathullah,1 David Solow-Cordero,2 Kwang-Min Kim,1 Aneesh V Samineni,1 Mansi B Parekh,1 Lobat Tayebi,3 Jayakumar Rajadas1 1Biomaterials and Advanced Drug Delivery Laboratory, Stanford Cardiovascular Pharmacology Division, Cardiovascular Institute, Stanford University School of Medicine, Palo Alto, 2Chemical & Systems Biology, Stanford University School of Medicine, Stanford, CA, 3Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI, USA Abstract: Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved that were screened are Library of Pharmacologically Active Compounds (LOPAC1280, the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150

  16. Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

    Science.gov (United States)

    Gul, Sheraz; Brown, Richard; May, Earl; Mazzulla, Marie; Smyth, Martin G; Berry, Colin; Morby, Andrew; Powell, David J

    2004-11-01

    DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

  17. Radio-immuno-assay for trypsin in newborn-screening for cystic fibrosis

    International Nuclear Information System (INIS)

    Sander, J.; Niehaus, C.

    1982-01-01

    In 4,956 infants the concentration of immunoreactive trypsin was measured in dried blood on filter paper using a double antibody radioimmuno assay. About 90% of all results were below 40 ng/ml. In 13 infants the concentration of immunoreactive trypsin exceeded 80 ng/ml. These infants were examined clinically, including sweationtophoresis. We found three children suffering from cystic fibrosis. One further child showing an elevated concentration of chloride in the sweat (60 mval/ml) could not be reexamined. The concentration of immunoreactive trypsin in the cystic fibrosis children was 230, 297, and in one case 108 ng/ml at the 76th day of life, whereas the values for the 9 other children were between 80 and 154 ng/ml. We believe these results justify to use this test for a much higher number of infants, especially because it is inexpensive and can easily be added to existing newborn screening programs for inborn errors of metabolism. (orig.) [de

  18. A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera.

    Science.gov (United States)

    Bivén, K; Erdal, H; Hägg, M; Ueno, T; Zhou, R; Lynch, M; Rowley, B; Wood, J; Zhang, C; Toi, M; Shoshan, M C; Linder, S

    2003-06-01

    We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

  19. Prospective assessment of early fetal loss using an immunoenzymometric screening assay for detection of urinary human chorionic gonadotropin.

    Science.gov (United States)

    Taylor, C A; Overstreet, J W; Samuels, S J; Boyers, S P; Canfield, R E; O'Connor, J F; Hanson, F W; Lasley, B L

    1992-06-01

    To develop an economical, nonradiometric immunoenzymometric assay (IEMA) for the detection of urinary human chorionic gonadotropin (hCG) in studies of early fetal loss. To be effective, the IEMA must have a sensitivity equal to the standard immunoradiometric assay (IRMA) and sufficient specificity to eliminate the need for screening most nonconceptive cycles with the expensive and labor-intensive IRMA. Two different assays were used to measure hCG in daily early morning urine samples from potential conceptive cycles. Women undergoing donor artificial insemination (AI) were evaluated in a prospective study. Ninety-two women volunteers were selected on the basis of apparent normal reproductive health. Artificial insemination with nonfrozen donor semen was performed by cervical cup twice each menstrual cycle at 48-hour intervals, and daily urine samples were self-collected throughout the menstrual cycle. An IEMA was developed to detect urinary hCG using the same antibodies as in the standard IRMA; a study was designed to determine whether this nonradiometric assay could successfully detect the early fetal loss that was detected by the IRMA. Of 224 menstrual cycles analyzed by both assays, a total of six early fetal losses were detected by the IRMA. When the tentative screening rule was set to allow all six of these losses and 95% of future losses to be detected by the IEMA, an additional 34 false-positive results were detected by the IEMA. The specificity of the IEMA with this rule was calculated to be 84%. An IEMA based on the same antibodies used for the standard IRMA can serve as an efficient screening assay for the detection of early fetal loss. When the IEMA is used in this manner, nearly 80% of screened menstrual cycles can be eliminated without further testing by the IRMA.

  20. 78 FR 24425 - Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug...

    Science.gov (United States)

    2013-04-25

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2008-D-0642] Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug Administration Staff; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food...

  1. A multiple biomarker assay for quality assessment of botanical drugs using a versatile microfluidic chip.

    Science.gov (United States)

    Li, Zhen-Hao; Ai, Ni; Yu, Lawrence X; Qian, Zhong-Zhi; Cheng, Yi-Yu

    2017-09-25

    Quality control is critical for ensuring the safety and effectiveness of drugs. Current quality control method for botanical drugs is mainly based on chemical testing. However, chemical testing alone may not be sufficient as it may not capture all constituents of botanical drugs. Therefore, it is necessary to establish a bioassay correlating with the drug's known mechanism of action to ensure its potency and activity. Herein we developed a multiple biomarker assay to assess the quality of botanicals using microfluidics, where enzyme inhibition was employed to indicate the drug's activity and thereby evaluate biological consistency. This approach was exemplified on QiShenYiQi Pills using thrombin and angiotensin converting enzyme as "quality biomarkers". Our results demonstrated that there existed variations in potency across different batches of the intermediates and preparations. Compared with chromatographic fingerprinting, the bioassay provided better discrimination ability for some abnormal samples. Moreover, the chip could function as "affinity chromatography" to identify bioactive phytochemicals bound to the enzymes. This work proposed a multiple-biomarker strategy for quality assessment of botanical drugs, while demonstrating for the first time the feasibility of microfluidics in this field.

  2. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations

    Science.gov (United States)

    Mott, Bryan T.; Eastman, Richard T.; Guha, Rajarshi; Sherlach, Katy S.; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R.; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D.; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A.; Ferrer, Marc; Renslo, Adam R.; Inglese, James; Yuan, Jing; Roepe, Paul D.; Su, Xin-zhuan; Thomas, Craig J.

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

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

    International Nuclear Information System (INIS)

    Taylor, David J; Parsons, Christine E; Han, Haiyong; Jayaraman, Arul; Rege, Kaushal

    2011-01-01

    Tumor Necrosis Factor-α 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. 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. 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 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward malignant cells over normal pancreatic epithelial cells

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

    Directory of Open Access Journals (Sweden)

    Taylor David J

    2011-11-01

    Full Text Available Abstract Background Tumor Necrosis Factor-α 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 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward

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

    Directory of Open Access Journals (Sweden)

    Ramasamy S

    2014-12-01

    Full Text Available Sakthivel Ramasamy,1 Devasier Bennet,1 Sanghyo Kim1,2 1Department of Bionanotechnology, Gachon University, Gyeonggi-Do, Republic of Korea; 2Graduate Gachon Medical Research Institute, Gil Medical Center, Incheon, Republic of Korea Abstract: 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. Keywords: screening of bioactive agents, impedance-based cell

  6. Screening for substance abuse risk in cancer patients using the Opioid Risk Tool and urine drug screen.

    Science.gov (United States)

    Barclay, Joshua S; Owens, Justine E; Blackhall, Leslie J

    2014-07-01

    The use of opioids for management of cancer-related pain has increased significantly and has been associated with a substantial rise in rates of substance abuse and diversion. There is a paucity of data not only on the prevalence of substance abuse in cancer patients, but also for issues of drug use and diversion in family caregivers. This study aimed to evaluate the frequency of risk factors for substance abuse and diversion, and abnormal urine drug screens in cancer patients receiving palliative care. A retrospective chart review was performed for patients with cancer who were seen in the University of Virginia Palliative Care Clinic during the month of September 2012. We evaluated Opioid Risk Tool variables and total scores, insurance status, and urine drug screen results. Of the 114 cancer patients seen in September 2012, the mean Opioid Risk Tool score was 3.79, with 43% of patients defined as medium to high risk. Age (16-45 years old, 23%) and a personal history of alcohol (23%) or illicit drugs (21%) were the most common risk factors identified. We obtained a urine drug screen on 40% of patients, noting abnormal findings in 45.65%. Opioids are an effective treatment for cancer-related pain, yet substantial risk for substance abuse exits in the cancer population. Screening tools, such as the Opioid Risk Tool, should be used as part of a complete patient assessment to balance risk with appropriate relief of suffering.

  7. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Directory of Open Access Journals (Sweden)

    Helton J Wiggers

    Full Text Available 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 (K i 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

  8. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Science.gov (United States)

    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 (K i) 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

  9. A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

    Directory of Open Access Journals (Sweden)

    Jing Lu

    Full Text Available Polo-like kinase 1 (PLK1, one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

  10. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang Leyu [Research Technology Center, Pfizer Global Research and Development, Cambridge, MA (United States); Moore, Jennifer; Kuo, Ming-Shang T. [Pfizer Global Research and Development, San Diego, CA (United States); LaMarr, William A.; Ozbal, Can C. [Biotrove, Inc., Woburn, MA (United States); Bhat, B. Ganesh [Pfizer Global Research and Development, San Diego, CA (United States)], E-mail: gbhat@gnf.org

    2008-10-03

    Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K{sub m} = 10.5 {mu}M). The assay was highly reproducible with an average Z' value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC{sub 50} values of 0.88 and 0.12 {mu}M, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 {mu}M - 14% conformation rate). Of the confirmed hits 172 had IC{sub 50} values of <10 {mu}M, including 111 <1 {mu}M and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable

  11. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    International Nuclear Information System (INIS)

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang Leyu; Moore, Jennifer; Kuo, Ming-Shang T.; LaMarr, William A.; Ozbal, Can C.; Bhat, B. Ganesh

    2008-01-01

    Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K m = 10.5 μM). The assay was highly reproducible with an average Z' value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC 50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM - 14% conformation rate). Of the confirmed hits 172 had IC 50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal

  12. In planta assay of hygromycin susceptibility and mutant screening in rice at heading stage

    International Nuclear Information System (INIS)

    Fu Haowei; Li Youfa; Ma Xinghua; Shen Shengquan; Shu Xiaoli; Shu Qingyao; Chen Yang

    2012-01-01

    Cells and tissue of plants carrying the hygromycin phosphotransferase gene (HPT) are tolerant to antibiotic hygromycin and hence become the common selection gene for genetic transformation of crop plants, particularly monocots. The present study aimed at establishing a rapid method for in planta screening of hygromycin tolerant plants in transgenic breeding. At heading stage transgenic and conventional rice plants were sprayed with hygromycin solutions of different concentrations (25 ∼ 100 mg/L) and toxic symptoms were observed in the following days. It was observed that yellow-brown necrotic spots appeared in the leaves and grains of conventional rice varieties after foliar spraying of 50 mg/L hygromycin, and the more spots were occurred with the higher hygromycin solution. There were 21.1 and 19.2 spots per cm 2 on flag leaves of indica varieties Jiayou 99 and C10, respectively; while 27.6% and 23.5% grains were yellow-brown in the varieties, respectively. The corresponding data for two japonica varieties Jiayou 5 and R5 were 11.8 and 10.7 for leave spots and 11.2% and 11.6% for yellow-brown grains, respectively. These results indicated that the two indica varieties are more sensitive to hygromycin than the two japonica varieties. In the treatment of hygromycin with above levels, the transgenic rice line KMD1 with the HPT gene showed no toxic symptoms on leaf or panicles. The γ-rays treated M 2 population of KMD1 was sprayed with 100 mg/L hygromycin solution at heading stage and 42 plants were screened out with toxic symptoms in leaves and panicles form 120000 plants. Among the 42 hygromycin susceptible plants, 14 plants with leaves available for in vitro feeding assay were subjected to the feeding of rice striped steam borers [Chilo supperssalis (Walker)] and all showed high resistance. The above results showed that spraying of 100 mg/L hygromycin solution at heading stage would lead to visually apparent toxic symptoms in susceptibility rice plants and hence

  13. An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

    Science.gov (United States)

    Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

    2017-06-01

    The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

  14. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  15. Work-related injuries in a state trauma registry: relationship between industry and drug screening.

    Science.gov (United States)

    Bunn, Terry L; Slavova, Svetla; Bernard, Andrew C

    2014-08-01

    Work-related injuries exert a great financial and economic burden on the US population. The study objectives were to identify the industries and occupations associated with worker injuries and to determine the predictors for injured worker drug screening in trauma centers. Work-related injury cases were selected using three criteria (expected payer source of workers' compensation, industry-related e-codes, and work-related indicator) from the Kentucky Trauma Registry data set for years 2008 to 2012. Descriptive analyses and multiple logistic regression were performed on the work-related injury cases. The "other services" and construction industry sectors accounted for the highest number of work-related cases. Drugs were detected in 55% of all drug-screened work-related trauma cases. Higher percentages of injured workers tested positive for drugs in the natural resources and mining, transportation and public utilities, and construction industries. In comparison, higher percentages of injured workers in the other services as well as transportation and public utilities industries were drug screened. Treatment at Level I trauma centers and Glasgow Coma Scale (GCS) scores indicating a coma or severe brain injury were both significant independent predictors for being screened for drugs; industry was not a significant predictor for being drug screened. The injured worker was more likely to be drug screened if the worker had a greater than mild injury, regardless of whether the worker was an interfacility transfer. These findings indicate that there may be elevated drug use or abuse in natural resources and mining, transportation and public utilities, as well as construction industry workers; improved identification of the specific drug types in positive drug screen results of injured workers is needed to better target prevention efforts. Epidemiologic study, level III.

