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Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery  

PubMed Central

Abstract The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z?-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z?-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery. PMID:22050430

Che, Pulin; Cui, Long; Kutsch, Olaf; Cui, Liwang



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

PubMed Central

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

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



Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes  

PubMed Central

Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays. PMID:24189262

Schoone, Gerard J.; England, Paul; Faber, Dorien; Orrling, Kristina M.; Dujardin, Jean-Claude; Sundar, Shyam; Schallig, Henk D. F. H.; Adams, Emily R.



Miniaturized Assay for Solubility and Residual Solid Screening (SORESOS) in Early Drug Development  

Microsoft Academic Search

\\u000a Purpose  The aim was to develop a miniaturized method for solubility and residual solid screening of drug compounds in aqueous and\\u000a non-aqueous vehicles in early drug development.\\u000a \\u000a \\u000a \\u000a Methods  Different crystal modifications of caffeine, carbamazepine, and piroxicam were added into 96-well filter plates and solubility\\u000a was determined in 100 ?l of 17 pharmaceutical vehicles. After filtration, drug concentration was determined by Ultra Performance\\u000a Liquid

Nicole Wyttenbach; Jochem Alsenz; Olaf Grassmann



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

E-print Network

Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity, WI, USA, 53706 Abstract Evaluation of drug cardiotoxicity is essential to the safe development identifying lead compounds from drug screens is to measure their cardiotoxicity. Numerous lead compounds

Beebe, David J.


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


Drug-induced liver injury (DILI) is a major cause of drug failures in both the preclinical and clinical phase. Consequently, improving prediction of DILI at an early stage of drug discovery will reduce the potential failures in the subsequent drug development program. In this regard, high-content screening (HCS) assays are considered as a promising strategy for the study of DILI; however, the predictive performance of HCS assays is frequently insufficient. In the present study, a new testing strategy was developed to improve DILI prediction by employing in vitro assays that was combined with the RO2 model (i.e., 'rule-of-two' defined by daily dose ?100 mg/day & logP ?3). The RO2 model was derived from the observation that high daily doses and lipophilicity of an oral medication were associated with significant DILI risk in humans. In the developed testing strategy, the RO2 model was used for the rational selection of candidates for HCS assays, and only the negatives predicted by the RO2 model were further investigated by HCS. Subsequently, the effects of drug treatment on cell loss, nuclear size, DNA damage/fragmentation, apoptosis, lysosomal mass, mitochondrial membrane potential, and steatosis were studied in cultures of primary rat hepatocytes. Using a set of 70 drugs with clear evidence of clinically relevant DILI, the testing strategy improved the accuracies by 10 % and reduced the number of drugs requiring experimental assessment by approximately 20 %, as compared to the HCS assay alone. Moreover, the testing strategy was further validated by including published data (Cosgrove et al. in Toxicol Appl Pharmacol 237:317-330, 2009) on drug-cytokine-induced hepatotoxicity, which improved the accuracies by 7 %. Taken collectively, the proposed testing strategy can significantly improve the prediction of in vitro assays for detecting DILI liability in an early drug discovery phase. PMID:24958025

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



Establishment of a novel experimental protocol for drug-induced seizure liability screening based on a locomotor activity assay in zebrafish.  


As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery. PMID:25056783

Koseki, Naoteru; Deguchi, Jiro; Yamashita, Akihito; Miyawaki, Izuru; Funabashi, Hitoshi



Evaluation and validation according to international standards of the Delvotest ® SP-NT screening assay for antimicrobial drugs in milk  

Microsoft Academic Search

Two new microbial inhibition assays for the screening of antibacterial substances in milk have been validated in accordance with the ISO\\/IDF 183 international standard. The detection limits for the 10 antimicrobial substances (penicillin, ampicillin, amoxicillin, cephapirin, ceftiofur, cloxacillin, sulphadiazine, oxytetracycline, neomycin and erythromycin) tested by the Delvotest® SP-NT (sulphur-penicillin test no tablet) (ampoule and multi-plate formats) were found to be

S. L. Stead; H. Ashwin; S. F. Richmond; M. Sharman; P. C. Langeveld; J. P. Barendse; J. Stark; B. J. Keely



GAPDH binders as potential drugs for the therapy of polyglutamine diseases: Design of a new screening assay.  


Proteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and -(-)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia. PMID:25625921

Lazarev, Vladimir F; Benken, Konstantin A; Semenyuk, Pavel I; Sarantseva, Svetlana V; Bolshakova, Olga I; Mikhaylova, Elena R; Muronetz, Vladimir I; Guzhova, Irina V; Margulis, Boris A



A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening  

PubMed Central

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. PMID:23300705

Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio



An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.  


Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. PMID:24054573

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K



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


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. PMID:25074925

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



A High Throughput Screening Assay for Fungicidal Compounds against  

PubMed Central

Cryptococcus neoformans is a pathogenic fungus that causes meningitis world-wide, particularly in HIV-infected individuals. Although amphotericin B is the “gold standard” treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasizes a need for development of new anti-cryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anti-cryptococcal drugs for this disease. A high throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening a library of pharmaceutically active compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4 that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high throughput screening of fungicidal compounds under nutrient-limiting conditions for new anti-cryptococcal drug development. PMID:23896686

Dehdashti, Jean; Henderson, Christina; Zelazny, Adrian; Metallo, Steven J; Zheng, Wei; Williamson, Peter R.



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

E-print Network

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

Hammock, Bruce D.


Protein Reporter Bioassay Systems for the Phenotypic Screening of Candidate Drugs: A Mouse Platform for Anti-Aging Drug Screening  

PubMed Central

Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery. PMID:22438730

Chiba, Takuya; Tsuchiya, Tomoshi; Mori, Ryoichi; Shimokawa, Isao



Screening of drug candidates for their drug–drug interaction potential  

Microsoft Academic Search

Within the past year, additional papers have been published that focus on higher-throughput drug-interaction screening. Some papers have described enzyme assays that can be used to evaluate inhibition or induction of the human cytochrome P450s. At the same time, numerous investigators have developed computational (in silico) methods to predict interactions and have validated the approach using in vitro (assay-derived) data.

A. David Rodrigues; Jiunn H Lin



A phenotypic compound screening assay for lysosomal storage diseases.  


The lysosome is a vital cellular organelle that primarily functions as a recycling center for breaking down unwanted macromolecules through a series of hydrolases. Functional deficiencies in lysosomal proteins due to genetic mutations have been found in more than 50 lysosomal storage diseases that exhibit characteristic lipid/macromolecule accumulation and enlarged lysosomes. Recently, the lysosome has emerged as a new therapeutic target for drug development for the treatment of lysosomal storage diseases. However, a suitable assay for compound screening against the diseased lysosomes is currently unavailable. We have developed a Lysotracker staining assay that measures the enlarged lysosomes in patient-derived cells using both fluorescence intensity readout and fluorescence microscopic measurement. This phenotypic assay has been tested in patient cells obtained from several lysosomal storage diseases and validated using a known compound, methyl-?-cyclodextrin, in primary fibroblast cells derived from Niemann Pick C disease patients. The results demonstrate that the Lysotracker assay can be used in compound screening for the identification of lead compounds that are capable of reducing enlarged lysosomes for drug development. PMID:23983233

Xu, Miao; Liu, Ke; Swaroop, Manju; Sun, Wei; Dehdashti, Seameen J; McKew, John C; Zheng, Wei



Virtual screening and in vitro assay of potential drug like inhibitors from spices against glutathione-S-transferase of filarial nematodes.  


Glutathione-S-transferase(s) (GST) enzyme from Brugia malayi has been exploited as a target in lymphatic filariasis therapeutics. An active GST is a homodimer of a 208 residue long monomer consisting of two domains, a smaller ?/? domain and a larger ? domain. The components of the glutathione (GSH) system, mainly GST enzymes, are critical antioxidant and detoxification system responsible for the long-term existence of filarial worms in mammalian host; hence they are major chemotherapeutic targets in filarial species. In the present study, 58 phytochemicals from 10 plants, predicted and reported to have potential nematicidal activity and ADMET satisfaction, have been docked to GST enzyme of B. malayi to assess their binding affinity and consequently their inhibitory activity. A comparative study has been made with commonly employed chemotherapeutic GST inhibitors such as cibacron-blue, butylated hydroxyanisole, hexyl glutathione and ethacrynic acid. In vitro effects of potential drug like compound from in silico results have been done for validation of docking studies. In vitro assay revealed efficacy in GST inhibition in the following compounds: linalool (97.50%), alpha-pinene (90.00%), strychnine (87.49%), vanillin (84.99%), piperine (79.99%), isoeugenol (62.49%), curcumin (57.49%), beta-caryophyllene (39.50%), cinnamic acid (27.49%), capsaicin (19.99%), citronellol (19.99%) and geraniol (17.49%). An online database ( ) has been developed, which will serve as a useful repository of information on GST inhibitors for future development of drugs against filarial nematodes. These findings thus suggest that the above phytochemicals could be potentially developed as lead molecules for targeting GST of lymphatic filarial parasites. PMID:21523552

Azeez, Shamina; Babu, Rosana O; Aykkal, Riju; Narayanan, Reena




EPA Science Inventory

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


Cell death assays for drug discovery  

Microsoft Academic Search

Cell death has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different

Oliver Kepp; Lorenzo Galluzzi; Marta Lipinski; Junying Yuan; Guido Kroemer



Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments  

Microsoft Academic Search

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was devel- oped. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochroma- tographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data

Weihua Lai; Yang Xu; Daniel YC Fung; Yonghua Xiong


Assessing the Maximum Predictive Validity for Neuropharmacological Anxiety Screening Assays  

E-print Network

the model, such as comparisons of drug efficacy and the effects of drugs on varying behavioral phenotypes, this evaluative standard does not have the ability to compare the level of drug effects between these models (12, such as the zebrafish, to demonstrate predictive validity for a specific set of drug classes. A popular assay used

Kalueff, Allan V.


How to translate a bioassay into a screening assay for natural products: general considerations and implementation of antimicrobial screens.  


Natural product sources have been a valuable provider of molecular diversity in many drug discovery programs and several therapeutically important drugs have been isolated from these. However, the screening of such materials can be very complicated due to the fact that they contain a complex mixture of secondary metabolites, but also the purified natural compounds exert a challenge for bioactivity screening. Success in identifying new therapeutics using in vitro bioassays is largely dependent upon the proper design, validation, and implementation of the screening assay. In this review, we discuss some aspects which are of significant concern when screening natural products in a microtiter plate-based format, being partly applicable to other assay formats as well, such as validation parameters, layouts for assay protocols, and common interferences caused by natural products samples, as well as various troubleshooting strategies. Examples from the field of natural product drug discovery of antibacterial compounds are discussed, and contributions from the realm of academic screenings are highlighted. PMID:25221978

Fallarero, Adyary; Hanski, Leena; Vuorela, Pia



Lactate as a novel quantitative measure of viability in Schistosoma mansoni drug sensitivity assays.  


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

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



Prospects for drug screening using the reverse two-hybrid system  

Microsoft Academic Search

Rational drug-screening strategies have been limited by the number of available protein targets. The fields of genomics and functional genomics are now merging into ‘chemical genomics’ approaches, in which large numbers of potential target proteins can be used in standardized high-throughput drug-screening assays. Because protein–protein interactions are critical to most biological processes and can be tested in standardized assays, they

Marc Vidal; Hideki Endoh



Adolescents and Drug Abuse: Clinical Use of Urine Drug Screening.  

ERIC Educational Resources Information Center

Study examines the use of urine screening as a clinical diagnostic procedure to assess and monitor adolescents in a school-based outpatient program (N=296). Random screening provides little information regarding diagnostic level and pattern of drug use; however it may be helpful in bringing about positive behavior change. (EMK)

James, William H.; Moore, David D.



10GG-2005-09 A Multinetwork, Multiwell Assay Platform for Drug Development and Toxicology  

E-print Network

10GG-2005-09 A Multinetwork, Multiwell Assay Platform for Drug Development and Toxicology Screening A major temporal and financial burden associated with drug development and toxicology assessment and toxicological responses. By employing many networks parallel in an automated robotic system, it is possible

Mohanty, Saraju P.


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

NASA Astrophysics Data System (ADS)

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

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



Rapid screening assay for calcium bioavailability studies  

SciTech Connect

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

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



Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging  

PubMed Central

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.



A fluorescence-based rapid screening assay for cytotoxic compounds  

Microsoft Academic Search

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

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



System models, assays and endpoint parameters to evaluate anticancer compounds during preclinical screening.  


The effectiveness of anticancer therapies relies on the ability of these substances to selectively eliminate the malignant cells with little or no toxicity to normal cells. The isolation in most human tumors of a rare subpopulation of cancer stem cells (CSCs) associated with chemo resistance leads to the "stem cell theory" (SCT). The SCT proposed that eliminating this fraction will eventually cure cancer but experimental data supporting this classical view are controversial and now being gradually replaced by other models. These novel models of cancer biology predict that to cure cancer only drugs or combination of drugs that eliminate all (CSCs and non-CSCs) cancer cells at once ("pankiller drugs") will be effective. The search for "pankiller drugs" will require tests to assess (i) the elimination of all cancer cells in in vitro systems (ii) the ability to eradicate the tumors and prevent tumor relapse in in vivo systems. However, at present, most drugs are being tested in assays that can only provide a picture of the short term activity of anticancer compounds. This in part explains why only a small fraction of the drugs that enter clinical trials are actually approved for clinical use. This article will provide a concise review of the systems, assays and endpoint parameters routinely used to screen for potential anticancer drugs and propose, based in the current knowledge of cancer biology, a more rationale anticancer drug screening program. PMID:23701499

Yakisich, Juan Sebastian



ASSAY and Drug Development Technologies Volume 5, Number 1, 2007  

E-print Network

in contemporary drug discovery. To generate meaningful data with the required throughput, high-quality assay emission is by far the dominant assay methodology due to its broad adaptability to biological targets

Goldman, Steven A.


Determination of Designer Drug Cross-Reactivity on Five Commercial Immunoassay Screening Kits.  


The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), ?-keto amphetamines, substituted amphetamines, piperazines, ?-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits. PMID:25492523

Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z



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


Abstract CD4?+?CD25+ regulatory T cells (Tregs) are believed to be pivotal in controlling chronic inflammation as well as in opposing the effect of cancer immunotherapy. Therefore, identification of novel drug compounds that interfere with Treg function is of high priority together with research 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 sorting (FACS) sorted CD4?+?CD25(high)CD127(dim/-)CD45RA+ naďve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use of highly pure Tregs in a functional assay based on Treg mediated inhibition of effector T cell proliferation. We report highly reproducible Treg function in assays that test the effect of well-known model compounds such as CpG-A, anti-IL-6R (tocilizumab), anti-TNF-? (adalimumab) or a combination of IL-6 and TNF-?. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology. PMID:25367176

Pedersen, Anders Elm; Holmstrřm, Kim; Jřrgensen, Flemming; Jensen, Simon S; Gad, Monika



Development and application of a screening assay for glycoside phosphorylases.  


Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity. PMID:20188057

De Groeve, M R M; Tran, G H; Van Hoorebeke, A; Stout, J; Desmet, T; Savvides, S N; Soetaert, W



Hepatic organoids for microfluidic drug screening.  


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

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



Statistical evaluation of several methods for cut point determination of immunogenicity screening assay.  


ABSTRACT Cut point of the immunogenicity screening assay is the level of response of the immunogenicity screening assay at or above which a sample is defined to be positive and below which it is defined to be negative. Food and Drug Administration Guidance for Industry on Assay Development for Immunogenicity Testing of Therapeutic recommends the cut point to be an upper 95 percentile of the negative control patients. In this article, we assume that the assay data is a random sample from a normal distribution. The sample normal percentile is a point estimate with variability that decreases with the increase of sample size. Therefore the sample percentile does not assure at least 5% false positive rate with high confidence level (e.g., 90%) when the sample size is not sufficiently enough. With this concern, we propose to use a lower confidence limit for a percentile as the cut point instead. We have conducted an extensive literature review on the estimation of the statistical cut point and compare several selected methods for the immunogenicity screening assay cut point determination in terms of bias, the coverage probability, and false positive rate. The selected methods evaluated for the immunogenicity screening assay cut point determination are sample normal percentile, the exact lower confidence limit of a normal percentile (Charkraborti and Li, 2007), and the approximate lower confidence limit of a normal percentile. It is shown that the actual coverage probability for the lower confidence limit of a normal percentile using approximate normal method is much larger than required confidence level with a small number of assays conducted in practice. We recommend using the exact lower confidence limit of a normal percentile for cut point determination. PMID:25356783

Shen, Meiyu; Dong, Xiaoyu; Tsong, Yi



Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy  

PubMed Central

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

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



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

Microsoft Academic Search

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

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



A novel microplate-based assay for screening radioprotectors and its validation based on DNA and membrane system.  


Ionizing radiation leads to damage at various cellular and sub-cellular levels and can be prevented by radioprotectors. There are many in vitro and in vivo but rather expensive assays for screening of radioprotectors from natural and synthetic sources. We have developed a cell free radioprotector screening assay which involves bleaching of crocin pigment, isolated from saffron by radiolytic products of water. Any molecules/compounds which can inhibit the bleaching of the crocin will act as a radioprotector. The developed assay was further validated by the existing in vitro assays. Different radioprotectors have different level for inhibition of bleaching of crocin. The trends of radioprotection offered by crocin bleaching assay, plasmid relaxation and lipid peroxidation are TMG>FA>VA>Amifos>Trox, TMG>VA>FA>Amifos>Trox, and TMG>FA>Trox>VA>Amifos, respectively. We are getting different trends for different assays. This is because different drugs have different mechanisms of radioprotection in different assay systems. In conclusion, the crocin bleaching assay developed here is a simple, fast and economical screening assay and it will have great value in radioprotection programme for screening many potential compounds for radioprotection. PMID:22989745

Maurya, Dharmendra Kumar; Nair, Cherupally Krishnan Krishnan; Devasagayam, Thomas Paul A



A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein  

PubMed Central

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.



[Development of fluorescence imaging based assay for screening compounds with anti-migration activity].  


In the present study, A fluorescent imaging-based high-throughput screening method was developed for identifying anti-migratory compounds with 96-well Transwell plates. The correlation, precision and stability of this method were examined and the incubation time of dye Hoechst 33342 in addition to migration time was optimized. In addition, The inhibitory activity of anti-cancer drug paclitaxel on tumor cell migration was assayed and an IC50 value of 0.717 micromol x L(-1) was obtained. Using this method, 24 components from Rhizoma Alismatis were screened and one component with anti-migration activity was found. These results show that the new proposed method with good precision, stability and linear range has the potential to assay the inhibitory activity of anticancer compounds. PMID:22010348

Nie, Xiao-Jing; Zhao, Xiao-Ping; Wang, Yi



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

PubMed Central

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

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



High Throughput Screening for Anti–Trypanosoma cruzi Drug Discovery  

PubMed Central

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

Alonso-Padilla, Julio; Rodríguez, Ana



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


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 (qPCR) as our method of allele quantification, but any method that can reliably distinguish the difference between one and two copies of the target gene can be used to develop an LOA assay. We have designed qPCR LOA assays for deletions, insertions, point mutations, domain swaps, conditional, and humanized alleles and have used the insert assays to quantify the copy number of random insertion BAC transgenics. Because of its quantitative precision, specificity, and compatibility with high throughput robotic operations, the LOA assay eliminates bottlenecks in ES cell screening and mouse genotyping and facilitates maximal speed and throughput for knockout mouse production. PMID:20691873

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



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


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

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



Developmental toxicity assay using high content screening of zebrafish embryos.  


Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2?days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. PMID:24871937

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



A fluorescence-based rapid screening assay for cytotoxic compounds.  


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

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



Comparison of rapid screening assays using organic chemicals  

SciTech Connect

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

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



Evaluation of enzyme inhibition data in screening for new drugs.  


Enzyme inhibitors selected from either natural product screening or synthetic chemistry programmes can be characterized according to a number of criteria to determine their usefulness as potential drug candidates. Inhibition may be classified as either irreversible or reversible. Irreversible inhibitors may be described with respect to first order rate constants or half-times for inactivation. Reversible inhibitors should be evaluated with respect to Ki values rather than I50 values. Assays should be designed using the simplest and most precise procedures available. If possible, assays utilizing continuously-recording methods should be selected. When reversible inhibitors are desired, greater sensitivity for inhibition is achieved using low substrate concentrations. For irreversible inhibitors or tight-binding competitive inhibitors, low enzyme concentrations also result in greater inhibitory sensitivity. The order of addition of enzyme and substrate should be varied to determine whether inhibition requires preincubation of enzyme and inhibitor for maximum effect. Time-dependence of inhibition should also be established. Enzyme inhibitors should be specific for the enzyme that is being targeted. Novel competitive inhibitors of angiotensin-converting enzyme were isolated in the author's laboratory using the above philosophy of screening for enzyme inhibitors. The properties of the muraceins, phenacein and aspergillomarasmine A' are discussed. PMID:3488892

Bush, K



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

Microsoft Academic Search

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

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



High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes.  


Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors. PMID:24870017

Seegers, Nicole; van Doornmalen, Antoon M; Uitdehaag, Joost C M; de Man, Jos; Buijsman, Rogier C; Zaman, Guido J R



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

Microsoft Academic Search

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

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



Hierarchical virtual screening approaches in small molecule drug discovery.  


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. PMID:25072167

Kumar, Ashutosh; Zhang, Kam Y J



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


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. PMID:24952337

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



Screening for drugs of abuse. I: Opiates, amphetamines and cocaine.  


(1) In order to provide an efficient and reliable service for drugs of abuse screening in urine, the laboratory should analyse 20-30 samples per week, and the staff should include a scientist with special expertise in the subject. (2) Turnaround times should be between 2-3 days of sample collection. To achieve this aim it may be necessary to make special arrangements for the delivery of samples to the laboratory. Results should preferably be transmitted by electronic mail or facsimile with the necessary precautions for security and confidentiality: hardcopy reports may also be required. (3) Good communications between the requesting clinician and the laboratory are essential. An advisory service should be provided by the laboratory and clinicians should be encouraged to discuss requests and results with laboratory staff. It is important that the laboratory inform doctors of the range of substances detected and the sensitivity and specificity of laboratory assays. (4) Assays should be performed according to the manufacturer's protocols, or by modified methods that have been rigorously validated. Quality control samples should be included in each analytical run and participation in an external quality assessment scheme, e.g. UKNEQAS, is essential to provide independent confirmation and confidence that results compare with those from other laboratories. Other requirements include adequate training and supervision of staff, and careful recording of samples and results. (5) Drugs to be tested will depend on the drug 'scene' in the area but should include those drugs regularly prescribed for maintenance therapy (e.g. methadone, dihydrocodeine, benzodiazepines), and drugs frequently misused (e.g. heroin, buprenorphine, amphetamines, cocaine). (6) Positive results obtained by preliminary screening methods e.g. EMIT, should be confirmed by another analytical technique, e.g. TLC, GC or GC-MS. If there are potentially serious or legal implications, and in employment and preemployment testing, confirmation of positive results is mandatory. In some cases, e.g. checking for methadone or benzodiazepine compliance, it may be considered unnecessary to confirm positive results although possible spiking of samples cannot be excluded without checking for the presence of metabolites by a chromatographic procedure. PMID:7785941

Braithwaite, R A; Jarvie, D R; Minty, P S; Simpson, D; Widdop, B



Urine Drug Screening of Adolescents on Request of Parents.  

ERIC Educational Resources Information Center

Of 100 adolescents screened for drug use, 43% tested positive for drugs of abuse. Twenty-five percent of these adolescents entered treatment, with 8% requiring medical detoxification or inpatient treatment. Urine screening, when done for clinical rather than punitive purposes, appeared to facilitate entry into treatment. (RJM)

Tennant, Forest



Potential impact of drug effects, availability, pharmacokinetics, and screening on estimates of drugs implicated in cases of assault.  


Drug-facilitated sexual assault (DFSA) is a serious and troubling crime. It is important to know if and how different drugs might be used to facilitate assault in order to deter such crime. There are a number of ways in which drugs that are used for DFSA might not be detected by routine screens. The purpose of this analysis was to draw reasonable inferences regarding drugs with a high likelihood of being used for DFSA and not being detected by routine screens. National data from poison control centres, hospital emergency rooms, and law enforcement seizures were used to evaluate the relative magnitude of problems and illicit availability associated with different classes of drugs. General drug classes were examined to include additional drugs that might be used for DFSA on the basis of their amnesic effects, widespread availability, and pharmacokinetics (i.e. short half-life). The benzodiazepine-site ligands zolpidem and eszopiclone, 'club drugs' GHB and ketamine, muscle relaxants such as carisoprodol, and antihistamines such as diphenhydramine were identified as drugs that might be used for DFSA and remain undetected by routine screens. Future studies that are designed to examine the role of these drugs in DFSA cases could provide better estimates of their use for DFSA. A better understanding of what is being missed in DFSA cases might help prioritize the development of new assays, provide rationale for the availability of particular assays for routine testing, and inform practitioners and the general public of the potential DFSA risks of certain drugs. PMID:21960542

Carter, Lawrence P



Convenient cell fusion assay for rapid screening for HIV entry inhibitors  

NASA Astrophysics Data System (ADS)

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

Jiang, Shibo; Radigan, Lin; Zhang, Li



A cell-based screen for drugs to treat Huntington's disease.  