  16. A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

    Science.gov (United States)

    Chiaravalli, Jeanne; Glickman, J Fraser

    2017-08-01

    We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

  17. Use of a single alcohol screening question to identify other drug use.

    Science.gov (United States)

    Smith, Peter C; Cheng, Debbie M; Allensworth-Davies, Donald; Winter, Michael R; Saitz, Richard

    2014-06-01

    People who consume unhealthy amounts of alcohol are more likely to use illicit drugs. We tested the ability of a screening test for unhealthy alcohol use to simultaneously detect drug use. Adult English speaking patients (n=286) were enrolled from a primary care waiting room. They were asked the screening question for unhealthy alcohol use "How many times in the past year have you had X or more drinks in a day?", where X is 5 for men and 4 for women, and a response of one or more is considered positive. A standard diagnostic interview was used to determine current (past year) drug use or a drug use disorder (abuse or dependence). Oral fluid testing was also used to detect recent use of common drugs of abuse. The single screening question for unhealthy alcohol use was 67.6% sensitive (95% confidence interval [CI], 50.2-82.0%) and 64.7% specific (95% CI, 58.4-70.6%) for the detection of a drug use disorder. It was similarly insensitive for drug use detected by oral fluid testing and/or self-report. Although a patient with a drug use disorder has twice the odds of screening positive for unhealthy alcohol use compared to one without a drug use disorder, suggesting patients who screen positive for alcohol should be asked about drug use, a single screening question for unhealthy alcohol use was not sensitive or specific for the detection of other drug use or drug use disorders in a sample of primary care patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Clinical Usefulness of the Histoculture Drug Response Assay for Prostate Cancer and Benign Prostate Hypertrophy (BPH).

    Science.gov (United States)

    Hoffman, Robert M

    2018-01-01

    The histoculture drug response assay (HDRA) has been adapted to determine androgen sensitivity in Gelfoam histoculture of human benign prostatic tissue as well as prostate cancer. Gelfoam histoculture was used to measure androgen-independent and androgen-dependent growth of benign and malignant prostate tissue. The androgen-sensitivity index was significantly higher in 23 paired specimens of prostate cancer compared to benign prostate hypertrophy (BPH). Genistein decreased the androgen-sensitivity index of BPH and prostate cancer in Gelfoam ® histoculture in a dose-dependent manner.

  19. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available 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.

  20. A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples

    Energy Technology Data Exchange (ETDEWEB)

    Metcalfe, Chris D., E-mail: cmetcalfe@trentu.ca [Environmental and Resource Studies, Trent University, Peterborough, ON, K9J 7B8 (Canada); Kleywegt, Sonya [Standards Development Branch, Ontario Ministry of the Environment, 40 St. Clair Ave. West, Toronto, ON, M4V 1M2 (Canada); Letcher, Robert J. [Ecotoxicology and Wildlife Health Division, Science and Technology Branch, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3 (Canada); Topp, Edward [Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, N5V 7T3 (Canada); Wagh, Purva; Trudeau, Vance L.; Moon, Thomas W. [Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, ON, K1N 6N5 (Canada)

    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

  1. A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples

    International Nuclear Information System (INIS)

    Metcalfe, Chris D.; Kleywegt, Sonya; Letcher, Robert J.; Topp, Edward; Wagh, Purva; Trudeau, Vance L.; Moon, Thomas W.

    2013-01-01

    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

  2. Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening

    Directory of Open Access Journals (Sweden)

    Yoon-Dong Park

    2016-08-01

    Full Text Available Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development.

  3. High throughput screening for small molecule enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery.

    Directory of Open Access Journals (Sweden)

    Dhara A Patel

    Full Text Available 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

  4. Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells

    Science.gov (United States)

    Nguyen, Minh M.; Dar, Javid A.; Ai, Junkui; Wang, Yujuan; Masoodi, Khalid Z.; Shun, Tongying; Shinde, Sunita; Camarco, Daniel P.; Hua, Yun; Huryn, Donna M.; Wilson, Gabriela Mustata; Lazo, John S.; Nelson, Joel B.; Wipf, Peter

    2016-01-01

    Abstract Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide. PMID:27187604

  5. Virtual screening methods as tools for drug lead discovery from large chemical libraries.

    Science.gov (United States)

    Ma, X H; Zhu, F; Liu, X; Shi, Z; Zhang, J X; Yang, S Y; Wei, Y Q; Chen, Y Z

    2012-01-01

    Virtual screening methods have been developed and explored as useful tools for searching drug lead compounds from chemical libraries, including large libraries that have become publically available. In this review, we discussed the new developments in exploring virtual screening methods for enhanced performance in searching large chemical libraries, their applications in screening libraries of ~ 1 million or more compounds in the last five years, the difficulties in their applications, and the strategies for further improving these methods.

  6. Screening the Drug Sensitivity Genes Related to GEM and CDDP in the Lung Cancer Cell-lines

    Directory of Open Access Journals (Sweden)

    Chunyu YANG

    2009-10-01

    Full Text Available Background and objective Screening of small-cell lung cancer (SCLC and non-small cell lung cancer (NSCLC cell lines with gemcitabine hydrochloride (GEM and cisplatin (CDDP related to drug sensitivity gene might clarify the action mechanism of anti-cancer drugs and provide a new clue for overcoming drug resistance and the development of new anti-cancer drugs, and also provide theoretical basis for the clinical treatment of individual. Methods The drug sensitivity of CDDP and GEM in 4 SCLC cell lines and 6 NSCLC cell lines was determined using MTT colorimetric assay, while the cDNA macroarray was applied to detect the gene expression state related to drug sensitivity of 10 lung cancer cell line in 1 291, and the correlation between the two was analysized. Results There were 6 genes showing significant positive correlation (r≥0.632, P < 0.05 with GEM sensitivity; 45 genes positively related to CDDP; another 41 genes related to both GEM and CDDP (r≥± 0.4. Lung cancer with GEM and CDDP sensitivity of two types of drugs significantly related genes were Metallothinein (Signal transduction molecules, Cathepsin B (Organization protease B and TIMP1 (Growth factor; the GEM, CDDP sensitivity associated genes of lung cancer cell lines mainly distributed in Metallothinein, Cathepsin B, growth factor TIMP1 categories. Conclusion There existed drug-related sensitive genes of GEM, CDDP in SCLC and NSCLC cell lines; of these genes, Metallothinein, Cathepsin B and TIMP1 genes presented a significant positive correlation with GEM drug sensitivity, a significant negative correlation with CDDP drug sensitivity.

  7. Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

    Science.gov (United States)

    Xu, Yan; Hadjiargyrou, M; Rafailovich, Miriam; Mironava, Tatsiana

    2017-07-11

    Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). Herein, we report that titanium dioxide (TiO 2 ) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO 2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

  8. Screening and brief intervention for unhealthy drug use: little or no efficacy

    Directory of Open Access Journals (Sweden)

    Richard eSaitz

    2014-09-01

    Full Text Available Unhealthy drug use ranges from use that risks health harms through severe drug use disorders. This narrative review addresses whether screening and brief intervention, 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 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 screening and brief intervention that has effects in primary care settings on risky alcohol use may not be efficacious for other drug use.

  9. Evaluation of the Roche prototype 454 HIV-1 ultradeep sequencing drug resistance assay in a routine diagnostic laboratory.

    Science.gov (United States)

    Garcia-Diaz, A; Guerrero-Ramos, A; McCormick, A L; Macartney, M; Conibear, T; Johnson, M A; Haque, T; Webster, D P

    2013-10-01

    Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings. To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory. 50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals. The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen. The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Prokofjeva Maria M

    2013-01-01

    Full Text Available Abstract 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.

  11. Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.

    Science.gov (United States)

    Li, Ying; Jortani, Saeed A; Ramey-Hartung, Bronwyn; Hudson, Elizabeth; Lemieux, Bertrand; Kong, Huimin

    2011-01-14

    The response to the anticoagulant drug warfarin is greatly affected by genetic polymorphisms in the VKORC1 and CYP2C9 genes. Genotyping these polymorphisms has been shown to be important in reducing the time of the trial and error process for finding the maintenance dose of warfarin thus reducing the risk of adverse effects of the drug. We developed a real-time isothermal DNA amplification system for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. For each SNP, real-time isothermal Helicase Dependent Amplification (HDA) reactions were performed to amplify a DNA fragment containing the SNP. Amplicons were detected by fluorescently labeled allele specific probes during real-time HDA amplification. Fifty clinical samples were analyzed by the HDA-based method, generating a total of 150 results. Of these, 148 were consistent between the HDA-based assays and a reference method. The two samples with unresolved HDA-based test results were repeated and found to be consistent with the reference method. The HDA-based assays demonstrated a clinically acceptable performance for genotyping the VKORC1 -1639G>A SNP and two SNPs (430C>T and 1075A>C) for the CYP2C9 enzyme (CYP2C9*2 and CYP2C9*3), all of which are relevant in warfarin pharmacogenentics. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    Science.gov (United States)

    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.

  13. Hydrophilic interaction chromatography with a focus on the drug-phosphate interaction in drug screening to determine the phospholipidosis induction risk.

    Science.gov (United States)

    Okamoto, Haruka; Hamaguchi, Ryohei; Kuroda, Yukihiro

    2017-04-15

    Cationic amphiphilic drugs (CADs) can induce the hyperaccumulation of phospholipids in cells and tissues. This side effect, which is known as drug-induced phospholipidosis, is sometimes problematic in the development and clinical use of CADs. It is known that CADs generally interact with phospholipids via both hydrophobic and acid-base interactions, and CADs with the larger affinity to phospholipid exhibit the larger induction risk. To develop a chromatographic assay system to predict the phospholipidosis-inducing potential with considering the acid-base interaction between CAD and phosphate group of phospholipid, hydrophilic interaction chromatographic (HILIC) methods were tested in this study. First, a PC HILIC column with phosphocholine groups on a packed material was used. The acid-base or other hydrophilic interactions to the stationary phase differed among basic drugs, and retention to the PC HILIC column did not accurately reflect the induction potential of phospholipidosis. As an alternative HILIC approach, the elution of CADs with the phosphate buffer from an amide column was tested. The elution effect, which is expressed as ratio of retention factors between different phosphate content in the mobile phase, closely correlated with the induction potential. Using the elution effect and retention factor to a reversed-phase HPLC column, the phospholipidosis-inducing drugs were clearly discriminated from the non-inducers. These results suggest that the proposed chromatographic approach can screen phospholipidosis-inducing drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. High-Throughput Screening of Ototoxic and Otoprotective Pharmacological Drugs

    Science.gov (United States)

    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…

  15. Referral population studies underestimate differences between human papillomavirus assays in primary cervical screening

    DEFF Research Database (Denmark)

    Rebolj, M.; Njor, S.; Lynge, E.

    2017-01-01

    with SurePath® cytology, and Hybrid Capture 2 (HC2), cobas, CLART and APTIMA HPV assays. Women with positive test results were offered a follow-up. For all detected HPV infections and HPV-positive high-grade cervical intraepithelial neoplasia (≥CIN2), we studied the distributions of assay-specific signal...

  16. Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development

    Directory of Open Access Journals (Sweden)

    Soh Byoung

    2013-02-01

    Full Text Available Abstract Background With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. Methods Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. Results Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. Conclusion The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite

  17. New approach for high-throughput screening of drug activity on Plasmodium liver stages.

    NARCIS (Netherlands)

    Gego, A.; Silvie, O.; Franetich, J.F.; Farhati, K.; Hannoun, L.; Luty, A.J.F.; Sauerwein, R.W.; Boucheix, C.; Rubinstein, E.; Mazier, D.

    2006-01-01

    Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an

  18. Comprehensive screening and quantification of veterinary drugs in milk using UPLC-ToF-MS

    NARCIS (Netherlands)

    Stolker, A.A.M.; Rutgers, P.; Oosterink, J.E.; Lasaroms, J.J.P.; Peters, R.J.B.; Rhijn, van J.A.; Nielen, M.W.F.

    2008-01-01

    Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC¿ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins,

  19. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    DEFF Research Database (Denmark)

    Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

    2017-01-01

    to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

  20. Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

    Science.gov (United States)

    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 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

  1. Drug-drug cocrystals of antituberculous 4-aminosalicylic acid: Screening, crystal structures, thermochemical and solubility studies.

    Science.gov (United States)

    Drozd, Ksenia V; Manin, Alex N; Churakov, Andrei V; Perlovich, German L

    2017-03-01

    Experimental multistage cocrystal screening of the antituberculous drug 4-aminosalicylic acid (PASA) has been conducted with a number of coformers (pyrazinamide (PYR), nicotinamide (NAM), isonicotinamide (iNAM), isoniazid (INH), caffeine (CAF) and theophylline (TPH)). The crystal structures of 4-aminosalicylic acid cocrystals with isonicotinamide ([PASA+iNAM] (2:1)) and methanol solvate with caffeine ([PASA+CAF+MeOH] (1:1:1)) have been determined by single X-ray diffraction experiments. For the first time for PASA cocrystals it has been found that the structural unit of the [PASA+iNAM] cocrystal (2:1) is formed by 2 types of heterosynthons: acid-pyridine and acid-amide. The desolvation study of the [PASA+CAF+MeOH] cocrystal solvate (1:1:1) has been conducted. The correlation models linking the melting points of the cocrystals with the melting points of the coformers used in this paper have been developed. The thermochemical and solubility properties for all the obtained cocrystals have been studied. Cocrystallization has been shown to lead not only to PASA solubility improving but also to its higher stability against the chemical decomposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The redox behaviour of diazepam (Valium®) using a disposable screen-printed sensor and its determination in drinks using a novel adsorptive stripping voltammetric assay.