We have developed a medium-throughput cell-based assay to screen drugs for Huntington's disease (HD). The assay measures the ability of drugs to protect cultured neuronal (PC12) cells from death caused by an expanded polyglutamine (poly Q) form of huntingtin exon 1. Using this assay, we have blindly screened a library of 1040 compounds compiled by the NINDS: the NIH Custom Collection (NCC). Each compound was tested at five concentrations for its ability to protect cells against huntingtin-induced cell death as well as for its toxicity. Of the compounds tested, 18 prevented cell death completely, and 51 partially. Some of these also exhibited toxicity at higher doses. The majority of drugs (81%) were ineffective. Caspase inhibitors and cannabinoids showed reproducible protection in our assay. We believe these compounds, and others in our hit list, are appealing candidates for further investigation. Additionally, this assay is amenable to scaling up to screen additional compounds for treating Huntington's disease. PMID:15262266

Aiken, Charity T; Tobin, Allan J; Schweitzer, Erik S



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

SciTech Connect

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.

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



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

PubMed Central

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

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



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


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. PMID:21819864

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



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

Microsoft Academic Search

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




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


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. PMID:21521801

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



Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes  

Microsoft Academic Search

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid® fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their

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



Miniaturization of ultra-high-throughput screening assays into 1536-well format  

NASA Astrophysics Data System (ADS)

Assay miniaturization and the implementation of high-density 1536 micro-well screening increases the speed and efficiency of screening and lead discovery. To serve this need, a platform of miniaturizable assay technologies has been assembled for specific biological targets. This platform will enable initiation and completion of uHTS screens in a straightforward and expeditious manner. For kinases, we have examined assays using several technologies including DELFIA, HTR-FRET, FP, EFC, and FMAT. This presentation compares these technologies for the measurement of typical tyrosine kinase activity in 1536-well format. Quality parameters such as assay reproducibility, signal: background ratio, Z factor, and assay sensitivity were calculated and compared. Additionally, the relative merits of each of these technologies were assessed in terms of assay miniaturization, ease of development, ultimate screening capability, efficiency, and cost.

Beasley, James R.; McCoy, Paul M.; Walker, Tiffany; Dunn, David A.



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

PubMed Central

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

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




E-print Network

NEW JERSEY INSTITUTE OF TECHNOLOGY DRUG SCREENING POLICY AND PROCEDURE FOR SAFETY SENSITIVE EMPLOYEES I. Introduction New Jersey Institute of Technology has a legal responsibility and managerial while abusing drugs according to the New Jersey Department of Law and Public Safety Division of Criminal


A Differential Drug Screen for Compounds That Select Against Antibiotic Resistance  

PubMed Central

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus “inverting” selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets. PMID:21209699

Chait, Remy; Michel, Jean-Baptiste; Kishony, Roy



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

PubMed Central

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

Ingólfsson, Helgi I.



A Different Approach to Validating Screening Assays for Developmental Toxicity  

EPA Science Inventory

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


Drug screening for hearing loss: using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss  

PubMed Central

Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause or prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. Identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection. PMID:20096805

Santos, Felipe; Raible, David W.; Simon, Julian A.; Rubel, Edwin W.



A Magnetic Bead-Based Ligand Binding Assay to Facilitate Human Kynurenine 3-Monooxygenase Drug Discovery.  


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. PMID:25296660

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



A high-throughput fluorescence chemical denaturation assay as a general screen for protein-ligand binding.  


Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range. PMID:21138727

Mahendrarajah, Kumaran; Dalby, Paul A; Wilkinson, Barrie; Jackson, Sophie E; Main, Ewan R G



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


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

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



High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation†,‡  

PubMed Central

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery. PMID:22566096

Wu, Angela R.; Kawahara, Tiara L.A.; Rapicavoli, Nicole A.; van Riggelen, Jan; Shroff, Emelyn H.; Xu, Liwen; Felsher, Dean W.; Chang, Howard Y.; Quake, Stephen R.



The beautiful cell: high-content screening in drug discovery  

Microsoft Academic Search

The term “high-content screening” has become synonymous with imaging screens using automated microscopes and automated image\\u000a analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established\\u000a itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments\\u000a and analyzing the data have

Marc Bickle



Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy.  


Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs. PMID:22876965

Ruan, Lingao; Xu, Zhancheng; Lan, Tao; Wang, Jinjie; Liu, Heng; Li, Chaodong; Dong, Chaoqing; Ren, Jicun



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

NASA Astrophysics Data System (ADS)

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

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




EPA Science Inventory

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



EPA Science Inventory

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


Genotoxicity screening via the ?H2AX by flow assay.  


The measurement of serine139-phosphorylated histone H2AX (?H2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for ?H2AX detection (hereafter termed the ?H2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in ?H2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold ?H2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the ?H2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). ?H2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that ?H2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on ?H2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the ?H2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins. PMID:21824484

Smart, D J; Ahmedi, K P; Harvey, J S; Lynch, A M



Development of a thyroperoxidase inhibition assay for high-throughput screening  

EPA Science Inventory

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...


Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors.  

PubMed Central

The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors. PMID:11389689

Luciani, N; de Rocquigny, H; Turcaud, S; Romieu, A; Roques, B P



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

Microsoft Academic Search

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

Lisbeth Elster; Christian Elling; Anders Heding



Robust regression for high throughput drug screening.  


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

Fomenko, Igor; Durst, Mark; Balaban, David



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

SciTech Connect

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

Zhu, Xiaoming; Fu, Afu [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)] [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Luo, Kathy Qian, E-mail: [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)



Reliability of the Drug Use Screening Inventory among Adolescent Alcoholics.  

ERIC Educational Resources Information Center

Examines psychometric reliability of Drug Use Screening Inventory (DUSI) utilizing adolescents with DSM-III-R diagnosis of Psychoactive Substance Use Disorder. Concludes that split-half, internal, and test-retest reliability is superior. Suggests that DUSI may be useful for identifying and quantifying substance use and related problems. Includes…

Tarter, Ralph E.; And Others



Screening Test Finds Drugs That Show Promise Against Ebola  


... Screening Test Finds Drugs That Show Promise Against Ebola Researchers uncover 53 potential treatments; all are already FDA approved but need more ... biocontainment," Garcia-Sastre said. Researchers used this fake Ebola virus ... treatment project, according to background information from the study. ...


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

PubMed Central

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

Bruni, Giancarlo; Lakhani, Parth; Kokel, David



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

PubMed Central

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

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



AlphaScreen selectivity assay for ?-catenin/B-cell lymphoma 9 inhibitors.  


The aberrant formation of the ?-catenin/B-cell lymphoma 9 (BCL9) protein-protein complex is the driving force for many diseases, including cancer. Crystallographic analyses demonstrate that the surface area in ?-catenin for interacting with BCL9 is overlapped with that for the ?-catenin/E-cadherin interaction. In this study, a robust AlphaScreen selectivity assay was developed to quantify inhibitor potency for the ?-catenin/BCL9 interaction and selectivity for ?-catenin/BCL9 over ?-catenin/E-cadherin interactions. A pilot screen was performed to demonstrat the feasibility of this assay. This selectivity assay is highly sensitive and suitable for adaptation to high-throughput screening. The establishment of this assay lays the foundation for the discovery of selective inhibitors specific for ?-catenin/BCL9 interactions. PMID:25312469

Zhang, Min; Wisniewski, John A; Ji, Haitao



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

SciTech Connect

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.

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



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


High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach. PMID:24383450

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



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

SciTech Connect

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

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



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

PubMed Central

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

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



siRNA Genome Screening Approaches to Therapeutic Drug Repositioning  

PubMed Central

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

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



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

PubMed Central

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

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



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


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

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



Blockade of the I(Ks) potassium channel: an overlooked cardiovascular liability in drug safety screening?  


The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I(Ks) channel, which along with the transient outward current (I(to)) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I(Ks) blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I(Ks) channel blockade and describe the discovery of a potent I(Ks) blocker whose activity was not detected by standard hERG or invitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) invivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I(Ks) blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I(Ks) blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I(Ks)) activity. Assay Drug Development Technology 5, 617-627]). Because of the presence of I(Ks) channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I(Ks) blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I(Ks) liability before testing in humans. The possibility of undetected I(Ks) blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427-1438] in the ICH S7B regulatory guidelines. PMID:19439185

Towart, Rob; Linders, Joannes T M; Hermans, An N; Rohrbacher, Jutta; van der Linde, Henk J; Ercken, Martine; Cik, Miroslav; Roevens, Peter; Teisman, Ard; Gallacher, David J



Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.  


Abstract Given the multiplicity of nanoparticles (NPs) there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study we fully characterized the panel of NP suspensions used in this project (PLGA-PEO, TiO2, SiO2, uncoated and oleic acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, 3H-Tymidine incorporation, cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6 and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interferences with testing systems as a first step of a screening strategy for biomedical NPs. PMID:23889211

Guadagnini, Rina; Halamoda Kenzaoui, Blanka; Cartwright, Laura; Pojana, Giulio; Magdolenova, Zuzana; Bilanicova, Dagmar; Saunders, Margaret; Juillerat, Lucienne; Marcomini, Antonio; Huk, Anna; Dusinska, Maria; Fjellsbř, Lise M; Marano, Fracelyne; Boland, Sonja



High-content screening of drug-induced mitochondrial impairment in hepatic cells: effects of statins.  


A frequent mechanism for drug-induced liver injury (DILI) is mitochondrial impairment, and early evaluation of new drugs for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. To this end, we designed a high-content screening assay to study mitochondrial-induced hepatotoxicity in HepG2 cells in detail. Simultaneous assessment of mitochondrial mass and cell viability in cells exposed for 24 h to compounds provides preliminary information on the mitochondrial- or nonmitochondrial-related hepatotoxic potential of compounds. To fully address the mechanisms implicated in mitochondrial impairment, prelethal changes in mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial permeability transition, intracellular calcium concentration and apoptotic cell death were studied in cells incubated for 1 h with compounds. The assay correctly classified a set of well-known mitochondrial toxicants and negative controls and revealed high sensitivity for the detection of mitochondrial DILI and the establishment of different mitochondrial toxicity risks (low to high). This procedure was used for analysing the potential mitochondrial impairment of six statins to determine their clinical risk. All the tested statins produced mitochondrial impairment, although they showed different levels of toxicity (low-medium toxicity risk). The results suggest that this cell-based assay is a promising in vitro approach to predict the potential of drug candidates to induce mitochondrial-associated hepatotoxicity. PMID:25160661

Tolosa, Laia; Carmona, Antonio; Castell, José V; Gómez-Lechón, M José; Donato, M Teresa



Drug Discovery for Human African Trypanosomiasis: Identification of Novel Scaffolds by the Newly Developed HTS SYBR Green Assay for Trypanosoma brucei.  


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

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



Fluorescent Cellular Assay for Screening Agents Inhibiting Pseudomonas aeruginosa Adherence.  


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

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



High throughput miniature drug-screening platform using bioprinting technology.  


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

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



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

ERIC Educational Resources Information Center

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)

Cannell, M. Barry; Favazza, Armando R.



A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.  


Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent essential aspects of in vivo pharmacology and toxicology. Several in vitro designs are optimized for high throughput to benefit screening efficiencies, allowing the entire libraries of potential pharmacologically relevant or possible toxin molecules to be screened for different types of cell signals relevant to tissue damage or to therapeutic goals. Creative approaches to multiplexed cell-based assay designs that select specific cell types, signaling pathways and reporters are routine. However, substantial percentages of new chemical and biological entities (NCEs/NBEs) that fail late-stage human drug testing, or receive regulatory "black box" warnings, or that are removed from the market for safety reasons after regulatory approvals all provide strong evidence that in vitro cell-based assays and subsequent preclinical in vivo studies do not yet provide sufficient pharmacological and toxicity data or reliable predictive capacity for understanding drug candidate performance in vivo. Without a reliable translational assay tool kit for pharmacology and toxicology, the drug development process is costly and inefficient in taking initial in vitro cell-based screens to in vivo testing and subsequent clinical approvals. Commonly employed methods of in vitro testing, including dissociated, organotypic, organ/explant, and 3-D cultures, are reviewed here with specific focus on retaining cell and molecular interactions and physiological parameters that determine cell phenotypes and their corresponding responses to bioactive agents. Distinct advantages and performance challenges for these models pertinent to cell-based assay and their predictive capabilities required for accurate correlations to in vivo mechanisms of drug toxicity are compared. PMID:22252140

Astashkina, Anna; Mann, Brenda; Grainger, David W



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

PubMed Central

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. PMID:24010766



AroER tri-screen is a biologically relevant assay for endocrine disrupting chemicals modulating the activity of aromatase and/or the estrogen receptor.  


Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17?-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals. PMID:24496634

Chen, Shiuan; Zhou, Dujin; Hsin, Li-Yu; Kanaya, Noriko; Wong, Cynthie; Yip, Richard; Sakamuru, Srilatha; Xia, Menghang; Yuan, Yate-Ching; Witt, Kristine; Teng, Christina



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


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. PMID:23676922

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



Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction.  


The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential. PMID:21832258

Doshi, Utkarsh; Li, Albert P



QDs Capped with Enterovirus As Imaging Probes for Drug Screening  

Microsoft Academic Search

The increasing threats of viral diseases have gained worldwide attention in recent years. Quite a few infectious diseases\\u000a are still lack of effective prevention or treatment. The pace of developing antiviral agents could be expedited by the availability\\u000a of quick and efficient drug screening platforms. Enterovirus 71 (EV71) is a highly infectious major causative agent of hand,\\u000a foot, and mouth

Chung-Hao Wang; Ching-An Peng


Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids.  


Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018?N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10?µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10?µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250?µg/L (r(2) ?=?0.992) with intra- and inter-plate imprecision of ?5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5?µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10?µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018?N-pentanoic acid. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25167963

Spinelli, Eliani; Barnes, Allan J; Young, Sheena; Castaneto, Marisol S; Martin, Thomas M; Klette, Kevin L; Huestis, Marilyn A



Using Multiple Phenotype Assays and Epistasis Testing to Enhance the Reliability of RNAi Screening and Identify Regulators of Muscle Protein Degradation  

PubMed Central

RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening. PMID:23152949

Lehmann, Susann; Shephard, Freya; Jacobson, Lewis A.; Szewczyk, Nathaniel J.



Biomimetic membrane platform containing hERG potassium channel and its application to drug screening.  


The hERG (human ether-ŕ-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions. PMID:23423263

Arslan Yildiz, Ahu; Kang, CongBao; Sinner, Eva-Kathrin



A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery  

PubMed Central

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. PMID:23825598

Jiménez-Díaz, María Belén; Viera, Sara; Ibáńez, 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, Ińigo



Screening for plasma cholinesterase deficiency: an automated succinylcholine based assay.  


We describe an automated kinetic method that uses a single aqueous reagent to measure the in vitro hydrolysis of the muscle relaxant succinylcholine. The substrate succinylcholine is hydrolyzed by plasma cholinesterase (EC, and the choline produced is oxidized by choline oxidase (EC 11.3.17) in the presence of peroxidase, 4-aminophenazone and phenol, to yield a chromagen with maximum absorbance at 500 nm. The method is reproducible (CV 1.3%), correlates well with a manual procedure using the same substrate (r = 0.994, y = 0.99x - 0.25), and is linear to 150 U/L. The method is well suited to pre-operative screening and detection of "at-risk" individuals, as illustrated by the family of one patient who had a prolonged succinylcholine apnea. PMID:3390904

George, P M; Joyce, S L; Abernethy, M H



Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples  

Microsoft Academic Search

An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH4+ is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease

Belen Bello Rodriguez; John A. Bolbot; Ibtisam E. Tothill



Drugs and Drug-Like Compounds: Discriminating Approved Pharmaceuticals from Screening-Library Compounds  

NASA Astrophysics Data System (ADS)

Compounds in drug screening-libraries should resemble pharmaceuticals. To operationally test this, we analysed the compounds in terms of known drug-like filters and developed a novel machine learning method to discriminate approved pharmaceuticals from “drug-like” compounds. This method uses both structural features and molecular properties for discrimination. The method has an estimated accuracy of 91% in discriminating between the Maybridge HitFinder library and approved pharmaceuticals, and 99% between the NATDiverse collection (from Analyticon Discovery) and approved pharmaceuticals. These results show that Lipinski’s Rule of 5 for oral absorption is not sufficient to describe “drug-likeness” and be the main basis of screening-library design.

Schierz, Amanda C.; King, Ross D.


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


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

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



Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery.  


Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons and stem cell-derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors. PMID:24450833

Kiris, Erkan; Kota, Krishna P; Burnett, James C; Soloveva, Veronica; Kane, Christopher D; Bavari, Sina



Image-based high-throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas' disease.  


Chagas' disease, caused by infection with the parasite Trypanosoma cruzi, is the major cause of heart failure in Latin America. Classic clinical manifestations result from the infection of heart muscle cells leading to progressive cardiomyopathy. To ameliorate disease, chemotherapy must eradicate the parasite. Current drugs are ineffective and toxic, and new therapy is a critical need. To expedite drug screening for this neglected disease, we have developed and validated a cell-based, high-throughput assay that can be used with a variety of untransfected T. cruzi isolates and host cells and that simultaneously measures efficacy against the intracellular amastigote stage and toxicity to host cells. T. cruzi-infected muscle cells were incubated in 96-well plates with test compounds. Assay plates were automatically imaged and analyzed based on size differences between the DAPI (4',6-diamidino-2-phenylindole)-stained host cell nuclei and parasite kinetoplasts. A reduction in the ratio of T. cruzi per host cell provided a quantitative measure of parasite growth inhibition, while a decrease in count of the host nuclei indicated compound toxicity. The assay was used to screen a library of clinically approved drugs and identified 55 compounds with activity against T. cruzi. The flexible assay design allows the use of various parasite strains, including clinical isolates with different biological characteristics (e.g., tissue tropism and drug sensitivity), and a broad range of host cells and may even be adapted to screen for inhibitors against other intracellular pathogens. This high-throughput assay will have an important impact in antiparasitic drug discovery. PMID:20547819

Engel, Juan C; Ang, Kenny K H; Chen, Steven; Arkin, Michelle R; McKerrow, James H; Doyle, Patricia S



A High-Throughput Screen for Antibiotic Drug Discovery  

PubMed Central

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

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




EPA Science Inventory

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


High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening  

E-print Network

: There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the avail- ability spots from 58 random newborns and lysosomal storage disease­affected patients showed that the assay online at DOI: 10.1373/clinchem.2012.189936 4 Nonstandard abbreviations: LSD, lysosomal storage disease

Gelb, Michael


TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors  

PubMed Central

UBC13 is a non-canonical Ubiquitin Conjugating Enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of Lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair, and thus a potential radiosensitizer and chemosensitizer target for oncology. We developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. We defined conditions for optimized performance of the TR-FRET assay in both 384 and 1536-well formats. Chemical library screens (total 456,865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically > 0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13. PMID:22034497

Madiraju, Charitha; Welsh, Kate; Cuddy, Michael P.; Godoi, Paulo; Pass, Ian; Ngo, Tram; Vasile, Stefan; Sergienko, Eduard A.; Diaz, Paul; Matsuzawa, Shu-Ichi; Reed, John C.



Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.  


An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening. PMID:11080690

Boisclair, M D; McClure, C; Josiah, S; Glass, S; Bottomley, S; Kamerkar, S; Hemmilä, I



Loop-mediated isothermal amplification assays for screening of bacterial integrons  

PubMed Central

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



Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors.  


Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD?) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD?. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction. PMID:23871998

Vanderporten, Erica; Frick, Lauren; Turincio, Rebecca; Thana, Peter; Lamarr, William; Liu, Yichin



A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line  

PubMed Central

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models. In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes. Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished). PMID:23299097

Jain, Surendra K.; Sahu, Rajnish; Walker, Larry A.; Tekwani, Babu L.



High-Throughput RT-PCR for small-molecule screening assays  

PubMed Central

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

Bittker, Joshua A.



Drug Screening of Preserved Oral Fluid by Liquid Chromatography-Tandem Mass Spectrometry  

Microsoft Academic Search

Background: Oral fluid is an alternative matrix with potential applications in road-side drug screening, work-place testing, drug treatment programs, and epi- demiological surveys. Development of methods for ex- tensive drug screening in oral fluid is warranted. Methods: We developed a liquid chromatography- tandem mass spectrometry (LC-MS\\/MS) method for drug screening of preserved oral fluid collected with the Intercept® collection device.

Elisabeth Leere Řiestad; Unni Johansen; Asbjorg Solberg Christophersen


Optimization of Fluorescence Assay of Cellular Manganese Status for High Throughput Screening  

PubMed Central

The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra-and inter-plate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport. PMID:23169769

Kumar, Kevin K.; Aboud, Asad A.; Patel, Devin K.; Aschner, Michael; Bowman, Aaron B.



Rapid in vitro screening of drug-metal ion interactions.  


ABSTRACT The toxic side effects of synthetic drugs may, in part, be arising due to their interactions with essential metal ions, especially when the metal ions are administered along with the drug as mineral supplements. In this paper we report the feasibility of establishing such drug-metal ion interactions through in vitro spectrophotometric studies, which are rapid and can be used for routine screening prior to clinical studies. The interaction of the drugs levothyroxine and ranitidine with eight metal ions, copper(II), chromium(III), molybdenum(VI), magnesium(II), calcium(II), iron(II), manganese(II), and zinc(II), commonly used in mineral supplements, was verified through in vitro UV-visible spectrophotometric studies. The experiments were carried out at the physiological pH values 1.5, 7.4, and 8.0 and the concentrations of the drugs and mineral supplements used were comparable to those in their usual doses. These studies indicated interaction between ranitidine and calcium(II), magnesium(II), and iron(II) ions and between levothyroxine and copper(II) and iron(II) ions. A comparison of the results with those reported from clinical studies demonstrated the efficacy of this method. PMID:20020882

Sridevi, N; Yusuff, K K Mohammed



Engineering Xenopus embryos for phenotypic drug discovery screening.  


Many rare human inherited diseases remain untreatable despite the fact that the disease causing genes are known and adequate mouse disease models have been developed. In vivo phenotypic drug screening relies on isolating drug candidates by their ability to produce a desired therapeutic phenotype in whole organisms. Embryos of zebrafish and Xenopus frogs are abundant, small and free-living. They can be easily arrayed in multi-well dishes and treated with small organic molecules. With the development of novel genome modification tools, such a zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas, it is now possible to efficiently engineer non-mammalian models of inherited human diseases. Here, we will review the rapid progress made in adapting these novel genome editing tools to Xenopus. The advantages of Xenopus embryos as in vivo models to study human inherited diseases will be presented and their utility for drug discovery screening will be discussed. Being a tetrapod, Xenopus complements zebrafish as an indispensable non-mammalian animal model for the study of human disease pathologies and the discovery of novel therapeutics for inherited diseases. PMID:24576445

Schmitt, Stefan M; Gull, Mazhar; Brändli, André W



Pluripotency and targeted reprogramming: strategies, disease modeling and drug screening.  


Pluripotent stem cell research has developed over the last fifty years from the study of embryonic development to a multifaceted discipline that encompasses development, epigenetics, reprogramming, cell therapy, disease modeling and chemical and drug screening. The idea of patient-specific therapies and disease modeling using human pluripotent stem cells has been the theoretical golden-egg of the field since the generation of human embryonic stem cells. With the advent of induced pluripotent stem cells (PSCs), the ability to generate patient-specific cells for therapeutic use, to model disease progression and to test drugs on disease relevant cells moved a large step closer to reality. While there still is a long way to go before the results of PSC research is found in the clinic or in the pharmacy, recent developments have demonstrated that it is possible to generate patient-specific pluripotent cells which can differentiate into disease relevant cell types, are amenable to gene correction, can phenocopy molecular and functional disease characteristics, at least in vitro, and can be used to validate the efficacy of therapeutic compounds. This review will cover recent developments in the generation and manipulation of pluripotent stem cells with a focus on the use of pluripotent stem cells for disease modeling and therapeutic drug screening. In addition, the latest developments in somatic cell reprogramming will also be discussed. PMID:23517625

Stewart, Morag H



Screening for Noise in Gene Expression Identifies Drug Synergies  

PubMed Central

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

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



Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay  

Microsoft Academic Search

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using

Gregory L. Erexson; M. V. Periago; Carol S. Spicer



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


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

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



Screening pharmaceuticals for possible carcinogenic effects: initial positive results for drugs not previously screened  

PubMed Central

Objective We screened commonly used prescription drugs for possible carcinogenic effects. Methods In a large health care program we identified 105 commonly used drugs, not previously screened. Recipients were followed for up to 12˝ years for incident cancer. Nested case-control analyses of 55 cancer sites and all combined included up to ten matched controls per case, with lag of at least two years between drug dispensing and cancer. Positive associations entailed a relative risk (RR) of 1.50, with p? 0.01 and higher risk for three or more, than for one prescription. Evaluation included further analyses, searches of the literature, and clinical judgment. Results There were 101 associations of interest for 61 drugs. Sixty-six associations were judged to have involved substantial confounding. We found evidence that of the remaining 35, the following associations may not be due to chance: sulindac with gallbladder cancer and leukemia, hyoscyamine with non-Hodgkin lymphoma, nortriptyline with esophageal and hepatic cancer, oxazepam with lung cancer, both fluoxetine and paroxetine with testicular cancer, hydrochlorothiazide with renal and lip cancer, and nifedipine with lip cancer. Conclusions These preliminary findings suggest that further studies are indicated regarding sulindac, hyoscyamine, nortriptyline, oxazepam, fluoxetine, paroxetine, hydrochlorothiazide and nifedipine. PMID:19582585

Friedman, Gary D.; Udaltsova, Natalia; Chan, James; Quesenberry, Charles P; Habel, Laurel A.