    Science.gov (United States)

    Honeychurch, Kevin C; Crew, Adrian; Northall, Hannah; Radbourne, Stuart; Davies, Owian; Newman, Sam; Hart, John P

    2013-11-15

    In this study we investigated the possibility of applying disposable electrochemical screen-printed carbon sensors for the rapid identification and quantitative determination of diazepam in beverages. This was achieved utilising a previously unreported oxidation peak. The origin of this peak was investigated further by cyclic voltammetry and gas chromatography/mass spectroscopy. At pH 6 the voltammetric behaviour of this oxidation process was found to involve adsorption of the drug allowing for the development of an adsorptive stripping voltammetric assay. Experimental conditions were then optimised for the determination of diazepam in a beverage sample using a medium exchange technique. It was shown that no elaborate extraction procedures were required as the calibration plots obtained in the absence and presence of the beverage were very similar. © 2013 Elsevier B.V. All rights reserved.

  3. DISIS: prediction of drug response through an iterative sure independence screening.

    Directory of Open Access Journals (Sweden)

    Yun Fang

    Full Text Available 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.

  4. Screening of potent anticancer drug taxol from Entophytic fungus ...

    African Journals Online (AJOL)

    Muthumary

    2011-02-21

    Feb 21, 2011 ... Isolation and detection of taxol, an anticancer drug produced from ... cancer cell line, taxol produced by the test fungus in MID culture medium was isolated for its .... then plotted on a graph. RESULTS AND ... Wavelength (nm).

  5. How Phenotypic Screening Influenced Drug Discovery: Lessons from Five Years of Practice.

    Science.gov (United States)

    Haasen, Dorothea; Schopfer, Ulrich; Antczak, Christophe; Guy, Chantale; Fuchs, Florian; Selzer, Paul

    Since 2011, phenotypic screening has been a trend in the pharmaceutical industry as well as in academia. This renaissance was triggered by analyses that suggested that phenotypic screening is a superior strategy to discover first-in-class drugs. Despite these promises and considerable investments, pharmaceutical research organizations have encountered considerable challenges with the approach. Few success stories have emerged in the past 5 years and companies are questioning their investment in this area. In this contribution, we outline what we have learned about success factors and challenges of phenotypic screening. We then describe how our efforts in phenotypic screening have influenced our approach to drug discovery in general. We predict that concepts from phenotypic screening will be incorporated into target-based approaches and will thus remain influential beyond the current trend.

  6. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  7. Stem cells: a model for screening, discovery and development of drugs.

    Science.gov (United States)

    Kitambi, Satish Srinivas; Chandrasekar, Gayathri

    2011-01-01

    The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.

  8. AN APPROACH FOR SCREENING CHOLINESTERASE INHIBITORS IN DRINKING WATER USING AN IMMOBILIZED ENZYME ASSAY

    Science.gov (United States)

    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 ...

  9. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

    Science.gov (United States)

    Binks, Michael J; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P; Dunne, Eileen M; Richmond, Peter C; Marsh, Robyn L; Leach, Amanda J; Smith-Vaughan, Heidi C

    2012-01-01

    Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

  10. Assessment of the microscreen phage-induction assay for screening hazardous wastes (1989)

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-01-01

    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 of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that were mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.

  11. Drug screening in clinical or forensic toxicology: are there differences?

    Science.gov (United States)

    Gerostamoulos, Dimitri; Beyer, Jochen

    2010-09-01

    Legal and medical practitioners need to remember that, with respect to drug analysis, there are two distinct disciplines in analytical toxicology concerned with human biological matrices, namely clinical and forensic toxicology. Both fields use similar analytical techniques designed to detect and quantify drugs, chemicals and poisons in fluids or tissues. In clinical toxicology, analytical results help to specify the appropriate treatment of a poisoned or intoxicated patient. In forensic toxicology, the results often play a vital role in determining the possible impairment or behavioural changes in an individual, or the contribution of drugs or poisons to death in a medico-legal investigation. This column provides an overview of the similarities and differences inherent in clinical and forensic toxicology.

  12. A static-cidal assay for Trypanosoma brucei to aid hit prioritisation for progression into drug discovery programmes.

    Directory of Open Access Journals (Sweden)

    Manu De Rycker

    Full Text Available Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency.

  13. The use of virtual laboratories and other web-based tools in a drug assay course.

    Science.gov (United States)

    Dunham, Marissa Waldman; Ghirtis, Konstantine; Beleh, Mustapha

    2012-06-18

    To determine students' perceptions of and performance in a drug assay laboratory course after the addition of Web-based multimedia tools. Video modules and other Web-based tools to deliver instructions and emulate the laboratory set up for experiments were implemented in 2005 to improve student preparation for laboratory sessions and eliminate the need for graduate students to present instructions live. Data gathered from quizzes, final examinations, and post-course surveys administered over 6 years were analyzed. Students' scores on online quizzes after implementation of the virtual laboratories reflected improved student understanding and preparation. Students' perception of the course improved significantly after the introduction of the tools and the new teaching model. Implementation of an active-learning model in a laboratory course led to improvement in students' educational experience and satisfaction. Additional benefits included improved resource use, student exposure to a variety of educational methods, and having a highly structured laboratory format that reduced inconsistencies in delivered instructions.

  14. Combinatorial drug screening identifies Ewing sarcoma-specific sensitivities

    OpenAIRE

    Radic-Sarikas, Branka; Tsafou, Kalliopi P.; Emdal, Kristina B.; Papamarkou, Theodore; Huber, Kilian V.M.; Mutz, Cornelia; Toretsky, Jeffrey A.; Bennett, Keiryn L.; Olsen, Jesper V.; Brunak, Søren; Kovar, Heinrich; Superti-Furga, Giulio

    2017-01-01

    Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three p...

  15. Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

    Science.gov (United States)

    Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

    2017-12-01

    Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

  16. Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities

    DEFF Research Database (Denmark)

    Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B.

    2017-01-01

    Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any...... including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong...

  17. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value...... culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause...... for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay....

  18. Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

    Science.gov (United States)

    Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham

    2016-03-01

    To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Screening for drugs of abuse (II): Cannabinoids, lysergic acid diethylamide, buprenorphine, methadone, barbiturates, benzodiazepines and other drugs.

    Science.gov (United States)

    Simpson, D; Braithwaite, R A; Jarvie, D R; Stewart, M J; Walker, S; Watson, I W; Widdop, B

    1997-09-01

    Requirements for the provision of an efficient and reliable service for drugs of abuse screening in urine have been summarized in Part I of this review. The requirements included rapid turn-around times, good communications between requesting clinicians and the laboratory, and participation in quality assessment schemes. In addition, the need for checking/confirmation of positive results obtained for preliminary screening methods was stressed. This aspect of the service has assumed even greater importance with widespread use of dip-stick technology and the increasing number of reasons for which drug screening is performed. Many of these additional uses of drug screening have possible serious legal implications, for example, screening school pupils, professional footballers, parents involved in child custody cases, persons applying for renewal of a driving licence after disqualification for a drug-related offence, doctors seeking re-registration after removal for drug abuse, and checking for compliance with terms of probation orders; as well as pre-employment screening and work-place testing. In many cases these requests will be received from a general practitioner or drug clinic with no indication of the reason for which testing has been requested. This also raises the serious problems of a chain of custody, provision of two samples, stability of samples, and secure and lengthy storage of samples in the laboratory-samples may be requested by legal authorities several months after the initial testing. The need for confirmation of positive results is now widely accepted but it may be equally important to confirm unexpected negative results. Failure to detect the presence of maintenance drugs may lead to the patient being discharged from a drug treatment clinic and, if attendance at the clinic is one of the terms of continued employment, to dismissal. It seems likely that increasing abuse of drugs and the efforts of regulatory authorities to control this, will lead to

  20. Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

    OpenAIRE

    Lewis, Russell E.; Liao, Guangling; Young, Katherine; Douglas, Cameron; Kontoyiannis, Dimitrios P.

    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-κB 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 f...

  1. Screening for alcohol and drug use disorders among adults in primary care: a review

    Directory of Open Access Journals (Sweden)

    Pilowsky DJ

    2012-04-01

    Full Text Available Daniel J Pilowsky1, Li-Tzy Wu21Departments of Epidemiology and Psychiatry, Columbia University, and the New York State Psychiatric Institute, New York City, NY, 2Department of Psychiatry and Behavioral Sciences, School of Medicine, Duke University Medical Center, Durham, NC, USABackground: The Patient Protection and Affordable Care Act of 2010 supports integration of substance abuse interventions and treatments into the mainstream health care system. Thus, effective screening and intervention for substance use disorders in health care settings is a priority.Objective: This paper reviews the prevalence of alcohol and drug use disorders (abuse or dependence in primary care settings and emergency departments, as well as current screening tools and brief interventions.Methods: MEDLINE was searched using the following keywords: alcohol use, alcohol use disorder, drug use, drug use disorder, screening, primary care, and emergency departments. Using the related-articles link, additional articles were screened for inclusion. This review focuses on alcohol and drug use and related disorders among adults in primary care settings.Conclusion: Screening, brief intervention, and referral for treatment are feasible and effective in primary care settings, provided that funding for screening is available, along with brief interventions and treatment facilities to which patients can be referred and treated promptly.Keywords: brief intervention, emergency departments

  2. SEMIAUTOMATED SOLID-PHASE EXTRACTION PROCEDURE FOR DRUG SCREENING IN BIOLOGICAL-FLUIDS USING THE ASPEC SYSTEM IN COMBINATION WITH CLEAN SCREEN DAU COLUMNS

    NARCIS (Netherlands)

    CHEN, XH; FRANKE, JP; ENSING, K; WIJSBEEK, J; DEZEEUW, RA

    1993-01-01

    The use of a semi-automated solid-phase extraction system (ASPEC) for the screening of drugs in plasma and urine on a single mixed-mode column (Clean Screen DAU) is described. The processes of column preconditioning, sample application, column wash, pH adjustment and elution of the drugs were

  3. Stem cells: a model for screening, discovery and development of drugs

    Directory of Open Access Journals (Sweden)

    Kitambi SS

    2011-09-01

    Full Text Available Satish Srinivas Kitambi1, Gayathri Chandrasekar21Department of Medical Biochemistry and Biophysics; 2Department of Biosciences, Karolinska Institutet, Stockholm, SwedenAbstract: The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.Keywords: therapeutics, stem cells, cancer stem cells, screening models, drug development, high throughput screening

  4. Screening for the drug-phospholipid interaction: correlation to phospholipidosis

    DEFF Research Database (Denmark)

    Alakoskela, Juha-Matti; Vitovic, Pavol; Kinnunen, Paavo K J

    2009-01-01

    Phospholipid bilayers represent a complex, anisotropic environment fundamentally different from bulk oil or octanol, for instance. Even "simple" drug association to phospholipid bilayers can only be fully understood if the slab-of-hydrocarbon approach is abandoned and the complex, anisotropic...

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

    Science.gov (United States)

    Dalecki, Alex G; Wolschendorf, Frank

    2016-07-01

    Facing totally resistant bacteria, traditional drug discovery efforts have proven to be of limited use in replenishing our depleted arsenal of therapeutic antibiotics. Recently, the natural anti-bacterial properties of metal ions in synergy with metal-coordinating ligands have shown potential for generating new molecule candidates with potential therapeutic downstream applications. We recently developed a novel combinatorial screening approach to identify compounds with copper-dependent anti-bacterial properties. Through a parallel screening technique, the assay distinguishes between copper-dependent and independent activities against Mycobacterium tuberculosis with hits being defined as compounds with copper-dependent activities. These activities must then be linked to a compound master list to process and analyze the data and to identify the hit molecules, a labor intensive and mistake-prone analysis. Here, we describe a software program built to automate this analysis in order to streamline our workflow significantly. We conducted a small, 1440 compound screen against M. tuberculosis and used it as an example framework to build and optimize the software. Though specifically adapted to our own needs, it can be readily expanded for any small- to medium-throughput screening effort, parallel or conventional. Further, by virtue of the underlying Linux server, it can be easily adapted for chemoinformatic analysis of screens through packages such as OpenBabel. Overall, this setup represents an easy-to-use solution for streamlining processing and analysis of biological screening data, as well as offering a scaffold for ready functionality expansion. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Application of a drug-induced apoptosis assay to identify treatment strategies in recurrent or metastatic breast cancer.