CYP2C9-mediated metabolic activation of losartan detected by a highly sensitive cell-based screening assay.  


Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4'-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatography-tandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9. PMID:21321060

Iwamura, Atsushi; Fukami, Tatsuki; Hosomi, Hiroko; Nakajima, Miki; Yokoi, Tsuyoshi



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

PubMed Central

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

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.



FRET-based calcium imaging: a tool for high-throughput/content phenotypic drug screening in Alzheimer disease.  


Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid ? (A?) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics. PMID:24221842

Honarnejad, Kamran; Kirsch, Achim K; Daschner, Alexander; Szybinska, Aleksandra; Kuznicki, Jacek; Herms, Jochen



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


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

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



Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei.  


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

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



Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.  


Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world. PMID:23482387

Bell, Sara C; Alford, Ross A; Garland, Stephen; Padilla, Gabriel; Thomas, Annette D



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


Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor ?(E) is critical for maintenance of the cell envelope. Because ?(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the ?(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a ?(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the ?(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify ?(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds ?(E), inhibits RNA polymerase holoenzyme formation, and inhibits ?(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the ?(E) pathway. PMID:25331704

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



Evaluation of the GADD45?-GFP GreenScreen HC assay for rapid and reliable in vitro early genotoxicity screening.  


Twenty-two of Galderma's proprietary compounds were tested in the GADD45?-GFP 'GreenScreen HC' assay (GS), the SOS-ChromoTest and the Mini-Ames to evaluate GSs performance for early genotoxicity screening purposes. Forty more characterized compounds were also tested, including antibiotics: metronidazole, clindamycin, tetracycline, lymecycline and neomycin; and catecholamines: resorcinol mequinol, hydroquinone, one aneugen carbendazim, one corticoid dexamethasone, one peroxisome proliferator-activated receptor rosiglitazone, one pesticide carbaryl and two further proprietary molecules with in vitro genotoxicity data. With proprietary molecules, this study concluded that the GS renders the SOS-ChromoTest obsolete for in vitro screening. The GS confirmed all results of the Mini-Ames test (100% concordance). Compared with the micronucleus test, the GS showed a concordance of 82%. With known compounds, the GS ranked the potency of positive results for catecholamines in accordance with other genotoxicity tests and showed very reproducible results. It confirmed positive results for carbendazim, for tetracycline antibiotics and for carbaryl. The GS produced negative results for metronidazole, a nitroreduction-specific bacterial mutagen, for dexamethasone (a non-genotoxic apoptosis inducer), for rosiglitazone (a GADD45? promoter inducer) and for clindamycin and neomycin (inhibitors of macromolecular synthesis in bacteria). As such, the GS appears to be a reproducible, robust, specific and sensitive test for genotoxicity screening. PMID:22806210

Luzy, Anne-Pascale; Orsini, Nicolas; Linget, Jean-Michel; Bouvier, Guy



A modified MS2 bacteriophage plaque reduction assay for the rapid screening of antiviral plant extracts  

PubMed Central

Introduction: Traditional methods of screening plant extracts and purified components for antiviral activity require up to a week to perform, prompting the need to develop more rapid quantitative methods to measure the ability of plant based preparations to block viral replication. We describe an adaption of an MS2 plaque reduction assay for use in S. aureus. Results: MS2 bacteriophage was capable of infecting and replicating in B. cereus, S. aureus and F + E. coli but not F- E. coli. Indeed, both B. cereus and S. aureus were more sensitive to MS2 induced lysis than F+ E. coli. When MS2 bacteriophage was mixed with Camellia sinensis extract (1 mg/ml), Scaevola spinescens extract (1 mg/ml) or Aloe barbadensis juice and the mixtures inoculated into S. aureus, the formation of plaques was reduced to 8.9 ± 3.8%, 5.4 ± 2.4% and 72.7 ± 20.9% of the untreated MS2 control values respectively. Conclusions: The ability of the MS2 plaque reduction assay to detect antiviral activity in these known antiviral plant preparations indicates its suitability as an antiviral screening tool. An advantage of this assay compared with traditionally used cytopathic effect reduction assays and replicon based assays is the more rapid acquisition of results. Antiviral activity was detected within 24 h of the start of testing. The MS2 assay is also inexpensive and non-pathogenic to humans making it ideal for initial screening studies or as a simulant for pathogenic viruses. PMID:21808571

Cock, Ian; Kalt, F. R.



Key Learnings from the Endocrine Disruptor Screening Program (EDSP) Tier 1 Rodent Uterotrophic and Hershberger Assays  

PubMed Central

In 2009, companies began screening compounds using the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). EDSP has two tiers: Tier 1 includes 11 assays to identify compounds with potential endocrine activity. This article describes two laboratories' experiences conducting Tier 1 uterotrophic and Hershberger assays. The uterotrophic assay detects estrogen receptor agonists through increases in uterine weight. The advantages of the uterotrophic rat models (immature vs. adult ovariectomized) and exposure routes are discussed. Across 29 studies, relative differences in uterine weights in the vehicle control group and 17?-ethynylestradiol–positive control group were reasonably reproducible. The Hershberger assay detects androgen receptor (AR) agonists, antagonists, and 5?-reductase inhibitors through changes in accessory sex tissue (AST) weights. Across 23 studies, AST weights were relatively reproducible for the vehicle groups (baseline), testosterone propionate (TP) groups (androgenic response), and flutamide + TP groups (antiandrogenic response). In one laboratory, one and four compounds were positive in the androgenic and antiandrogenic portions of the assay, respectively. Each compound was also positive for AR binding. In the other laboratory, three compounds showed potential antiandrogenic activity, but each compound was negative for AR binding and did not fit the profile for 5?-reductase inhibition. These compounds induced hepatic enzymes that enhanced testosterone metabolism/clearance, resulting in lower testosterone and decreased capacity to maintain AST weights. The Hershberger androgenic and antiandrogenic performance criteria were generally attainable. Overall, the uterotrophic and Hershberger assays were easily adopted and function as described for EDSP screening, although the mode of action for positive results may not be easily determined. PMID:24515841

Marty, M Sue; O'Connor, John C



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

PubMed Central

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

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



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


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

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



Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores.  


In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple ?-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays. PMID:23672540

Schneider, Ralf; Gohla, Anne; Simard, Jeffrey R; Yadav, Dharmendra B; Fang, Zhizhou; van Otterlo, Willem A L; Rauh, Daniel



Dictyostelium discoideum developmental cycle (DDDC) assay: a tool for Hg toxicity assessment and soil health screening.  


The social amoeba Dictyostelium discoideum has been proposed for assessing stress responses to pollutants in soil and it has already been successfully employed in the aquatic environment. Presently, we developed the DDDC assay (D. discoideum developmental cycle assay) for both soil toxicity assessment and soil health screening. The DDDC assay is primarily aimed at determining the capacity of D. discoideum to undergo its developmental programme forming a fruiting body, measured in terms of fruiting body formation inhibition and fruiting body size factor, which may be considered an indication of its ecological fitness (potential for spore dispersal). A second objective of the solid phase DDDC assay is to identify potential mechanisms of toxic action on the developmental cycle, for which three checkpoints are examined: (a) aggregation arrest, (b) migration arrest, and (c) culmination arrest. Presently, conditions for the DDDC assay such as soil texture, soil water content, soil pH, food availability and incubation time were investigated and optimized. In addition, both solid and liquid phase variants of the DDDC assay were applied to assess the toxicity of Hg, at regulatory concentrations. The developmental cycle and ecological fitness were affected from the exposure to 0.3 mg Hg/kg dry-wt soil onwards. The DDDC assay has been shown to be a high sensitivity test. PMID:23454908

Rodríguez-Ruiz, Amaia; Marigómez, Ionan; Boatti, Lara; Viarengo, Aldo



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

PubMed Central

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

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



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


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

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



Application of the Mirrorball High-Sensitivity Cytometer to Multiplexed Assays for Antibody Drug Discovery.  


Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time. The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays. We have developed bead- and cell-based binding assays that demonstrate how the multilaser capability of the mirrorball can be exploited to enhance assay sensitivity. In addition, using the multilaser simultaneous scanning capability, we have established multiplexed cytokine quantitation assays and antibody-cell binding assays. Our results demonstrate the potential utility of this technology to improve the sensitivity and efficiency of biologics screening, resulting in streamlining of the lead antibody selection process. PMID:25381256

England, Elizabeth; Newton, Philip; Neal, Frances; Kitching, Lisa; Colley, Caroline; Rossant, Christine J



On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases†  

PubMed Central

Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones. PMID:23339304

Naik, Subhashchandra; Zhang, Na; Gao, Phillip; Fisher, Mark T.



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

PubMed Central

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

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



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

PubMed Central

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

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



High-Throughput Screening Assay for Embryoid Body Differentiation of Human Embryonic Stem Cells  

PubMed Central

Serum-free human pluripotent stem cell media offer the potential to develop reproducible clinically applicable differentiation strategies and protocols. The vast array of possible growth factor and cytokine combinations for media formulations makes differentiation protocol optimization both labor and cost-intensive. This unit describes a 96-well plate, 4-color flow cytometry-based screening assay to optimize pluripotent stem cell differentiation protocols. We provide conditions both to differentiate human embryonic stem cells (hESCs) to the three primary germ layers, ectoderm, endoderm, and mesoderm, and to utilize flow cytometry to distinguish between them. This assay exhibits low inter-well variability and can be utilized to efficiently screen a variety of media formulations, reducing cost, incubator space, and labor. Protocols can be adapted to a variety of differentiation stages and lineages. PMID:22415836

Outten, Joel T.; Gadue, Paul; French, Deborah L.; Diamond, Scott L.



Inhibition of Thioredoxin Reductase 1 by Porphyrins and Other Small Molecules Identified By a High Throughput Screening Assay  

PubMed Central

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC1280 compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKC delta inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1 with a Ki = 2.7 ?M with regards to Trx1, and in the absence of Trx1 displayed time dependent irreversible inhibition with an apparent second-order rate constant (kinact) of 0.73×10?3 ± 0.07×10?3 ?M?1min?1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels; such as, reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high throughput TrxR1 assay presented here. PMID:21262347

Prast-Nielsen, Stefanie; Dexheimer, Thomas S.; Schultz, Lena; Stafford, William C.; Cheng, Qing; Xu, Jianqiang; Jadhav, Ajit; Arnér, Elias S.J.; Simeonov, Anton



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

PubMed Central

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

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



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


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 mono-ARTDs. PMID:23485441

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



Application of luciferase assay for ATP to antimicrobial drug susceptibility  

NASA Technical Reports Server (NTRS)

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.

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



Investigation of dissociation enhanced lanthanide fluoroimmunoassay as an alternative screening test for veterinary drug residues.  


Both the number of drugs used in veterinary medicine and the diversity of procedures employed to detect their residues are ever increasing. Many laboratories that carry out such testing have employed a variety of immunoassays to serve as rapid screening tests. Owing to the high sensitivity, good reproducibility and availability of multi-analyte assays dissociation enhanced time-resolved fluorescence immunoassay (dissociation enhanced lanthanide fluoroimmunoassay, DELFIA) kits have become a market leader in the human clinical field. No commercial DELFIA kits are presently available for veterinary drug analysis. A DELFIA method was developed for the quantitative analysis of residues of medroxyprogesterone (MP) in bovine bile. Commercially available DELFIA reagents were used in a microtitre assay format. Within- and between-assay sr values (relative standard deviation) were 4.2-9.9 and 5.1-8.5%, respectively. Detection limits were calculated (mean + 3 s of a known negative population) as being 0.52 and 4.91 micrograms l-1 for male and female animals, respectively. A second study was carried out to optimize the labelling of haptens with a range of lanthanide metals. Purification of the hapten-lanthanide conjugates produced was achieved using fast protein liquid chromatography. Sensitive standard curves were produced for nortestosterone and diethylstilbestrol (curve mid-points of 30 and 40 pg, respectively). By using two different lanthanide labels (Eu and Tb) the simultaneous measurement of these compounds in a single assay system was achieved. Use of a third lanthanide label (Sm) yielded a product of low signal intensity and sensitivity. PMID:7879855

Elliott, C T; Francis, K S; McCaughey, W J



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2010 CFR

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



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2013 CFR

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



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2014 CFR

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



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2011 CFR

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



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2012 CFR

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



Protocols for the Routine Screening of Drug Sensitivity in the Human Parasite Trichomonas vaginalis.  