    Directory of Open Access Journals (Sweden)

    Linda Bosserman

    Full Text Available A drug-induced apoptosis assay has been developed to determine which chemotherapy drugs or regimens can produce higher cell killing in vitro. This study was done to determine if this assay could be performed in patients with recurrent or metastatic breast cancer patients, to characterize the patterns of drug-induced apoptosis, and to evaluate the clinical utility of the assay. A secondary goal was to correlate assay use with clinical outcomes.In a prospective, non-blinded, multi institutional controlled trial, 30 evaluable patients with recurrent or metastatic breast cancer who were treated with chemotherapy had tumor samples submitted for the MiCK drug-induced apoptosis assay. After receiving results within 72 hours after biopsy, physicians could use the test to determine therapy (users, or elect to not use the test (non-users.The assay was able to characterize drug-induced apoptosis in tumor specimens from breast cancer patients and identified which drugs or combinations gave highest levels of apoptosis. Patterns of drug activity were also analyzed in triple negative breast cancer. Different drugs from a single class of agents often produced significantly different amounts of apoptosis. Physician frequently (73% used the assay to help select chemotherapy treatments in patients, Patients whose physicians were users had a higher response (CR+PR rate compared to non-users (38.1% vs 0%, p = 0.04 and a higher disease control (CR+PR+Stable rate (81% vs 25%, p<0.01. Time to relapse was longer in users 7.4 mo compared to non-users 2.2 mo (p<0.01.The MiCK assay can be performed in breast cancer specimens, and results are often used by physicians in breast cancer patients with recurrent or metastatic disease. These results from a good laboratory phase II study can be the basis for a future larger prospective multicenter study to more definitively establish the value of the assay.Clinicaltrials.gov NCT00901264.

  7. Radioimmunoassay screening and GC/MS confirmation of whole blood samples for drugs of abuse

    Energy Technology Data Exchange (ETDEWEB)

    Spiehler, V.R.; Sedgwick, P.

    From 1981 to 1984, an average of 300 radioimmunoassay screens on whole blood were performed each week in the authors laboratory. Most samples were screened for opiates phencyclidine and its analogs, barbiturates, and cocaine or its metabolite benzoylecgonine. A commercially available radioimmunoassay was used with modifications to facilitate screening of whole blood. Increasing sample size increased the sensitivity of the assay. Changing reagent concentration (1:1 dilution), incubation time, sample matrix (water, urine, or blood), or fraction counted (precipitate or supernatant) did not affect the utility of the standard curve or the sensitivity of the assay. All positive results for phencyclidine, opiates, cocaine, and related compounds were confirmed by GC/MA. Barbiturate positives were confirmed by UV spectrophotometry.

  8. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  9. Riboflavin-mediated photosensitization of Vinca alkaloids distorts drug sensitivity assays.

    Science.gov (United States)

    Granzow, C; Kopun, M; Kröber, T

    1995-11-01

    Poor reproducibility of cytotoxicity tests with Vinca alkaloids has frequently been reported. A commonly presumed light sensitivity of the drugs could not be confirmed. However, we found that they are photosensitized by riboflavin (vitamin B2), an obligatory component of cell culture media. Light of wavelengths below 500 nm triggered rapid photoreactions of riboflavin with vinblastine, vincristine, and vindesine in aqueous solutions. The photoreactions altered the absorption spectra of these alkaloids and yielded degradation products that could be separated by TLC. In cell cultures, both immediate and persisting, riboflavin-mediated photoreactivity could be distinguished. They preclude reliable determinations of sensitivity and resistance to Vinca alkaloids, as exemplified on chemosensitive and multidrug-resistant mouse ascites cells. In experiments involving photosensitization, the 50% inhibitory concentration values of sensitive and resistant cells were overlapping and fluctuated in the ranges from 3 to 30 nM and 15 to 360 nM vinblastine, respectively. Corresponding values from series of experiments protected from photosensitization were 1.02 +/- 0.22 nM and 18.5 +/- 3.42 nM. Hence, riboflavin-mediated photoreactions must be fully prevented in assays of cellular drug sensitivity. Procedures for eliminating immediate as well as persisting photoreactivity were established.

  10. The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine.

    Science.gov (United States)

    Kisser, Beatrice; Mangelsen, Eva; Wingolf, Caroline; Partecke, Lars Ivo; Heidecke, Claus-Dieter; Tannergren, Christer; Oswald, Stefan; Keiser, Markus

    2017-06-22

    The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step-by-step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  11. Characterisation of powerful antioxidants and synthetic iron ligands, as protective agents against oxidative damages, using new high throughput screening assays

    International Nuclear Information System (INIS)

    Meunier, St.

    2002-12-01

    This work was devoted to the development of pertinent high throughput screening assays in the aim of studying oxidative stress. Three screening assays have been developed for the evaluation of protective agents toward ROS generated by gamma irradiation, UV or by a Fenton-like system. 24 natural extracts and a library of 120 pure compounds, containing among the most powerful antioxidants known to date, have been readily studied using, these new techniques. We found that two pulvinic acid derivatives possess excellent protective properties, and especially a pigment of fungus named norbadione A. Beyond its in vitro activity, this molecule displays remarkable biological properties. In the aim of studying an alternative pathway of protection against oxidation induced by iron, ligands able to modify the redox properties of this metal, have been synthesised. We have developed a parallel synthesis allowing the variation of the architecture, denticity, chelating moieties and hydrophobicity of iron chelates. Using this strategy, 47 potential Fe(III) ligands were obtained. Their protective capacities have been studied using a fourth screening assay, demonstrating the effectiveness of some ligands. Finally, the immunoassay technique called SPI-RAD has been used in order to study a particular consequence of drastic oxidative stress, namely covalent crosslinks between proteins. Our results demonstrate that these linkages occur in the presence of metals (FeII or CuII) and hydrogen peroxide, as well as in the presence of NO . radical. Moreover, it has been demonstrated that tyrosines residues and disulfide bridges play an important role in these phenomena. (author)

  12. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

    Directory of Open Access Journals (Sweden)

    Michael J Binks

    Full Text Available BACKGROUND: Unambiguous identification of nontypeable Haemophilus influenzae (NTHi is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh; however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. METHODOLOGY/PRINCIPAL FINDINGS: Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22, Hh (n = 27 or equivocal (n = 11, were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3 and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. CONCLUSIONS/SIGNIFICANCE: Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

  13. Screening of Riboflavin-Producing Lactobacilli by a Polymerase-Chain-Reaction-Based Approach and Microbiological Assay.

    Science.gov (United States)

    Thakur, Kiran; Tomar, Sudhir Kumar; Brahma, Biswajit; De, Sachinandan

    2016-03-09

    Riboflavin has an important role in various cellular metabolic activities through its participation in oxidation-reduction reactions. In this study, as many as 60 lactobacilli were screened for the presence or absence of riboflavin biosynthesis genes and riboflavin production. Of these, only 14 strains were able to grow in a commercial riboflavin-free medium. We observed that the presence of riboflavin biosynthesis genes is strain-specific across different species of lactobacilli. The microbiological assay was found to be appreciably reproducible, sensitive, rapid, and inexpensive and, hence, can be employed for screening the riboflavin-producing strains. The study thus represents a convenient and efficient method for selection of novel riboflavin producers. These riboflavin(+) strains thus identified and characterized could be explored as potent candidates for the development of a wide range of dairy- and cereal-based foods for the delivery of in situ riboflavin to consumers.

  14. Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening

    International Nuclear Information System (INIS)

    Druwe, Ingrid; Freudenrich, Theresa M.; Wallace, Kathleen; Shafer, Timothy J.; Mundy, William R.

    2015-01-01

    High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery

  15. Cost-effectiveness of interferon-γ release assay versus chest X-ray for tuberculosis screening of employees.

    Science.gov (United States)

    Kowada, Akiko

    2011-12-01

    Currently, an annual chest X-ray examination (CXR) for detection of active tuberculosis (TB) in employees aged ≥40 years is recommended in the guidelines of the Japan Industrial Safety and Health Law. Interferon-γ release assays are new alternatives to the tuberculin skin test for detecting Mycobacterium tuberculosis infection, with higher specificity than the tuberculin skin test and without cross-reactivity with the Bacille Calmette-Guérin vaccine. This study aimed to assess the cost-effectiveness of employee TB screening using QuantiFERON-TB Gold In-Tube (QFT) versus CXR. Markov models were constructed. The target population was a hypothetical cohort of immunocompetent 40-year-old individuals, using a societal perspective and a lifetime horizon. All costs and clinical benefits were discounted at a fixed annual rate of 3%. In a base-case analysis, the QFT strategy was the most cost-effective ($US 262.84; 22.87049 quality-adjusted life-years [QALYs]) compared with no screening ($448.38; 22.85452 QALYs) and CXR ($543.50; 22.85453 QALYs) [year 2009 values]. The QFT strategy is currently robust for screening Bacille Calmette-Guérin- vaccinated employees in Japan. There appears to be little role for CXR. These findings may be applicable to other countries in terms of choosing optimal TB screening for employees. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  16. SCREENING FOR TOXIC INDUSTRIAL CHEMICALS USING SEMIPERMEABLE MEMBRANE DEVICES WITH RAPID TOXICITY ASSAYS

    Science.gov (United States)

    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...

  17. VAPOR SAMPLING DEVICE FOR INTERFACE WITH MICROTOX ASSAY FOR SCREENING TOXIC INDUSTRIAL CHEMICALS

    Science.gov (United States)

    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...

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

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

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

  19. Screening assays to find out late latent syphilis cases – which is the ...

    African Journals Online (AJOL)

    non-treponemal) such as Rapid Plasma Reagin (RPR), and specific (treponemal) such as Treponema pallidum hemagglutination test (TPHA) and Enzyme-linked immunosorbent assay IgG, IgM (ELISA IgG, IgM) tests. The aim of this ...

  20. Diagnostic accuracy of a two-item Drug Abuse Screening Test (DAST-2).

    Science.gov (United States)

    Tiet, Quyen Q; Leyva, Yani E; Moos, Rudolf H; Smith, Brandy

    2017-11-01

    Drug use is prevalent and costly to society, but individuals with drug use disorders (DUDs) are under-diagnosed and under-treated, particularly in primary care (PC) settings. Drug screening instruments have been developed to identify patients with DUDs and facilitate treatment. The Drug Abuse Screening Test (DAST) is one of the most well-known drug screening instruments. However, similar to many such instruments, it is too long for routine use in busy PC settings. This study developed and validated a briefer and more practical DAST for busy PC settings. We recruited 1300 PC patients in two Department of Veterans Affairs (VA) clinics. Participants responded to a structured diagnostic interview. We randomly selected half of the sample to develop and the other half to validate the new instrument. We employed signal detection techniques to select the best DAST items to identify DUDs (based on the MINI) and negative consequences of drug use (measured by the Inventory of Drug Use Consequences). Performance indicators were calculated. The two-item DAST (DAST-2) was 97% sensitive and 91% specific for DUDs in the development sample and 95% sensitive and 89% specific in the validation sample. It was highly sensitive and specific for DUD and negative consequences of drug use in subgroups of patients, including gender, age, race/ethnicity, marital status, educational level, and posttraumatic stress disorder status. The DAST-2 is an appropriate drug screening instrument for routine use in PC settings in the VA and may be applicable in broader range of PC clinics. Published by Elsevier Ltd.

  1. GENIUS In Silico Screening Technology for HCV Drug Discovery.

    Science.gov (United States)

    Patil, Vaishali M; Masand, Neeraj; Gupta, Satya P

    2016-01-01

    The various reported in silico screening protocols such as molecular docking are associated with various drawbacks as well as benefits. In molecular docking, on interaction with ligand, the protein or receptor molecule gets activated by adopting conformational changes. These conformational changes cannot be utilized to predict the 3D structure of a protein-ligand complex from unbound protein conformations rigid docking, which necessitates the demand for understanding protein flexibility. Therefore, efficiency and accuracy of docking should be achieved and various available/developed protocols may be adopted. One such protocol is GENIUS induced-fit docking and it is used effectively for the development of anti-HCV NS3-4A serine protease inhibitors. The present review elaborates the GENIUS docking protocol along with its benefits and drawbacks.

  2. Comparison of a fluoroquinolone surface plasmon resonance biosensor screening assay with established methods

    NARCIS (Netherlands)

    Weigel, S.; Pikkemaat, M.G.; Elferink, J.W.A.; Mulder, P.P.J.; Huet, A.C.; Delahaut, P.; Schittko, S.; Flerus, R.; Nielen, M.W.F.

    2009-01-01

    The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue

  3. Development of a PCR assay suitable for Campylobacter spp. mass screening programs in broiler production

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Pedersen, Karl; Madsen, Mogens

    2001-01-01

    Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide. In order to comply with the demands of consumers for food free of bacterial pathogens, a mass screening program for Campylobacter in Danish broilers has been carried out based on conventional bacterial...

  4. Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

    Science.gov (United States)

    Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

  5. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

  6. Setting up a Bioluminescence Resonance Energy Transfer high throughput screening assay to search for protein/protein interaction inhibitors in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Cyril eCouturier

    2012-09-01

    Full Text Available 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 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 protein-protein interactions 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 substrate concentration, number of cells and medium composition used on the Z’ factor, and expected interferences for colored or fluorescent compounds.

  7. Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.

    Science.gov (United States)

    Martins da Silva, Sarah J; Brown, Sean G; Sutton, Keith; King, Louise V; Ruso, Halil; Gray, David W; Wyatt, Paul G; Kelly, Mark C; Barratt, Christopher L R; Hope, Anthony G

    2017-05-01

    Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Grid Based Technologies for in silico Screening and Drug Design.

    Science.gov (United States)

    Potemkin, Vladimir; Grishina, Maria

    2018-03-08

    Various techniques for rational drug design are presented in the paper. The methods are based on a substitution of antipharmacophore atoms of the molecules of training dataset by new atoms and/or group of atoms increasing the atomic bioactivity increments obtained at a SAR study. Furthermore, a design methodology based on the genetic algorithm DesPot for discrete optimization and generation of new drug candidate structures is described. Additionally, wide spectra of SAR approaches (3D/4D QSAR interior and exterior-based methods - BiS, CiS, ConGO, CoMIn, high-quality docking method - ReDock) using MERA force field and/or AlteQ quantum chemical method for correct prognosis of bioactivity and bioactive probability is described. The design methods are implemented now at www.chemosophia.com web-site for online computational services. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. In silico structure-based drug screening of novel antimycobacterial pharmacophores by DOCK-GOLD tandem screening

    Directory of Open Access Journals (Sweden)

    Junichi Taira

    2017-01-01

    Full Text Available Background: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1 is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. Materials and Methods: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS. Following the selection of such compounds, their antimycobacterial activity was examined. Results: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. Conclusion: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

  11. ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    McGown, Alexander; Shaw, Dame Pamela J; Ramesh, Tennore

    2016-07-26

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2-3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well suited for the study of diseasepathophysiology and also for high throughput (HT) drug screening. The mutant sod1 zebrafish model of ALS mimics the hallmark features of ALS. Using a fluorescence based readout of neuronal stress, we developed a high throughput (HT) screen to identify neuroprotective compounds. Here we show that the zebrafish screen is a robust system that can be used to rapidly screen thousands ofcompounds and also demonstrate that riluzole is capable of reducing neuronal stress in this model system. The screen shows optimal quality control, maintaining a high sensitivity and specificity withoutcompromising throughput. Most importantly, we demonstrate that many compounds previously failed in human clinical trials, showed no stress reducing activity in the zebrafish assay. We conclude that HT drug screening using a mutant sod1 zebrafish is a reliable model system which supplemented with secondary assays would be useful in identifying drugs with potential for neuroprotective efficacy in ALS.

  12. The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

    Science.gov (United States)

    Kimber, Ian; Dearman, Rebecca J; Betts, Catherine J; Gerberick, G Frank; Ryan, Cindy A; Kern, Petra S; Patlewicz, Grace Y; Basketter, David A

    2006-04-01

    The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.

  13. Focused use of drug screening in overdose patients increases impact on management.

    Science.gov (United States)

    Erdmann, Andreas; Werner, Dominique; Hugli, Olivier; Yersin, Bertrand

    2015-01-01

    Drug poisoning is a common cause for attendance in the emergency department. Several toxicology centres suggest performing urinary drug screens, even though they rarely influence patient management. Measuring the impact on patient management, in a University Emergency Department with approximately 40 000 admissions annually, of a rapid urinary drug screening test using specifically focused indications. Drug screening was restricted to patients having a first psychotic episode or cases demonstrating respiratory failure, coma, seizures, a sympathomimetic toxidrome, severe opiate overdose necessitating naloxone, hypotension, ventricular arrhythmia, acquired long QT or QRS >100 ms, and high-degree heart block. Retrospective analysis of Triage® TOX drug screen tests performed between September 2009 and November 2011, and between January 2013 and March 2014. A total of 262 patients were included, mean age 35 ± 14.6 (standard deviation) years, 63% men; 29% poisoning with alcohol, and 2.3% deaths. Indications for testing were as follows: 34% were first psychotic episodes; 20% had acute respiratory failure; 16% coma; 8% seizures; 8% sympathomimetic toxidromes; 7% severe opioid toxidromes; 4% hypotension; 3% ventricular arrhythmias or acquired long QT intervals on electrocardiogram. A total of 78% of the tests were positive (median two substances, maximum five). The test resulted in drug-specific therapy in 6.1%, drug specific diagnostic tests in 13.3 %, prolonged monitoring in 10.7% of methadone-positive tests, and psychiatric admission in 4.2%. Overall, 34.3% tests influenced patient management. In contrast to previous studies showing modest effects of toxicological testing, restricted use of rapid urinary drug testing increases the impact on management of suspected overdose patients in the ED.

  14. Fertility drug use and mammographic breast density in a mammography screening cohort of premenopausal women

    OpenAIRE

    Sprague, Brian L.; Trentham-Dietz, Amy; Terry, Mary Beth; Nichols, Hazel B.; Bersch, Andy J.; Buist, Diana S. M.

    2008-01-01

    The widespread use of ovulation-inducing drugs to enhance fertility has raised concerns regarding potential effects on breast cancer risk, as ovarian stimulation is associated with increases in estrogen and progesterone levels. We investigated the short-term relation between fertility drug use and mammographic breast density, a strong marker of breast cancer risk, among participants in the Group Health Breast Cancer Screening Program. Data linkage with Group Health’s automated pharmacy record...

  15. Using a 3-d model system to screen for drugs effective on solid tumors

    OpenAIRE

    Fayad, Walid

    2011-01-01

    There is a large medical need for the development of effective anticancer agents with minimal side effects. The present thesis represents an attempt to identify potent drugs for treatment of solid tumors. We used a strategy where 3-D multicellular tumor spheroids (cancer cells grown in three dimensional culture) were utilized as in vitro models for solid tumors. Drug libraries were screened using spheroids as targets and using apoptosis induction and loss of cell viability as endpoints. The h...

  16. Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

    Science.gov (United States)

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. iScreen: world's first cloud-computing web server for virtual screening and de novo drug design based on TCM database@Taiwan.

    Science.gov (United States)

    Tsai, Tsung-Ying; Chang, Kai-Wei; Chen, Calvin Yu-Chian

    2011-06-01

    The rapidly advancing researches on traditional Chinese medicine (TCM) have greatly intrigued pharmaceutical industries worldwide. To take initiative in the next generation of drug development, we constructed a cloud-computing system for TCM intelligent screening system (iScreen) based on TCM Database@Taiwan. iScreen is compacted web server for TCM docking and followed by customized de novo drug design. We further implemented a protein preparation tool that both extract protein of interest from a raw input file and estimate the size of ligand bind site. In addition, iScreen is designed in user-friendly graphic interface for users who have less experience with the command line systems. For customized docking, multiple docking services, including standard, in-water, pH environment, and flexible docking modes are implemented. Users can download first 200 TCM compounds of best docking results. For TCM de novo drug design, iScreen provides multiple molecular descriptors for a user's interest. iScreen is the world's first web server that employs world's largest TCM database for virtual screening and de novo drug design. We believe our web server can lead TCM research to a new era of drug development. The TCM docking and screening server is available at http://iScreen.cmu.edu.tw/.

  18. iScreen: world's first cloud-computing web server for virtual screening and de novo drug design based on TCM database@Taiwan

    Science.gov (United States)

    Tsai, Tsung-Ying; Chang, Kai-Wei; Chen, Calvin Yu-Chian

    2011-06-01

    The rapidly advancing researches on traditional Chinese medicine (TCM) have greatly intrigued pharmaceutical industries worldwide. To take initiative in the next generation of drug development, we constructed a cloud-computing system for TCM intelligent screening system (iScreen) based on TCM Database@Taiwan. iScreen is compacted web server for TCM docking and followed by customized de novo drug design. We further implemented a protein preparation tool that both extract protein of interest from a raw input file and estimate the size of ligand bind site. In addition, iScreen is designed in user-friendly graphic interface for users who have less experience with the command line systems. For customized docking, multiple docking services, including standard, in-water, pH environment, and flexible docking modes are implemented. Users can download first 200 TCM compounds of best docking results. For TCM de novo drug design, iScreen provides multiple molecular descriptors for a user's interest. iScreen is the world's first web server that employs world's largest TCM database for virtual screening and de novo drug design. We believe our web server can lead TCM research to a new era of drug development. The TCM docking and screening server is available at http://iScreen.cmu.edu.tw/.

  19. In vitro determination of cytotoxic drug response in ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Csóka, K; Tholander, B; Gerdin, E; de la Torre, M; Larsson, R; Nygren, P

    1997-09-17

    The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated.

  20. Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Larsson, R; Kristensen, J; Sandberg, C; Nygren, P

    1992-01-21

    An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia.

  1. A rapid in vitro screening system for the identification and evaluation of anticancer drugs

    International Nuclear Information System (INIS)

    Kao, J.W.; Collins, J.L.

    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

  2. DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

    Czech Academy of Sciences Publication Activity Database

    Navrátil, Václav; Schimer, Jiří; Tykvart, Jan; Knedlík, Tomáš; Vik, V.; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2017-01-01

    Roč. 45, č. 2 (2017), č. článku e10. ISSN 0305-1048 R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : quantitative PCR * enzyme detection * inhibitor screening Subject RIV: CE - Biochemistry OBOR OECD: Biochemical research methods Impact factor: 10.162, year: 2016 https:// academic .oup.com/nar/article-lookup/doi/10.1093/nar/gkw853

  3. Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

    Science.gov (United States)

    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

  4. The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

    Science.gov (United States)

    Song, Lele; Jia, Jia; Peng, Xiumei; Xiao, Wenhua; Li, Yuemin

    2017-06-08

    The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

  5. An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

    Science.gov (United States)

    Nelson, Emily V; Pacheco, Jennifer R; Hume, Adam J; Cressey, Tessa N; Deflubé, Laure R; Ruedas, John B; Connor, John H; Ebihara, Hideki; Mühlberger, Elke

    2017-10-01

    Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  7. Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

    Science.gov (United States)

    Heintz-Buschart, Anna; Eickhoff, Holger; Hohn, Erwin; Bilitewski, Ursula

    2013-03-10

    Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Beyond reverse pharmacology: Mechanism-based screening of Ayurvedic drugs.

    Science.gov (United States)

    Lele, R D

    2010-10-01

    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.

  9. Human Vascular Microphysiological System for in vitro Drug Screening.

    Science.gov (United States)

    Fernandez, C E; Yen, R W; Perez, S M; Bedell, H W; Povsic, T J; Reichert, W M; Truskey, G A

    2016-02-18

    In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

  10. Toxicological screening of basic drugs in whole blood using UPLC-TOF-MS

    DEFF Research Database (Denmark)

    Dalsgaard, Petur Weihe; Rasmussen, Brian Schou; Müller, Irene Breum

    2012-01-01

    Ultra performance liquid chromatography (UPLC) coupled with time-of-flight (TOF) mass spectrometry (MS) was established for toxicological screening of basic drugs in whole blood and tested on authentic samples. Whole blood samples (0.2 ml) were extracted using a Gilson apparatus equipped with Bond...

  11. Screening for mental illness: the merger of eugenics and the drug industry.

    Science.gov (United States)

    Sharav, Vera Hassner

    2005-01-01

    The implementation of a recommendation by the President's New Freedom Commission (NFC) to screen the entire United States population--children first--for presumed, undetected, mental illness is an ill-conceived policy destined for disastrous consequences. The "pseudoscientific" methods used to screen for mental and behavioral abnormalities are a legacy from the discredited ideology of eugenics. Both eugenics and psychiatry suffer from a common philosophical fallacy that undermines the validity of their theories and prescriptions. Both are wed to a faith-based ideological assumption that mental and behavioral manifestations are biologically determined, and are, therefore, ameliorated by biological interventions. NFC promoted the Texas Medication Algorithm Project (TMAP) as a "model" medication treatment plan. The impact of TMAP is evident in the skyrocketing increase in psychotropic drug prescriptions for children and adults, and in the disproportionate expenditure for psychotropic drugs. The New Freedom Commission's screening for mental illness initiative is, therefore, but the first step toward prescribing drugs. The escalating expenditure for psychotropic drugs since TMAP leaves little doubt about who the beneficiaries of TMAP are. Screening for mental illness will increase their use.

  12. Drug screening in biological fluids - The need for a systematic approach

    NARCIS (Netherlands)

    de Zeeuw, R.A

    1997-01-01

    In this paper the key steps towards drug screening in biological fluids are considered: (i) sample work up-isolation-concentration: (ii) differentiation-detection; (iii) identification. For (i) solid-phase extraction has very good potential; for (ii) thin-layer chromatography, gas chromatography and

  13. Are Peripheral Blood Mononuclear Cells Derived from Patients with Certain Myopathies Suitable for Personalized Drug Screening?

    Directory of Open Access Journals (Sweden)

    Andriy V. Shatillo

    2014-12-01

    Full Text Available Background: Limb girdle muscular dystrophies (LGMDs and several other disorders which share their specific phenotype are rare, predominantly hereditary conditions with no curative treatment. Differential diagnosis of these myopathies is quite challenging and expensive in many cases. Therefore, a significant proportion of patients remains undiagnosed and untreated for a long time. At the same time there is a huge amount of drugs and supplements potentially able to modify the course of some of these muscular dystrophies. That is why a simple empirical approach able to define a patient’s reaction to a specific compound seems rational. Because most common basic pathogenetic mechanisms for these quite different disorders increase the vulnerability of muscle cells (or decrease ability for reparation during mechanical stress, we propose a simple, noninvasive and inexpensive approach for individualized drug screening based on the drug’s influence on the mechanical vulnerability of peripheral blood mononuclear cells (PBMC. Methods: PBMC derived from 8 patients with Duchenne muscular dystrophy (DMD, 2 patients with LGMD2A, 1 patient with LGMD2B, 1 with MERRF syndrome, 1 with facioscapulohumeral muscular dystrophy (FSHD and 13 matched control subjects were irradiated by ultrasound in the presence of several compounds (lisinopril, vitamin D3, prednisolon, tocopherol, topiramate, glutargin, α-lipoic acid, essentiale, and physiological solution. Then viability indexes of the samples were detected by citotoxic assays based on vital dye (neutral red and resazurin metabolism. Results: In cytotoxicity tests with active transport of neutral red into PBMC derived from DMD patients, the cells showed signs of destruction at 1.06±0.52 minutes of ultrasounding compared to 1.75±0.6 minutes in control. PBMCs from patients with other myopathies have either normal or decreased resistance to ultrasound. The addition of tocopherol significantly changes the PBMC

  14. A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

    DEFF Research Database (Denmark)

    Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune

    2015-01-01

    suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed...... plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. RESULTS: The assay detected specific and saturable immunoglobulin G (IgG) binding to each...... cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients...