Trichomonas vaginalis is a sexually transmitted protozoan parasite of humans. Treatment of trichomoniasis is almost completely dependent on the old drug metronidazole and is hampered by resistance. New drug development, like routine screening for drug resistance, has however been hampered by the lack of reliable screening protocols with sufficient throughput. Here we report on two separate in vitro protocols that use fluorescent dyes and allow for standardized drug sensitivity testing on the required scale. PMID:25618339

Natto, Manal J; Eze, Anthonius A; de Koning, Harry P



The impact of diversity-based, high-throughput screening on drug discovery: "chance favours the prepared mind".  


Since its modest beginnings in support of natural product discovery in the early 1980s, diversity-based high-throughput screening (dHTS) has developed within the pharmaceutical, biotechnology and academic sectors to become one of the most widely used hit identification screening paradigms in early drug discovery. Advances in key component technologies, specifically in diversity collection design, high-throughput assay development and screening informatics, continue to improve the economics and successes of dHTS hit discovery from large screening collections. Through the application of these components in concert, dHTS has evolved from an expensive technology-centric process that was used to screen collections of randomly acquired compounds, into a process that balances chemical, biological and technological inputs to purposefully build diverse compound collections through planned synthesis and purchasing strategies, and that screens these collections efficiently and economically. As a backlash to the 1990s hype that placed the HTS paradigm at the center of attempts to improve overall R&D productivity, sceptics predicted an undignified demise for this approach. Nevertheless, the use of key component technologies in tandem with sophisticated process and quality control systems is now beginning to deliver the success rates promised by the early proponents of the approach. These results indicate that, given continued 'preparedness', dHTS will remain as the principal hit identification tool for early drug discovery well into the next decade. PMID:18600571

Snowden, Michael; Green, Darren Vs



A spatiotemporally defined in vitro microenvironment for controllable signal delivery and drug screening.  


Cancer metastasis and drug resistance are important malignant tumor phenotypes that cause roughly 90% mortality in human cancers. Current therapeutic strategies, however, face substantial challenges partially due to a lack of applicable pre-clinical models and drug-screening platforms. Notably, microscale and three-dimensional (3D) tissue culture platforms capable of mimicking in vivo microenvironments to replicate physiological conditions have become vital tools in a wide range of cellular and clinical studies. Here, we present a microfluidic device capable of mimicking a configurable tumor microenvironment to study in vivo-like cancer cell migration as well as screening of inhibitors on both parental tumors and migratory cells. In addition, a novel evaporation-based paper pump was demonstrated to achieve adaptable and sustainable concentration gradients for up to 6 days in this model. This straightforward modeling approach allows for fast patterning of a wide variety of cell types in 3D and may be further integrated into biological assays. We also demonstrated cell migration from tumor spheroids induced by an epidermal growth factor (EGF) gradient and exhibited lowered expression of an epithelial marker (EpCAM) compared with parental cells, indicative of partial epithelial-mesenchymal transition (EMT) in this process. Importantly, pseudopodia protrusions from the migratory cells - critical during cancer metastasis - were demonstrated. Insights gained from this work offer new opportunities to achieve active control of in vitro tumor microenvironments on-demand, and may be amenable towards tailored clinical applications. PMID:25089836

Kuo, Ching-Te; Liu, Hao-Kai; Huang, Guan-Syuan; Chang, Chi-Hao; Chen, Chen-Lin; Chen, Ken-Chao; Huang, Ruby Yun-Ju; Lin, Ching-Hung; Lee, Hsinyu; Huang, Chiun-Sheng; Wo, Andrew M



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

PubMed Central

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

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



Screening Current and Future Diagnosis of Psychiatric Disorders Using the Revised Drug Use Screening Inventory1  

PubMed Central

The revised Drug Use Screening Inventory (DUSI-R) is a valid and reliable self-report questionnaire used for quantifying problems that frequently precede and co-occur with substance abuse. The present investigation determined whether the DUSI-R's items can be aggregated into scales that implicate current and future psychiatric disorders. Scales were derived to screen for attention deficit, conduct, antisocial, anxiety, depression, and substance use disorders in a longitudinally tracked cohort of 328 boys. Evaluations were conducted when the boys were 12–14, 16, 19, and 22 years of age. All of the scales identified youths qualifying for current DSM-IV diagnosis with excellent accuracy. Predictive validity of the scales ranged from good to excellent. Accordingly cut-off scores were determined for each scale for use in practical settings to identify youths who require comprehensive diagnostic evaluation. Thus in addition to its utility for detecting problems that precede and correlate with substance abuse, the DUSI-R is cost-efficient for screening youths for mental disorders. PMID:18821459

Kirisci, Levent; Tarter, Ralph; Mezzich, Ada; Reynolds, Maureen



Bioluminescent bacteria assay of veterinary drugs in excreta of food-producing animals  

Microsoft Academic Search

The residues of pharmacological treatments on food-producing animals, present in the manure dispersed on agricultural land, can impact environmental and human health through toxic, genotoxic, and drug-resistance development effects. Biotoxicity assays can easily reveal the presence of noxious substances and those based on bioluminescent bacteria (BLB) are particularly simple and rapid. A BLB assay was developed as microplate format by

L. Bolelli; Z. Bobrovová; E. Ferri; F. Fini; S. Menotta; S. Scandurra; G. Fedrizzi; S. Girotti



Progesterone receptor chaperone complex-based high-throughput screening assay: identification of capsaicin as an inhibitor of the hsp90 machine.  


Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as antitumor agents have unfortunately shown disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with a different mechanism of action are urgently needed. We report here the development of a novel high-throughput screening assay platform to identify small-molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor, a physiological client of Hsp90, in a 96-well plate format. We screened the National Institutes of Health clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is a Food and Drug Administration-approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin. PMID:25184514

Patwardhan, Chaitanya A; Alfa, Eyad; Lu, Su; Chadli, Ahmed



Estrogenic activity of constituents of underarm deodorants determined by E-Screen assay.  


The purpose of this study was to ascertain whether different kinds of underarm deodorants commercially available in Germany might contain substances with estrogenic potential which after use enter the aquatic environment via wastewater. Twenty five deodorants produced by ten different manufacturers in the form of sprays, roll-ons and sticks were investigated using an in vitro-test system (E-Screen assay) for the determination of estrogenic activity based on the human breast cancer cell line MCF-7. Seven out of ten spray deodorant samples showed a quantifiable estrogenic activity. In the case of the sticks and roll-ons it was only one out of six and one out of nine, respectively. The 17?-estradiol equivalent concentrations (EEQs) of the samples ranged from 0.1 ng g(-1) to 9 ng g(-1) deodorant. Spray deodorant samples showed the highest activities in the E-Screen assay compared to the stick and roll-on deodorants. In order to identify substances possibly contributing to the observed biological activity the samples were additionally analyzed by GC/MS. The obtained results of this non-target screening led to the selection of 62 single substances present in the deodorants which for their part were analyzed by E-Screen assay. Eight of these single substances, all of them fragrances, showed estrogenic effects with estradiol equivalence factors (EEFs) similar to parabens, a group of 4-hydroxybenzoic acid esters commonly used as preservatives in personal care products, which are known to have a slight estrogenic effect. Thus, these fragrances are obviously responsible to a substantial degree for the observed estrogenic activity of the deodorants. PMID:24875918

Lange, Claudia; Kuch, Bertram; Metzger, Jörg W



Genome-wide screening of loci associated with drug resistance to 5-fluorouracil-based drugs.  


Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome-wide correlation analysis between DNA copy number and antitumor activity against 5-fluorouracil (5-FU)-based drugs (S-1, tegafur + uracil [UFT], 5'-DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nucleotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5-FU-based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5-FU-based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response. PMID:17425594

Ooyama, Akio; Okayama, Yoshihiro; Takechi, Teiji; Sugimoto, Yoshikazu; Oka, Toshinori; Fukushima, Masakazu



High-throughput Assay to Identify New Cancer Drugs

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


Brain Receptors for Antipsychotic Drugs and Dopamine: Direct Binding Assays  

Microsoft Academic Search

In order to test the suggestion that antipsychotic drugs act by blocking dopamine receptors in the brain, the direct effects of such neuroleptic drugs were tested on the stereospecific binding of [3H]dopamine and of [3H]haloperidol to rat brain striata and their subfractions. The stereospecific component of binding was defined as that amount of [3H]dopamine or [3H]haloperidol bound in the presence

P. Seeman; M. Chau-Wong; J. Tedesco; K. Wong



In vitro screening assay for teratogens using growth inhibition of human embryonic cells  

SciTech Connect

The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

Pratt, R.M.; Willis, W.D.



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


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 ?M, respectively) and microscopy (400.3 nM and 9.4 ?M, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 ?M) was similar to the recently described microscopy-based value (21.7 ?M) for B. bovis. Additionally, the Z' factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay. PMID:24914124

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



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

PubMed Central

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

Paul Khurana, S. M.



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

PubMed Central

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



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

NASA Astrophysics Data System (ADS)

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

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



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

PubMed Central

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

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



Indeterminate human immunodeficiency virus Western blot profiles in ethiopians with discordant screening-assay results.  


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

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



A Cellular Screening Assay Using Analysis of Metal-Modified Fluorescence Lifetime  

PubMed Central

Abstract Current methods for screening cell receptor internalization often require complex image analysis with limited sensitivity. Here we describe a novel bioassay based on detection of changes in global fluorescence lifetime above a gold substrate, with superresolution axial sensitivity and no need for image analysis. We show that the lifetime of enhanced green fluorescent protein expressed in a cellular membrane is greatly reduced in close proximity to the gold, resulting in a distance-dependent lifetime distribution throughout the cell. We demonstrate the application of this phenomenon in a screening assay by comparing the efficacies of two small molecule inhibitors interfering with the internalization process of a G protein-coupled receptor. PMID:20513420

Cade, Nicholas I.; Fruhwirth, Gilbert; Archibald, Stephen J.; Ng, Tony; Richards, David



Detection of hemolytic bacteria from Palythoa caribaeorum (Cnidaria, Zoantharia) using a novel palytoxin-screening assay.  


Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7% showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na+/K+-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum. PMID:19504172

Seemann, Petra; Gernert, Christine; Schmitt, Susanne; Mebs, Dietrich; Hentschel, Ute



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

USGS Publications Warehouse

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

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



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


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

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



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


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

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



A fluorescence-based screening assay for identification of hepatitis C virus NS3 helicase inhibitors and characterization of their inhibitory mechanism.  


Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism. PMID:25579589

Furuta, Atsushi; Salam, Kazi Abdus; Tani, Hidenori; Tsuneda, Satoshi; Sekiguchi, Yuji; Akimitsu, Nobuyoshi; Noda, Naohiro



A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries.  


Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group alpha-(1-->3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and alpha-L-Fucp-(1-->2)-beta-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen alpha-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-R. A closely related alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen alpha-D-Galp-NAc-(1-->3)-[alpha-L-Fuc(1-->2)]-beta-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the color change of the pH indicator bromothymol blue when a proton is released during the transfer of Gal/GalNAc from UDP-Gal/UDP-GalNAc to the acceptor substrate. Saturation mutagenesis of GTB enzyme at M214, a hot spot adjacent to the (211)DVD(213) metal binding motif, was performed and the resulting library was screened for increases in UDP-GalNAc transfer activity. Two novel mutants, M214G and M214S, identified by pH indicator screening, were purified and kinetically characterized. M214S and M214G both exhibited two-fold higher k(cat) and specific activity than wild-type GTB for UDP-GalNAc. The results confirm the importance of residue M214 for donor enzyme specificity. PMID:18405657

Persson, Mattias; Palcic, Monica M



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

PubMed Central

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

Ramasamy, Sakthivel; Bennet, Devasier; Kim, Sanghyo



Reduction of the diagnostic window with a new combined p24 antigen and human immunodeficiency virus antibody screening assay  

Microsoft Academic Search

In order to reduce the window phase between time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new fourth generation screening assays which permit a simultaneous detection of HIV antigen and antibody have been developed. In a multicenter study, a new automated fourth generation assay, Enzymun-Test® HIV Combi (Boehringer Mannheim GmbH) was compared to third generation assay, p24 antigen

Lutz Gürtler; Annelies Mühlbacher; Ulrike Michl; Hanns Hofmann; G Giancarlo Paggi; Vincenzo Bossi; Rigmor Thorstensson; Roberto G.-Villaescusa; Adolfo Eiras; JoseManuel Hernandez; Walter Melchior; Frédéric Donie; Bernard Weber



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


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

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



Evaluating the 3C-like protease activity of SARS-Coronavirus: Recommendations for Standardized Assays for Drug Discovery  

PubMed Central

Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution x-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide. PMID:17397958

Grum-Tokars, Valerie; Ratia, Kiira; Begaye, Adrian; Baker, Susan C.; Mesecar, Andrew D.



High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45  

PubMed Central

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157–177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107–137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors. PMID:22891353

Stanford, Stephanie M.; Panchal, Rekha G.; Walker, Logan M.; Wu, Dennis J.; Falk, Matthew D.; Mitra, Sayantan; Damle, Sagar S.; Ruble, David; Kaltcheva, Teodora; Zhang, Sheng; Zhang, Zhong-Yin; Bavari, Sina; Barrios, Amy M.; Bottini, Nunzio



The Drug Use Screening Inventory: School Adjustment Correlates of Substance Abuse.  

ERIC Educational Resources Information Center

The Drug Use Screening Inventory's (DUSI) sensitivity in detecting adolescents (N=706) who abuse drugs is demonstrated. Severity of drug involvement and psychiatric disturbance correlated with scores on the School Adjustment domain. Recommends additional research be conducted to determine the predictive validity and temporal stability of DUSI…

Tarter, Ralph E.; And Others



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


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

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



AlphaScreen HTS and live-cell bioluminescence resonance energy transfer (BRET) assays for identification of Tau-Fyn SH3 interaction inhibitors for Alzheimer disease.  


Alzheimer disease (AD) is the most common neurodegenerative disease, and with Americans' increasing longevity, it is becoming an epidemic. There are currently no effective treatments for this disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid-?, but the optimal strategy for targeting Tau has not yet been identified. On the basis of considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell bioluminescence resonance energy transfer (BRET) assay using a novel BRET pair for quantifying the Tau-Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the fifth and sixth PXXP motifs to show that AD-associated phosphorylation at microtubule affinity regulating kinase sites increases the affinity of the Tau-Fyn interaction and to identify Tau-Fyn interaction inhibitors by high-throughput screening. This screen has identified a variety of chemically tractable hits, suggesting that the Tau-Fyn interaction may represent a good drug target for AD. PMID:25156556

Cochran, J Nicholas; Diggs, Pauleatha V; Nebane, N Miranda; Rasmussen, Lynn; White, E Lucile; Bostwick, Robert; Maddry, Joseph A; Suto, Mark J; Roberson, Erik D



Fluorometric assay of erythrocyte protoporphyrin: simple screening test for lead poisoning and iron deficiency.  

PubMed Central

Erythrocyte protoporphyrin levels are high in lead poisoning, iron deficiency and erythropoietic porphyria. On-site fluorometric assay was used to screen for raised blood levels in three groups of children in one city: 166 who were severely mentally retarded and lived in an institution, 88 who were moderately to severely mentally retarded and attending special schools but lived at home, and 128 who were of normal intelligence and attended a regular school. High erythrocyte protoporphyrin levels (40 micrograms/dl [0.7 mumol/l] or greater) were found in 14 of the children, each of whom was tested for further evidence of lead poisoning and iron deficiency. The two children found to have high blood lead levels (above 30 micrograms/dl [1.5 mumol/l]) were both living in the institution, were ambulatory and had pica. Of the other 12 children 8 had evidence of iron deficiency, though in 4 the probability of a true deficiency was low. The fluorometric assay of erythrocyte protoporphyrin may prove to be a simple method of screening for lead poisoning and iron deficiency. PMID:7139505

Paton, T. J.; Cembrowski, G. S.



A High-throughput Screening Assay using Krabbe Disease Patient Cells  

PubMed Central

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

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



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


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

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



The E-screen assay: a comparison of different MCF7 cell stocks.  

PubMed Central

MCF7 human breast cancer cells have been studied extensively as a model for hormonal effects on breast cancer cell growth and specific protein synthesis. Because the proliferative effect of natural estrogen is considered the hallmark of estrogen action, it was proposed that this property be used to determine whether a substance is an estrogen. The E-screen assay, developed for this purpose, is based on the ability of MCF7 cells to proliferate in the presence of estrogens. The aim of our study was to characterize the response of four MCF7 cell stocks (BUS, ATCC, BB, and BB104) and determine which of them performed best in the E-screen test. The four stocks assayed were distinguishable by their biological behavior. In the absence of estrogen, MCF7 BUS cells stopped proliferating and accumulated in the G0/G1 phase of the cell cycle; estrogen receptors increased, progesterone receptors decreased, and small amounts of pS2 protein were secreted. Of all the MCF7 stocks tested, MCF7 BUS cells showed the highest proliferative response to estradiol-17 beta: cell yields increased up to sixfold over those of nontreated cells in a 144-hr period. The differences between estrogen-supplemented and nonsupplemented MCF7 BUS cells were due mostly to G0/G1 proliferative arrest mediated by charcoal dextran-stripped serum. MCF7 BUS cell stocks and others showing a similar proliferative pattern should be chosen for use in the E-screen test, or whenever a proliferative effect of estrogen is to be demonstrated. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 2. A Figure 2. B Figure 2. C Figure 2. D Figure 3. A Figure 3. B Figure 4. A Figure 4. B Figure 5. A Figure 5. B Figure 5. C Figure 5. D PMID:7498097

Villalobos, M; Olea, N; Brotons, J A; Olea-Serrano, M F; Ruiz de Almodovar, J M; Pedraza, V



Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples.  


An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH(4)(+) is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH(4)(+) production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with > +600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10-100 microgl(-1) with a detection limit of 7.2 microgl(-1) and 8.5 microgl(-1), respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1-30 mgl(-1), with limits of detection of 0.3 mgl(-1) for Cd(II) and 0.2 mgl(-1) for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mgl(-1)). Coefficients of variation (CV) values were 6-9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection. PMID:15046746

Rodriguez, Belen Bello; Bolbot, John A; Tothill, Ibtisam E



Detached leaf assay to screen for host plant resistance to Helicoverpa armigera.  


The noctuid Helicoverpa armigera (Hübner) is a major insect pest of chickpea Cicer arietinum L., pigeonpea Cajanus cajan (L.) Millsp., peanut Arachis hypogaea L., and cotton Gossypium spp., and host plant resistance is an important component for managing this pest in different crops. Because of variations in insect density and staggered flowering of the test material, it is difficult to identify cultivars with stable resistance to H. armigera across seasons and locations. To overcome these problems, we standardized the detached leaf assay to screen for resistance to this pest in chickpea, pigeonpea, peanut, and cotton under uniform insect pressure under laboratory conditions. Terminal branch (three to four fully expanded leaves) of chickpea, first fully expanded leaf of cotton, trifoliate of pigeonpea, or quadrifoliate of peanut, embedded in 3% agar-agar in a plastic cup/jar of appropriate size (250-500-ml capacity) infested with 10-20 neonate larvae can be used to screen for resistance to H. armigera. This technique keeps the leaves in a turgid condition for approximately 1 wk. The experiments can be terminated when the larvae have caused > 80% leaf damage in the susceptible check or when differences in leaf feeding between the resistant and susceptible checks are maximum. Detached leaf assay can be used as a rapid screening technique to evaluate germplasm, segregating breeding materials, and mapping populations for resistance to H. armigera in a short span of time with minimal cost, and under uniform insect infestation. It also provides useful information on antibiosis component of resistance to the target insect pest. PMID:15889750

Sharma, Hari C; Pampapathy, G; Dhillon, Mukesh K; Ridsdill-Smith, James T



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


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

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



High-throughput functional screening using a homemade dual-glow luciferase assay.  


We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

Baker, Jessica M; Boyce, Frederick M



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

PubMed Central

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



LCP-FRAP Assay for Pre-Screening Membrane Proteins for in Meso Crystallization  

PubMed Central

Fluorescence recovery after photobleaching was used to study the diffusion of two integral membrane proteins, bacteriorhodopsin and beta2-adrenergic receptor, in lipidic cubic phase (LCP). We found that the diffusion properties within the LCP matrix strongly depend on the protein construct and applied screening conditions. Common precipitants often induce restriction on diffusion of proteins in LCP and thereby impede their chances for crystallization. A high protein mobile fraction and a fast diffusion rate correlate very well with known crystallization conditions. Using this knowledge, one can now pre-screen precipitant conditions with microgram quantities of material to rule out conditions that are not conducive to diffusion, nucleation, and crystal growth. The results of this assay will narrow membrane protein crystallization space by identifying suitable protein constructs, stabilizing compounds and precipitant conditions amenable to in meso crystallization. Crystallization pre-screening will significantly increase the chances of obtaining initial crystal hits, expediting efforts in generating high-resolution structures of challenging membrane protein targets. PMID:19234616

Cherezov, Vadim; Liu, Jeffrey; Griffith, Mark; Hanson, Michael A.; Stevens, Raymond C.



Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay.  


A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis™ SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 ?g kg(-1). The detection capability was below 0.5 ?g kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 ?g kg(-1) for SEM. IC(50) values ranged from 0.14 ?g kg(-1) (AMOZ) to 2.19 ?g kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology. PMID:21515040

O'Mahony, John; Moloney, Mary; McConnell, Robert I; Benchikh, El O; Lowry, Philip; Furey, Ambrose; Danaher, Martin



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

PubMed Central

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

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



Antimalarial drug discovery: efficacy models for compound screening  

Microsoft Academic Search

Increased efforts in antimalarial drug discovery are urgently needed. The goal must be to develop safe and affordable new drugs to counter the spread of malaria parasites that are resistant to existing agents. Drug efficacy, pharmacology and toxicity are important parameters in the selection of compounds for development, yet little attempt has been made to review and standardize antimalarial drug-efficacy

Philip J. Rosenthal; Simon L. Croft; Reto Brun; Solomon Nwaka; David A. Fidock



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

PubMed Central

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

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



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

PubMed Central

Background Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann–Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid ?-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann–Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5–9 heterozygous carriers were approximately one-half those measured with 15–32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers. PMID:15292070

Li, Yijun; Scott, C. Ronald; Chamoles, Nestor A.; Ghavami, Ahmad; Pinto, B. Mario; Turecek, Frantisek; Gelb, Michael H.



Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts  

PubMed Central

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

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



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

NASA Astrophysics Data System (ADS)

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

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



Inclusion Fluorescent-Antibody Test as a Screening Assay for Detection of Antibodies to Chlamydia pneumoniae  

PubMed Central

A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the “gold standard.” In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of ?16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of ?512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (?16) or elevated (?512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae). PMID:11986260

Tapia, Olga; Slepenkin, Anatoly; Sevrioukov, Evgueni; Hamor, Kathi; de la Maza, Luis M.; Peterson, Ellena M.



Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants.  


The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited. PMID:15324747

Johansson, Fredrik; Allkvist, Annika; Erixon, Klaus; Malmvärn, Anna; Nilsson, Robert; Bergman, Ake; Helleday, Thomas; Jenssen, Dag



Detection, characterization, and screening of heme-binding molecules by mass spectrometry for malaria drug discovery.  


Drug screening for antimalarials uses heme biocrystallization inhibition methods as an alternative to parasite cultures, but they involve complex processes and cannot detect artemisinin-like molecules. The described method detects heme-binding compounds by mass spectrometry, using dissociation of the drug-heme adducts to evaluate putative antiplasmodial activity. Applied to a chemical library, it showed a good hit-to-lead ratio and is an efficient early stage screening for complex mixtures like natural extracts. PMID:22409647

Muńoz-Durango, Katalina; Maciuk, Alexandre; Harfouche, Abha; Torijano-Gutiérrez, Sandra; Jullian, Jean-Christophe; Quintin, Jérôme; Spelman, Kevin; Mouray, Elisabeth; Grellier, Philippe; Figadčre, Bruno



Rapid throughput solubility screening assay development and its applications in preformulation;.  

E-print Network

??Drug solubility is an important physicochemical property for orally administered drugs because it can significantly influence drug dissolution and absorption profiles. Investigative drugs need to… (more)

Guo, Jeremy



Cellular assays as portals to seven-transmembrane receptor-based drug discovery.  


As technology advances to the point at which various behaviours of seven-transmembrane (7TM) receptors (also known as G protein-coupled receptors (GPCRs)) can be observed individually, it is clear that, rather than being 'on-off' switches, 7TM receptors are more akin to 'microprocessors' of information. This has introduced the phenomenon of functional selectivity, whereby certain ligands initiate only portions of the signalling mechanisms mediated by a given receptor, which has opened new horizons for drug discovery. The need to discover new 7TM receptor-ligand behaviours and quantify the effect of the drug on these complex systems, to guide medicinal chemistry, puts the pharmacological assay into the spotlight. This Perspective outlines the return to whole-system assays from reductionist recombinant systems, and discusses how the efficacy of a drug is linked to the particular assay used to observe its effects. It also highlights how these new assays are adding value to the drug discovery process. PMID:19609267

Kenakin, Terry P



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

PubMed Central

Mis-sense mutations in the ?-subunit of the G-protein, Gs?, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gs? and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gs? protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gs? proteins (R201C and R201H). Stable cell lines with equivalent transfected Gs? protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses. PMID:24667240

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



Comprehensive drug screening by integrated use of gas chromatography/mass spectrometry and Remedi HS.  