  15. Beyond reverse pharmacology: Mechanism-based screening of Ayurvedic drugs

    Directory of Open Access Journals (Sweden)

    R D Lele

    2010-01-01

    Full Text Available 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.

  16. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    Science.gov (United States)

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

    2016-08-05

    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  17. Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Csoka, K; Larsson, R; Tholander, B; Gerdin, E; de la Torre, M; Nygren, P

    1994-08-01

    The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

  18. Prediction of phospholipidosis-inducing potential of drugs by in vitro biochemical and physicochemical assays followed by multivariate analysis.

    Science.gov (United States)

    Kuroda, Yukihiro; Saito, Madoka

    2010-03-01

    An in vitro method to predict phospholipidosis-inducing potential of cationic amphiphilic drugs (CADs) was developed using biochemical and physicochemical assays. The following parameters were applied to principal component analysis, as well as physicochemical parameters: pK(a) and clogP; dissociation constant of CADs from phospholipid, inhibition of enzymatic phospholipid degradation, and metabolic stability of CADs. In the score plot, phospholipidosis-inducing drugs (amiodarone, propranolol, imipramine, chloroquine) were plotted locally forming the subspace for positive CADs; while non-inducing drugs (chlorpromazine, chloramphenicol, disopyramide, lidocaine) were placed scattering out of the subspace, allowing a clear discrimination between both classes of CADs. CADs that often produce false results by conventional physicochemical or cell-based assay methods were accurately determined by our method. Basic and lipophilic disopyramide could be accurately predicted as a nonphospholipidogenic drug. Moreover, chlorpromazine, which is often falsely predicted as a phospholipidosis-inducing drug by in vitro methods, could be accurately determined. Because this method uses the pharmacokinetic parameters pK(a), clogP, and metabolic stability, which are usually obtained in the early stages of drug development, the method newly requires only the two parameters, binding to phospholipid, and inhibition of lipid degradation enzyme. Therefore, this method provides a cost-effective approach to predict phospholipidosis-inducing potential of a drug. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  19. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  20. Cardiac Arrhythmia: In vivo screening in the zebrafish to overcome complexity in drug discovery.

    Science.gov (United States)

    Macrae, Calum A

    2010-07-01

    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

  1. Using label-free screening technology to improve efficiency in drug discovery.

    Science.gov (United States)

    Halai, Reena; Cooper, Matthew A

    2012-02-01

    Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

  2. Novel heparan sulfate assay by using automated high-throughput mass spectrometry: Application to monitoring and screening for mucopolysaccharidoses.

    Science.gov (United States)

    Shimada, Tsutomu; Kelly, Joan; LaMarr, William A; van Vlies, Naomi; Yasuda, Eriko; Mason, Robert W; Mackenzie, William; Kubaski, Francyne; Giugliani, Roberto; Chinen, Yasutsugu; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji E; Fukao, Toshiyuki; Orii, Tadao; Tomatsu, Shunji

    2014-01-01

    Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity

  3. Validation of the direct analysis in real time source for use in forensic drug screening.

    Science.gov (United States)

    Steiner, Robert R; Larson, Robyn L

    2009-05-01

    The Direct Analysis in Real Time (DART) ion source is a relatively new mass spectrometry technique that is seeing widespread use in chemical analyses world-wide. DART studies include such diverse topics as analysis of flavors and fragrances, melamine in contaminated dog food, differentiation of writing inks, characterization of solid counterfeit drugs, and as a detector for planar chromatography. Validation of this new technique for the rapid screening of forensic evidence for drugs of abuse, utilizing the DART source coupled to an accurate mass time-of-flight mass spectrometer, was conducted. The study consisted of the determination of the lower limit of detection for the method, determination of selectivity and a comparison of this technique to established analytical protocols. Examples of DART spectra are included. The results of this study have allowed the Virginia Department of Forensic Science to incorporate this new technique into their analysis scheme for the screening of solid dosage forms of drugs of abuse.

  4. A screen to identify drug resistant variants to target-directed anti-cancer agents

    Directory of Open Access Journals (Sweden)

    Azam Mohammad

    2003-01-01

    Full Text Available The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec, a specific inhibitor of the Chronic Myeloid Leukemia (CML-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair.

  5. Exploiting pluripotent stem cell technology for drug discovery, screening, safety, and toxicology assessments.

    Science.gov (United States)

    McGivern, Jered V; Ebert, Allison D

    2014-04-01

    In order for the pharmaceutical industry to maintain a constant flow of novel drugs and therapeutics into the clinic, compounds must be thoroughly validated for safety and efficacy in multiple biological and biochemical systems. Pluripotent stem cells, because of their ability to develop into any cell type in the body and recapitulate human disease, may be an important cellular system to add to the drug development repertoire. This review will discuss some of the benefits of using pluripotent stem cells for drug discovery and safety studies as well as some of the recent applications of stem cells in drug screening studies. We will also address some of the hurdles that need to be overcome in order to make stem cell-based approaches an efficient and effective tool in the quest to produce clinically successful drug compounds. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. The effect of screening for cardio-renal risk factors on drug use in the general population

    NARCIS (Netherlands)

    Atthobari, J.; Gansevoort, R.T.; Visser, S.T.; De Jong, P.E.; de Jong-van den Berg, L.T.

    2007-01-01

    Aims To evaluate the effect of a cardio-renal screening programme on desired and undue drug use. Methods Data from the PREVEND cohort (Prevention of REnal and Vascular ENd-stage Disease) were used. The drug use of screened (randomly) selected subjects (n = 2650) was compared with unscreened

  7. An interferon-gamma release assay test performs well in routine screening for tuberculosis

    DEFF Research Database (Denmark)

    Vestergaard Danielsen, Allan; Fløe, Andreas; Lillebæk, Troels

    2014-01-01

    Introduction: A positive interferon-gamma release assay (IGRA) is regarded as proof of latent Mycobacterium tuberculosis infection. We conducted an evaluation of the IGRA test “T-SPOT.TB” to test its performance during clinical routine use by analysing the positivity rate and odds, effect of season...... and sensitivity. Material and methods: Data from T-SPOT.TB testing together with age and test indications (anti-tumour necrosis factor alpha (TNFα) candidate, contact investigation or suspicion of tuberculosis (TB)) were combined with mycobac­teria culture results. Results: A total of 1,809 patients were tested....... Conclusive results were achieved for 1,780 patients (98.4%). Among these, 4.6% of anti-TNFα candidates, 19.3% of contacts and 24.4% of TB suspects tested positive. Compared with anti-TNFα candidates, the odds for a positive result were significantly higher for contact investigations (odds ratio (OR), mean...

  8. Pharmacogenetic screening for polymorphisms in drug-metabolizing enzymes and drug transporters in a Dutch population.

    Science.gov (United States)

    Bosch, T M; Doodeman, V D; Smits, P H M; Meijerman, I; Schellens, J H M; Beijnen, J H

    2006-01-01

    A possible explanation for the wide interindividual variability in toxicity and efficacy of drug therapy is variation in genes encoding drug-metabolizing enzymes and drug transporters. The allelic frequency of these genetic variants, linkage disequilibrium (LD), and haplotype of these polymorphisms are important parameters in determining the genetic differences between patients. The aim of this study was to explore the frequencies of polymorphisms in drug-metabolizing enzymes (CYP1A1, CYP2C9, CYP2C19, CYP3A4, CYP2D6, CYP3A5, DPYD, UGT1A1, GSTM1, GSTP1, GSTT1) and drug transporters (ABCB1[MDR1] and ABCC2[MRP2]), and to investigate the LD and perform haplotype analysis of these polymorphisms in a Dutch population. Blood samples were obtained from 100 healthy volunteers and genomic DNA was isolated and amplified by PCR. The amplification products were sequenced and analyzed for the presence of polymorphisms by sequence alignment. In the study population, we identified 13 new single nucleotide polymorphisms (SNPs) in Caucasians and three new SNPs in non-Caucasians, in addition to previously recognized SNPs. Three of the new SNPs were found within exons, of which two resulted in amino acid changes (A428T in CYP2C9 resulting in the amino acid substitution D143V; and C4461T in ABCC2 in a non-Caucasian producing the amino acid change T1476M). Several LDs and haplotypes were found in the Caucasian individuals. In this Dutch population, the frequencies of 16 new SNPs and those of previously recognized SNPs were determined in genes coding for drug-metabolizing enzymes and drug transporters. Several LDs and haplotypes were also inferred. These data are important for further research to help explain the interindividual pharmacokinetic and pharmacodynamic variability in response to drug therapy.

  9. Use of a screening method to determine excipients which optimize the extent and stability of supersaturated drug solutions and application of this system to solid formulation design.

    Science.gov (United States)

    Vandecruys, Roger; Peeters, Jef; Verreck, Geert; Brewster, Marcus E

    2007-09-05

    Assessing the effect of excipients on the ability to attain and maintain supersaturation of drug-based solution may provide useful information for the design of solid formulations. Judicious selection of materials that affect either the extent or stability of supersaturating drug delivery systems may be enabling for poorly soluble drug candidates or other difficult-to-formulate compounds. The technique suggested herein is aimed at providing a screening protocol to allow preliminary assessment of these factors based on small to moderate amounts of drug substance. A series of excipients were selected that may, by various mechanisms, affect supersaturation including pharmaceutical polymers such as HMPC and PVP, surfactants such as Polysorbate 20, Cremophor RH40 and TPGS and hydrophilic cyclodextrins such as HPbetaCD. Using a co-solvent based method and 25 drug candidates, the data suggested, on the whole, that the surfactants and the selected cyclodextrin seemed to best augment the extent of supersaturation but had variable benefits as stabilizers, while the pharmaceutical polymers had useful effect on supersaturation stability but were less helpful in increasing the extent of supersaturation. Using these data, a group of simple solid dosage forms were prepared and tested in the dog for one of the drug candidates. Excipients that gave the best extent and stability for the formed supersaturated solution in the screening assay also gave the highest oral bioavailability in the dog.

  10. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    Science.gov (United States)

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

  11. Estimating Fitness by Competition Assays between Drug Susceptible and Resistant Mycobacterium tuberculosis of Predominant Lineages in Mumbai, India

    Science.gov (United States)

    Bhatter, Purva; Chatterjee, Anirvan; D'souza, Desiree; Tolani, Monica; Mistry, Nerges

    2012-01-01

    Background Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages. Methodology Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system. Conclusion/Significance While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains. PMID:22479407

  12. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India

    Directory of Open Access Journals (Sweden)

    Anand Kumar Maurya

    2013-01-01

    Full Text Available Background: The problem of multi-drug resistance tuberculosis (MDR-TB is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%, 52 (94.5% and 17 (30.9% strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05. The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3% in S531L, 52 (94.5% in S315T1 and 11 (20% in C15T regions respectively (P < 0.05. Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

  13. Enantioselective assay for therapeutic drug monitoring of eslicarbazepine acetate: no interference with carbamazepine and its metabolites.

    Science.gov (United States)

    Alves, Gilberto; Fortuna, Ana; Sousa, Joana; Direito, Rosa; Almeida, Anabela; Rocha, Marília; Falcão, Amílcar; Soares-da-Silva, Patrício

    2010-08-01

    As add-on therapy, phase III clinical trials of eslicarbazepine acetate (ESL) conducted in patients with refractory partial-onset seizures have shown good efficacy, safety, and tolerability, even in patients taking carbamazepine (CBZ) at baseline (approximately 60% of the enrolled patients). Thus, considering the pharmacological disadvantages of CBZ and the similar efficacy spectrum of CBZ and ESL, switching to ESL may be successful in many patients. As ESL is a prodrug almost instantaneously converted to S-licarbazepine (S-Lic; approximately 95%), an interest in therapeutic drug monitoring (TDM) of S-Lic is likely to develop in the future. This study investigated the plasma concentrations of S-Lic and R-licarbazepine (R-Lic) enantiomers in patients under CBZ long-term treatment to assess the potential interference of CBZ or its metabolites in the enantioselective TDM of ESL (using S-Lic concentrations). A chiral high-performance liquid chromatography assay with ultraviolet detection (HPLC-UV) previously developed and validated by our research group was used. Twenty-four patients admitted to the Coimbra University Hospital and supposedly receiving CBZ long-term treatment were identified. Blood samples were collected from patients and serum CBZ concentrations were measured by the usual TDM protocol. Aliquots of plasma from such patients were also submitted to a chiral HPLC-UV analysis. The bioanalytical data indicated that S-Lic and R-Lic were not present at detectable concentrations in plasma samples of the CBZ-treated patients. The chromatograms generated by the analysis of patient plasma samples, when compared with those obtained from blank plasma samples spiked with S-Lic and R-Lic, clearly showed the absence of interferences at the retention times of both Lic enantiomers. These data support the usefulness of the chiral HPLC-UV method used for the enantioselective TDM of ESL (using S-Lic) for programs in which switching from CBZ to ESL is implemented.

  14. Transgenic Plants as Low-Cost Platform for Chemotherapeutic Drugs Screening

    Directory of Open Access Journals (Sweden)

    Daniele Vergara

    2015-01-01

    Full Text Available 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.

  15. Tackling heterogeneity: a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco.

    Directory of Open Access Journals (Sweden)

    Natalia Piotrzkowski

    Full Text Available Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

  16. Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

    Science.gov (United States)

    Matavulj, M; Molitoris, H P

    1992-12-01

    The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Drug Repurposing Screening Identifies Novel Compounds That Effectively Inhibit Toxoplasma gondii Growth

    Science.gov (United States)

    Dittmar, Ashley J.; Drozda, Allison A.

    2016-01-01

    ABSTRACT The urgent need to develop new antimicrobial therapies has spawned the development of repurposing screens in which well-studied drugs and other types of compounds are tested for potential off-label uses. As a proof-of-principle screen to identify compounds effective against Toxoplasma gondii, we screened a collection of 1,120 compounds for the ability to significantly reduce Toxoplasma replication. A total of 94 compounds blocked parasite replication with 50% inhibitory concentrations of parasite invasion and replication but did so independently of inhibition of dopamine or other neurotransmitter receptor signaling. Tamoxifen, which is an established inhibitor of the estrogen receptor, also reduced parasite invasion and replication. Even though Toxoplasma can activate the estrogen receptor, tamoxifen inhibits parasite growth independently of this transcription factor. Tamoxifen is also a potent inducer of autophagy, and we find that the drug stimulates recruitment of the autophagy marker light chain 3-green fluorescent protein onto the membrane of the vacuolar compartment in which the parasite resides and replicates. In contrast to other antiparasitic drugs, including pimozide, tamoxifen treatment of infected cells leads to a time-dependent elimination of intracellular parasites. Taken together, these data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this obligate intracellular parasite. IMPORTANCE There is an urgent need to develop new therapies to treat microbial infections, and the repurposing of well-characterized compounds is emerging as one approach to achieving this goal. Using the protozoan parasite Toxoplasma gondii, we screened a library of 1,120 compounds and identified several compounds with significant antiparasitic activities. Among these were pimozide and tamoxifen, which are well-characterized drugs prescribed to treat patients with psychiatric disorders and breast cancer

  18. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    2010-08-01

    Full Text Available High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  19. Evaluation of laser diode thermal desorption (LDTD) coupled with tandem mass spectrometry (MS/MS) for support of in vitro drug discovery assays: increasing scope, robustness and throughput of the LDTD technique for use with chemically diverse compound libraries.

    Science.gov (United States)

    Beattie, Iain; Smith, Aaron; Weston, Daniel J; White, Peter; Szwandt, Simon; Sealey, Laura

    2012-02-05

    Within the drug discovery environment, the key process in optimising the chemistry of a structural series toward a potential drug candidate is the design, make and test cycle, in which the primary screens consist of a number of in vitro assays, including metabolic stability, cytochrome P450 inhibition, and time-dependent inhibition assays. These assays are often carried out using multiple drug compounds with chemically diverse structural features, often in a 96 well-plate format for maximum time-efficiency, and are supported using rapid liquid chromatographic (LC) sample introduction with a tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) endpoint, taking around 6.5 h per plate. To provide a faster time-to-decision at this critical point, there exists a requirement for higher sample throughput and a robust, well-characterized analytical alternative. This paper presents a detailed evaluation of laser diode thermal desorption (LDTD), a relatively new ambient sample ionization technique, for compound screening assays. By systematic modification of typical LDTD instrumentation and workflow, and providing deeper understanding around overcoming a number of key issues, this work establishes LDTD as a practical, rapid alternative to conventional LC-MS/MS in drug discovery, without need for extensive sample preparation or expensive, scope-limiting internal standards. Analysis of both the five and three cytochrome P450 competitive inhibition assay samples by LDTD gave improved sample throughput (0.75 h per plate) and provided comparable data quality as the IC₅₀ values obtained were within 3 fold of those calculated from the LC-MS/MS data. Additionally when applied generically to a chemically diverse library of over 250 proprietary compounds from the AstraZeneca design, make and test cycle, LDTD demonstrated a success rate of 98%. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. A comprehensive review of assay methods to determine drugs in breast milk and the safety of breastfeeding when taking drugs.

    Science.gov (United States)

    Fríguls, Bibiana; Joya, Xavier; García-Algar, Oscar; Pallás, C R; Vall, Oriol; Pichini, Simona

    2010-06-01

    Most of the licit and illicit drugs consumed by the breastfeeding woman pass into the milk and can modify the production, volume and composition of the milk, as well as hypothetically have short- and long-term harmful effects on the infant. There is much confusion in the scientific community regarding this issue: should a woman breastfeed her baby while continuing to use prescription drugs and/or drugs of abuse? There are many case reports of clinically significant toxicity in breast-fed infants from some substances used by mothers (such as irritability, vomiting, sedation, respiratory depression, shock), but there are too few data on studies conducted in breastfeeding women and their infants to make a realistic risk assessment. The objective measurement of a drug and/or metabolites in maternal milk is the first step when investigating the amount of drug excreted in milk and subsequently calculating the daily dose administered to the breast-fed infant. The present review reports the analytical methods developed to detect different drugs in the breast milk, listing the principal characteristics and validation parameters, advantages and disadvantages. Furthermore, the mechanisms of drug transfer into breast milk are discussed, the correlation between the concentration of the drug in breast milk and potential adverse outcomes on the infant are described for each drug, and suggested harm minimization strategies and approved breastfeeding recommendations are indicated.

  1. In vitro testing of chemotherapeutic drug combinations in acute myelocytic leukaemia using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Larsson, R; Fridborg, H; Kristensen, J; Sundström, C; Nygren, P

    1993-05-01

    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.

  2. Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays.

    Science.gov (United States)

    Hau, Jean Christophe; Fontana, Patrizia; Zimmermann, Catherine; De Pover, Alain; Erdmann, Dirk; Chène, Patrick

    2011-06-01

    The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

  3. [Comparison of the clinical performance of the ECLusys HIV combi assay with the Lumipulse f and HISCL 2000-i HIV-1/2 ab screening assays].

    Science.gov (United States)

    Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

    2012-04-01

    We compared the ECLusys HIV combi assay (ECL HIV Ag/Ab) to the Lumipulse Forte (LPf HIV 1/2 Ab) and HISCL (HIS HIV 1/2 Ab) assays. In a dilution sensitivity test using dilution panels of WHO HIV antibody international reference panel (HIV-1 Subtype A, B, C, E, HIV-1 Group O, HIV-2) and HIV-1/2 Ab CE marked material(HIV-1, HIV-2) parent specimens, the ECL assay enabled detection at a higher level of sensitivity than either the LPf assay or the HIS assay for all dilution panels. In an early detection test in the early phase of infection in which a BBI HIV seroconversion panel was used, the ECL assay enabled detection 7 days after initial blood sample collection, whereas the LPf and HIS assays enabled detection after 27 days. In a specificity test using high RF positive specimens (n=33), pregnancy specimens (n=35), cytomegalovirus antibody positive specimens (n=36), and high M protein positive specimens (n=21) that were confirmed negative for HIV-1/2 antibodies by the LPf assay, negative results were obtained for all specimens on both the ECL assay and the HIS assay. In a correlation test using routinely collected clinical specimens (n=121), including positive stock specimens, the ECL and HIS assays demonstrated the highest agreement rate 98.3%. The above results confirmed that the fourth-generation reagent ECL assay, which simultaneously detects both HIV-1/2 antibodies and p24 antigens, is both highly sensitive and specific, and is a suitable assay for use in routine testing.

  4. Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Strange, Mette; Bräuner-Osborne, Hans; Jensen, Anders A.

    2006-01-01

    We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay...... assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors....

  5. Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

    Science.gov (United States)

    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. Copyright 2009 Wiley-Liss, Inc.

  6. Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

    Science.gov (United States)

    Meng, Juncai; Lai, Ming-Tain; Munshi, Vandna; Grobler, Jay; McCauley, John; Zuck, Paul; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Adam, Gregory C

    2015-06-01

    HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. © 2015 Society for Laboratory Automation and Screening.

  7. Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Zor, Kinga; Canepa, Silvia

    2015-01-01

    We investigated the combined effect of the initial cell density (12 500, 35 000, 75 000, and 100 000 cells cm−2) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing timedependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation...... between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell–substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity...... was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell...

  8. Evaluation of California's Alcohol and Drug Screening and Brief Intervention Project for Emergency Department Patients

    Directory of Open Access Journals (Sweden)

    Susan I Woodruff

    2013-05-01

    Full Text Available Introduction: Visits to settings such as emergency departments (EDs may present a “teachable moment” in that a patient may be more open to feedback and suggestions regarding their risky alcohol and illicit drug-use behaviors. Screening, Brief Intervention, and Referral to Treatment (SBIRT is an ’opportunistic’ public health approach that targets low-risk users, in addition to those already dependent on alcohol and/or drugs. SBIRT programs provide patients with comprehensive screening and assessments, and deliver interventions of appropriate intensity to reduce risks related to alcohol and drug use. Methods: This study used a single group pre-post test design to assess the effect of the California SBIRT service program (i.e., CASBIRT on 6 substance-use outcomes (past-month prevalence and number of days of binge drinking, illegal drug use, and marijuana use. Trained bilingual/bicultural Health Educators attempted to screen all adult patients in 12 EDs/trauma centers (regardless of the reason for the patient’s visit using a short instrument, and then delivered a brief motivational intervention matched to the patient’s risk level. A total of 2,436 randomly selected patients who screened positive for alcohol and/or drug use consented to be in a 6-month telephone follow-up interview. Because of the high loss to follow-up rate, we used an intention-to-treat approach for the data analysis. Results: Results of generalized linear mixed models showed modest reductions in all 6 drug- and alcohol-use outcomes. Men (versus women, those at relatively higher risk status (versus lower risk, and those with only one substance of misuse (versus both alcohol and illicit drug misuse tended to show more positive change. Conclusion: These results suggest that SBIRT services provided in acute care settings are associated with modest changes in self-reported recent alcohol and illicit drug use. [West J Emerg Med. 2013;14(3:263–270.

  9. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

  10. [Comparison of the clinical performance of the ECLusys HBsAg II assay with the Lumipulse f and HISCL 2000-i HBsAg screening assays].

    Science.gov (United States)

    Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

    2012-02-01

    We compared the ECLusys HBsAgII (ECL HBsAg) assay to the Lumipulse Forte (LPf HBsAg) and HISCL (HIS HBsAg) assays. Measurement of dilution panels for which the WHO HBsAg international reference panel was the parent specimen revealed that the ECL and HIS assays enabled detection to a theoretical level of 0.04 IU/mL, whereas the LPf assay enabled detection to a level of 0.08 IU/mL. In a specificity test using high RF positive specimens (n = 33), pregnancy specimens (n = 35), cytomegalovirus antibody positive specimens (n = 36), and high M protein positive specimens (n = 21) that were confirmed negative for HBsAg by the LPf assay, negative results were obtained for all specimens on the HIS assay, but the ECL assay yielded a positive result for one of the high RF positive specimens. This individual was suggested on further testing to be an HBV carrier who was strongly positive for HBc antibody. In HBsAg mutants detection test, the detection rate was 92.3% with the ECL assay and 69.2% with the HIS assay. In a correlation test using routinely collected clinical specimens (n = 155), including positive stock specimens, aside from the one case where the LPf assay gave a negative result but both the ECL and HIS assays gave positive results, all of the results were consistent for all specimens. The above results confirmed that the ECL assay is both highly sensitive and specific, and also enables a high rate of HBsAg mutant detection.

  11. Feasibility and accuracy evaluation of three human papillomavirus assays for FTA card-based sampling: a pilot study in cervical cancer screening

    OpenAIRE

    Wang, Shao-Ming; Hu, Shang-Ying; Chen, Wen; Chen, Feng; Zhao, Fang-Hui; He, Wei; Ma, Xin-Ming; Zhang, Yu-Qing; Wang, Jian; Sivasubramaniam, Priya; Qiao, You-Lin

    2015-01-01

    Background Liquid-state specimen carriers are inadequate for sample transportation in large-scale screening projects in low-resource settings, which necessitates the exploration of novel non-hazardous solid-state alternatives. Studies investigating the feasibility and accuracy of a solid-state human papillomavirus (HPV) sampling medium in combination with different down-stream HPV DNA assays for cervical cancer screening are needed. Methods We collected two cervical specimens from 396 women, ...

  12. Rapid screening of pharmaceutical drugs using thermal desorption – SALDI mass spectrometry

    International Nuclear Information System (INIS)

    Grechnikov, A A; Kubasov, A E; Borodkov, A S; Georgieva, V B; Nikiforov, S M; Simanovsky, Ya O; Alimpiev, S S

    2012-01-01

    A novel approach to the rapid screening of pharmaceutical drugs by surface assisted laser desorption-ionization (SALDI) mass spectrometry with the rotating ball interface coupled with temperature programmed thermal desorption has been developed. Analytes were thermally desorbed and deposited onto the surface of amorphous silicon substrate attached to the rotating ball. The ball was rotated and the deposited analytes were analyzed using SALDI. The effectiveness of coupling SALDI mass spectrometry with thermal desorption was evaluated by the direct and rapid analysis of tablets containing lidocaine, diphenhydramine and propranolol without any sample pretreatment. The overall duration of the screening procedure was 30÷40 sec. Real urine samples were studied for drug analysis. It is shown that with simple preparation steps, urine samples can be quantitatively analyzed using the proposed technique with the detection limits in the range of 0.2÷0.5 ng/ml.

  13. Alcohol screening and brief intervention among drug users in primary care: a discussion paper.

    LENUS (Irish Health Repository)

    Field, C A

    2011-08-24

    BACKGROUND: Problem alcohol use is common among problem drug users (PDU) and associated with adverse health outcomes. Primary care has an important role in the overall stepped approach to alcohol treatment, especially screening and brief intervention (SBI). AIM: To discuss three themes that emerged from an exploration of the literature on SBI for problem alcohol use in drug users attending primary care. METHODS: Material for this discussion paper was gathered from three biomedical databases (PubMed, PsycINFO and Cochrane library), conference proceedings and online resources of professional organisations or national health agencies. RESULTS: Themes discussed in this paper are: (a) the potential of primary care for delivery of alcohol SBIs to PDUs, (b) screening methods and (c) application of brief interventions to PDUs. CONCLUSIONS: Although SBI improves health outcomes associated with problem alcohol use in the general population, further research is needed among high-risk patient groups, especially PDUs.

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

    Directory of Open Access Journals (Sweden)

    Atsuya Yamashita

    2015-11-01

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

  15. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    Science.gov (United States)

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

  16. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  17. Data mining a small molecule drug screening representative subset from NIH PubChem.

    Science.gov (United States)

    Xie, Xiang-Qun; Chen, Jian-Zhong

    2008-03-01

    PubChem is a scientific showcase of the NIH Roadmap Initiatives. It is a compound repository created to facilitate information exchange and data sharing among the NIH Roadmap-funded Molecular Library Screening Center Network (MLSCN) and the scientific community. However, PubChem has more than 10 million records of compound information. It will be challenging to conduct a drug screening of the whole database of millions of compounds. Thus, the purpose of the present study was to develop a data mining cheminformatics approach in order to construct a representative and structure-diverse sublibrary from the large PubChem database. In this study, a new chemical diverse representative subset, rePubChem, was selected by whole-molecule chemistry-space matrix calculation using the cell-based partition algorithm. The representative subset was generated and was then subjected to evaluations by compound property analyses based on 1D and 2D molecular descriptors. The new subset was also examined and assessed for self-similarity analysis based on 2D molecular fingerprints in comparing with the source compound library. The new subset has a much smaller library size (540K compounds) with minimum similarity and redundancy without loss of the structural diversity and basic molecular properties of its parent library (5.3 million compounds). The new representative subset library generated could be a valuable structure-diverse compound resource for in silico virtual screening and in vitro HTS drug screening. In addition, the established subset generation method of using the combined cell-based chemistry-space partition metrics with pairwised 2D fingerprint-based similarity search approaches will also be important to a broad scientific community interested in acquiring structurally diverse compounds for efficient drug screening, building representative virtual combinatorial chemistry libraries for syntheses, and data mining large compound databases like the PubChem library in general.

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

    OpenAIRE

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

    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 dyst...

  19. Stem cells: a model for screening, discovery and development of drugs

    OpenAIRE

    Kitambi, Satish Srinivas; Chandrasekar, Gayathri

    2011-01-01

    Satish Srinivas Kitambi1, Gayathri Chandrasekar21Department of Medical Biochemistry and Biophysics; 2Department of Biosciences, Karolinska Institutet, Stockholm, SwedenAbstract: The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficac...

  20. NMR screening in fragment-based drug design: a practical guide.

    Science.gov (United States)

    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 (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.

  1. The value of microscopic-observation drug susceptibility assay in the diagnosis of tuberculosis and detection of multidrug resistance.

    Science.gov (United States)

    Sertel Şelale, Denİz; Uzun, Meltem

    2018-01-01

    Inexpensive, rapid, and reliable tests for detecting the presence and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are urgently needed to control the transmission of tuberculosis. In this study, we aimed to assess the accuracy and speed of the microscopic-observation drug susceptibility (MODS) assay in the identification of MTBC and detection of multidrug resistance. Sputum samples from patients suspected to have tuberculosis were simultaneously tested with MODS and conventional culture [Löwenstein-Jensen (LJ) culture, BACTEC MGIT™ 960 (MGIT) system], and drug susceptibility testing (MGIT system) methods. A total of 331 sputum samples were analyzed. Sensitivity and specificity of MODS assay for detection of MTBC strains were 96% and 98.8%, respectively. MODS assay detected multidrug resistant MTBC isolates with 92.3% sensitivity and 96.6% specificity. Median time to culture positivity was similar for MGIT (8 days) and MODS culture (8 days), but was significantly longer with LJ culture (20 days) (p tuberculosis and detection of multidrug resistance. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  2. Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

    Directory of Open Access Journals (Sweden)

    Robson L. F. Amaral

    2017-08-01

    Full Text Available Introduction: Cell-based assays using three-dimensional (3D cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better.Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates, and hanging drop (HD methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL, and then, subjected to drug resistance evaluation.Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D cultures. The optimal range of spheroid diameter (300–500 μm was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively compared to 2D cultures (IC50 ranging from 0.39 to 0.43.Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

  3. Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

    Science.gov (United States)

    Amaral, Robson L F; Miranda, Mariza; Marcato, Priscyla D; Swiech, Kamilla

    2017-01-01

    Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 10 4 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 10 4 cells/mL for the ULA method and 2.5 and 3.75 × 10 4 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC 50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC 50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

  4. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

    Science.gov (United States)

    Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

    2002-08-01

    Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

  6. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions.

    Science.gov (United States)

    Corti, Daniele; Galbiati, Valentina; Gatti, Nicolò; Marinovich, Marina; Galli, Corrado L; Corsini, Emanuela

    2015-10-01

    Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1-2mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-β and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure-activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Resolution V fractional factorial Design for Screening of factors affecting weakly basic drugs liposomal systems.

    Science.gov (United States)

    El-Helaly, Sara Nageeb; Habib, Basant A; Abd El-Rahman, Mohamed K

    2018-04-21

    This study aims to investigate factors affecting weakly basic drugs liposomal systems. Resolution V fractional factorial design (2 V 5-1 ) is used as an example of screening designs that would better be used as a wise step before proceeding with detailed factors effects or optimization studies. Five factors probable to affect liposomal systems of weakly basic drugs were investigated using Amisulpride as a model drug. Factors studied were; A: Preparation technique B: Phosphatidyl choline (PhC) amount (mg) C: Cholesterol: PhC molar ratio, D: Hydration volume (ml) and E: Sonication type. Levels investigated were; Ammonium sulphate-pH gradient technique or Transmembrane zinc chelation-pH gradient technique, 200 or 400 mg, 0 or 0.5, 10 or 20 ml and bath or probe sonication for A, B, C, D and E respectively. Responses measured were Particle size (PS) (nm), Zeta potential (ZP) (mV) and Entrapment efficiency percent (EE%). Ion selective electrode was used as a novel method for measuring unentrapped drug concentration and calculating entrapment efficiency without the need for liposomal separation. Factors mainly affecting the studied responses were Cholesterol: PhC ratio and hydration volume for PS, preparation technique for ZP and preparation technique and hydration volume for EE%. The applied 2 V 5-1 design enabled the use of only 16 trial combinations for screening the influence of five factors on weakly basic drugs liposomal systems. This clarifies the value of the use of screening experiments before extensive investigation of certain factors in detailed optimization studies. Copyright © 2017. Published by Elsevier B.V.

  8. Stem cells as a novel tool for drug screening and treatment of degenerative diseases.

    Science.gov (United States)

    Zuba-Surma, Ewa K; Wojakowski, Wojciech; Madeja, Zbigniew; Ratajczak, Mariusz Z

    2012-01-01

    Degenerative diseases similarly as acute tissue injuries lead to massive cell loss and may cause organ failure of vital organs (e.g., heart, central nervous system). Therefore, they belong to a group of disorders that may significantly benefit from stem cells (SCs)-based therapies. Several stem and progenitor cell populations have already been described as valuable tools for developing therapeutic strategies in regenerative medicine. In particular, pluripotent stem cells (PSCs), including adult-tissue-derived PSCs, neonatal-tissue-derived SCs, embryonic stem cells (ESCs), and recently described induced pluripotent stem cells (iPSCs), are the focus of particular attention because of their capacity to differentiate into all the cell lineages. Although PSCs are predominantly envisioned to be applied for organ regeneration, they may be also successfully employed in drug screening and disease modeling. In particular, adult PSCs and iPSCs derived from patient tissues may not only be a source of cells for autologous therapies but also for individual customized in vitro drug testing and studies on the molecular mechanisms of disease. In this review, we will focus on the potential applications of SCs, especially PSCs i) in regenerative medicine therapies, ii) in studying mechanisms of disease, as well as iii) in drug screening and toxicology tests that are crucial in new drug development. In particular, we will discuss the application of SCs in developing new therapeutic approaches to treat degenerative diseases of the neural system and heart. The advantage of adult PSCs in all the above-mentioned settings is that they can be directly harvested from patient tissues and used not only as a safe non-immunogenic source of cells for therapy but also as tools for personalized drug screening and pharmacological therapies.

  9. Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: collaborative study

    International Nuclear Information System (INIS)

    Charm, S.E.; Chi, R.

    1988-01-01

    A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. In this method, microbial cells added to a milk sample provide specific binding sites for which 14 C or 3 H labeled drug competes with drug resides in the sample. The 14 C or 3 H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-amino-benzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action

  10. A multilayer microdevice for cell-based high-throughput drug screening

    International Nuclear Information System (INIS)

    Liu, Chong; Wang, Lei; Li, Jingmin; Ding, Xiping; Chunyu, Li; Xu, Zheng; Wang, Qi

    2012-01-01

    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. (paper)

  11. Toxicophore exploration as a screening technology for drug design and discovery: techniques, scope and limitations.

    Science.gov (United States)

    Singh, Pankaj Kumar; Negi, Arvind; Gupta, Pawan Kumar; Chauhan, Monika; Kumar, Raj

    2016-08-01

    Toxicity is a common drawback of newly designed chemotherapeutic agents. With the exception of pharmacophore-induced toxicity (lack of selectivity at higher concentrations of a drug), the toxicity due to chemotherapeutic agents is based on the toxicophore moiety present in the drug. To date, methodologies implemented to determine toxicophores may be broadly classified into biological, bioanalytical and computational approaches. The biological approach involves analysis of bioactivated metabolites, whereas the computational approach involves a QSAR-based method, mapping techniques, an inverse docking technique and a few toxicophore identification/estimation tools. Being one of the major steps in drug discovery process, toxicophore identification has proven to be an essential screening step in drug design and development. The paper is first of its kind, attempting to cover and compare different methodologies employed in predicting and determining toxicophores with an emphasis on their scope and limitations. Such information may prove vital in the appropriate selection of methodology and can be used as screening technology by researchers to discover the toxicophoric potentials of their designed and synthesized moieties. Additionally, it can be utilized in the manipulation of molecules containing toxicophores in such a manner that their toxicities might be eliminated or removed.

  12. Screens

    OpenAIRE

    2016-01-01

    This Sixth volume in the series The Key Debates. Mutations and Appropriations in European Film Studies investigates the question of screens in the context both of the dematerialization due to digitalization and the multiplication of media screens. Scholars offer various infomations and theories of topics such as the archeology of screen, film and media theories, contemporary art, pragmatics of new ways of screening (from home video to street screening).

  13. Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

    Directory of Open Access Journals (Sweden)

    Kamau Edwin

    2012-01-01

    Full Text Available Abstract Background Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. Methods TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD for each assay. Results Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. Conclusion TaqMan Allelic Discrimination assay provides a good alternative tool in

  14. A Clinical Drug Library Screen Identifies Tosufloxacin as Being Highly Active against Staphylococcus aureus Persisters

    Directory of Open Access Journals (Sweden)

    Hongxia Niu

    2015-07-01

    Full Text Available To identify effective compounds that are active against Staphylococcus aureus (S. aureus persisters, we screened a clinical drug library consisting of 1524 compounds and identified six drug candidates that had anti-persister activity: tosufloxacin, clinafloxacin, sarafloxacin, doxycycline, thiostrepton, and chlorosalicylanilide. Among them, tosufloxacin had the highest anti-persister activity, which could completely eradicate S. aureus persisters within 2 days in vitro. Clinafloxacin ranked the second with very few persisters surviving the drug exposure. Interestingly, we found that both tosufloxacin and trovafloxacin that had high activity against persisters contained at the N-1 position the 2,4-difluorophenyl group, which is absent in other less active quinolones and may be associated with the high anti-persister activity. Further studies are needed to evaluate tosufloxacin in animal models and to explain its unique activity against bacterial persisters. Our findings may have implications for improved treatment of persistent bacterial infections.

  15. Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

    Science.gov (United States)

    Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko

    2017-01-01

    In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umb