The authors evaluated an integrated approach for the screening of drugs in biosamples consisting of gas chromatography/mass spectrometry analysis of serum or whole blood (SB/GC-MS) and of high-performance liquid chromatographic and ultraviolet (HPLC-UV) analysis of urine with the REMEDi HS Biorad system (U/REM) (Bio-rad; Segrate, MI, Italy). Urine and blood samples from 26 suspected intoxicated patients and from 22 suspected lethal poisoning cases were examined. Eighty-one of the 99 parent drugs/main metabolites detected were identified by SB/GC-MS and 54 with U/REM. Thirty-six drugs/metabolites were identified with both methods, 45 by SB/GC-MS alone, and 18 by U/REM alone. Absence of the mass spectrometry (MS) spectra in the reference library and high polarity of the analytes were the main reasons for failed identification by SB/GC-MS. Unsuccessful identifications with U/REM were basically caused by the absence of the UV spectra in the reference library or by low chromatographic and spectroscopic selectivity as in the case of barbiturates and benzodiazepines (BZD), which represented 11% and 51%, respectively, of the 45 SB/GC-MS unique identifications. Urine samples of 14 BZD-positive cases were also submitted to enzymatic hydrolysis and analyzed with the REMEDi UBz assay, and results were compared with those obtained by SB/GC-MS: 14 of the 22 identified BZD were detected with both methods, three by U/REM only, and five by SB/GC-MS only. In conclusion, the integrated use of SB/GC-MS and U/REM approaches greatly enhances the amount and quality of analytical information obtainable by applying either method alone. PMID:11360040

Valli, A; Polettini, A; Papa, P; Montagna, M



Long term monitoring of three automated HIV 4th generation combined antibody/antigen screening assays.  


In a diagnostic laboratory performing analyses for about 40 hospitals and 2,000 physicians treating outpatients results of HIV 4th generation combined antibody/antigen screening assays were monitored over a period of 50 months. In period A (Jan 2007 - Mar 2009) 37,986 serum samples were examined by Architect and in period B (Apr 2009 - Feb 2011) 38,178 samples by Modular system. In period B1 (Apr 2009 - Jun 2010) 24,756 samples were analyzed only by Modular system while in period B2 (July 2010 - February 2011) 13,422 samples were examined in parallel by Modular and Axsym system. Sensitivity and negative predictive value of each was 100%. Specificities ranged from 99.69-99.88% and positive predictive values (ppv) from 35.1-65.9%. Architect test results obtained a better reliability than Modular test results while Axsym test results were similar to that of Architect system. However, specificity and ppv of Modular system was markedly improved in period B2. In summary our study shows that long term monitoring of HIV combined antibody/antigen screening test results allows discovering of impairment and improvement of HIV testing quality. We also show that in a low prevalence region specificities of > 99% are accompanied by relatively low ppv. Increase of cut off values to define reactivity of the tests will increase specificities and ppv without affecting sensitivity. PMID:22998235

Franz, Mirjam; Sagel, Ulrich; Borgmann, Stefan



Implementation of an interferon-gamma release assay to screen for tuberculosis in refugees and immigrants.  


Despite increased use and accuracy of interferon-gamma release assays to detect latent tuberculosis infection (LTBI) in foreign-born arrivals in the United States, risk characteristics associated with positive results are not well characterized. We conducted a retrospective record review of 541 refugees and immigrants screened for LTBI with QuantiFERON(®)-TB Gold In-Tube (QFT-IT) at the Spokane Public Health Clinic from January 2, 2008, through June 5, 2009. Overall, 24 % of the arrivals had a positive QFT-IT, with the greatest frequency of positive results occurring in arrivals from Liberia (100 %) and Bhutan (39 %). More than the expected number of Burmese had indeterminate QFT-IT results. A positive QFT-IT was associated with age, race, ethnicity, and extent of TB burden in the country of origin. QFT-IT is useful to screen for LTBI in foreign-born arrivals, particularly middle-aged adults from high-burden countries. However, the QFT-IT may not yield meaningful results in groups with significant immunocompromise. PMID:23179470

Simpson, Terri; Tomaro, Julie; Jobb, Cynthia



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


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

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



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

PubMed Central

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

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



Intracellular Mechanics-Based Drug Screening for Cancer Metastasis  

NASA Astrophysics Data System (ADS)

In 2007 alone, close to 1.5 million new cancer cases and over half of a million deaths from cancer are projected to occur in US. In general, cancer is much easier to be successfully treated before metastasis; the five-year survival rates for most of the cancers in the metastatic stage are lower than 10%. The origin of cancer is due to genomic instability; however, the genomics or proteomics studies focus on this phenomenon cannot thoroughly elucidate how cancer metastasis proceeds. During this process, cancer cells protrude and conquer their physical barriers, resist shear stress, establish anchorages and finally settle in a new environment. Each development in this process involves mechanical forces. Thus, whether force generation and cancer cells' mechanical properties can be integrated into the current mainstream of cancer research and offer new insight is worthy of being investigated. To measure the change of cell mechanics, specifically intracellular mechanics, a tool that least disrupts the probed cell's behavior and, simultaneously, can obtain real time quantitative measurement is necessary. To satisfy these criteria, we have developed a technique, ballistic intracellular nanorheology (BIN), in which we trace and analyze the trajectories of nanospheres that have been ballistically bombarded into the cytoplasm of individual cells. This technique allows us to probe the effects of chemical or mechanical stimuli on intracellular mechanics in various types of cells, on culture dishes or in a three-dimensional matrix. BIN is, currently, the first and only method available that can be applied to perform such tasks. Using this technique, we have gained detailed information about how the cytoskeletal remodeling pathways control the intracellular mechanics. We have also obtained information on the tempo-correlation between agonists and intracellular mechanics and how cells utilize their intracellular mechanics to react extracellular shear stress. These studies have set the framework for us to understand the mechanical mechanism of cancer cell metastasis on a molecular level. In this talk, I will describe the working principal using this technique to screen cancer drugs that prevent cancer metastasis.

Tseng, Yilder



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

SciTech Connect

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

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



Antimycobacterial screening of traditional medicinal plants using the microplate resazurin assay.  


Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guérin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate resazurin assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics. PMID:20657619

Webster, Duncan; Lee, Timothy D G; Moore, Jill; Manning, Tracy; Kunimoto, Dennis; LeBlanc, Darren; Johnson, John A; Gray, Christopher A



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

PubMed Central

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

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



CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells.  


Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo. PMID:24343869

Steuwe, Christian; Patel, Imran I; Ul-Hasan, Mahmud; Schreiner, Alexander; Boren, Joan; Brindle, Kevin M; Reichelt, Stefanie; Mahajan, Sumeet



Development of a biomimetic phospholipid vesicle-based permeation assay for the estimation of intestinal drug permeability.  


Permeability is a crucial property of orally administered drugs. Therefore, in drug discovery, it is important to employ methods suitable for rapidly and reliably screening the permeability of large numbers of new drug candidates. The phospholipid vesicle-based permeation assay (PVPA), a model consisting of a tight layer of liposomes immobilized on a filter, offers potential advantages unmet by other methods and has been successfully used in permeability testing of novel active substances as well as formulations. In this study, the PVPA was developed into a more robust, biomimetic model by employing a lipid composition matching that of the intestinal permeation barrier and performing the experiments at the more biologically relevant pH 6.2. As expected, positively charged basic compounds demonstrated increased permeability through the negatively charged biomimetic barriers, and the degree of correct classification according to in vivo absorption was comparable between the original PVPA and the biomimetic PVPA. The biomimetic PVPA further proved to be tremendously more robust toward the presence of tensides compared with the original PVPA; this is a promising finding that renders the biomimetic PVPA an enhanced ability to estimate the permeability of poorly soluble compounds. Hence, the PVPA model developed in this study has evolved an important step forward. PMID:24665049

Naderkhani, Elenaz; Isaksson, Johan; Ryzhakov, Alexey; Flaten, Gřril Eide



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

NSDL National Science Digital Library

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

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



The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants.  

PubMed Central

Estrogens are defined by their ability to induce the proliferation of cells of the female genital tract. The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure. Rodent bioassays are not suited for the large-scale screening of chemicals before their release into the environment because of their cost, complexity, and ethical concerns. The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 beta-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Among the compounds tested, several "new" estrogens were found; alkylphenols, phthalates, some PCB congeners and hydroxylated PCBs, and the insecticides dieldrin, endosulfan, and toxaphene were estrogenic by the E-SCREEN assay. In addition, these compounds competed with estradiol for binding to the estrogen receptor and increased the levels of progesterone receptor and pS2 in MCF-7 cells, as expected from estrogen mimics. Recombinant human growth factors (bFGF, EGF, IGF-1) and insulin did not increase in cell yields. The aims of the work summarized in this paper were a) to validate the E-SCREEN assay; b) to screen a variety of chemicals present in the environment to identify those that may be causing reproductive effects in wildlife and humans; c) to assess whether environmental estrogens may act cumulatively; and finally d) to discuss the reliability of this and other assays to screen chemicals for their estrogenicity before they are released into the environment. PMID:8593856

Soto, A M; Sonnenschein, C; Chung, K L; Fernandez, M F; Olea, N; Serrano, F O



An in silico screen links gene expression signatures to drug response in glioblastoma stem cells.  


Cancer stem cells (CSCs) are thought to promote resistance to chemotherapeutic drugs in glioblastoma, the most common and aggressive primary brain tumor. However, the use of high-throughput drug screens to discover novel small-molecule inhibitors for CSC has been hampered by their instability in long-term cell culture. We asked whether predictive models of drug response could be developed from gene expression signatures of established cell lines and applied to predict drug response in glioblastoma stem cells. Predictions for active compounds were confirmed both for 185 compounds in seven established glioma cell lines and 21 compounds in three glioblastoma stem cells. The use of established cell lines as a surrogate for drug response in CSC lines may enable the large-scale virtual screening of drug candidates that would otherwise be difficult or impossible to test directly in CSCs.The Pharmacogenomics Journal advance online publication, 2 December 2014; doi:10.1038/tpj.2014.61. PMID:25446780

Riddick, G; Song, H; Holbeck, S L; Kopp, W; Walling, J; Ahn, S; Zhang, W; Fine, H A



Hydration of nail plate: a novel screening model for transungual drug permeation enhancers.  


Drug delivery by topical route for the treatment of onychomycosis, a nail fungal infection, is challenging due to the unique barrier properties of the nail plate which imparts high resistance to the passage of antifungal drugs. Permeation enhancers are used in transungual formulations to improve the drug flux across the nail plate. Selection of the effective permeation enhancer among the available large pool of permeation enhancers is a difficult task. Screening the large number of permeation enhancers using conventional Franz diffusion cells is laborious and expensive. The objective of present study was to evolve a simple, accurate and rapid method for screening of transungual drug permeation enhancers based on the principle of hydration of nail plate. The permeation enhancer which affects the structural or physicochemical properties of nail plate would also affect their hydration capacity. Two screening procedures namely primary and secondary screenings were evolved wherein hydration and uptake of ciclopirox olamine by nail plates were measured. Hydration enhancement factor, HEF(24) and drug uptake enhancement factor, UEF(24) were determined for screening of 23 typical permeation enhancers. The Pearson's correlation coefficient between HEF(24) and UEF(24) was determined. A good agreement between the HEF(24) and UEF(24) data proved the validity of the proposed nail plate hydration model as a screening technique for permeation enhancers. PMID:22705091

Chouhan, P; Saini, T R



Highly sensitive photoelectrochemical assay for DNA methyltransferase activity and inhibitor screening by exciton energy transfer coupled with enzyme cleavage biosensing strategy.  


Highly sensitive DNA methyltransferase (MTase) activity and inhibitor screening photoelectrochemical (PEC) assay was developed based on the exciton energy transfer (EET) effect coupled with site-specific cleavage of restriction endonuclease (HpaII). The assay was designed by integrating the Au nanoparticles (NPs) labeled probe DNA (pDNA-Au) with CdSe quantum dots (QDs). The strong EET effect between Au NPs and CdSe QDs resulted in the dramatic decrease of photocurrent signal. The pDNA carried a sensing region for specifically recognizing target DNA (tDNA) and hybridizing with it to form a DNA duplex. With the site-specific cleavage of HpaII, the DNA duplex could be cleaved and Au NPs would be released, which broke the EET and resulted in the restoration of photocurrent signal. However, when the DNA duplex was methylated by M.SssI MTase, this cleavage of HpaII was blocked, and therefore the unbroken EET effect kept the lower photocurrent signal. That was, the restored photocurrent was inversely proportional to the MTase activity. Based on this strategy, the PEC assay could determine as low as ~0.0042U/mL of M.SssI MTase with a linear range from 0.01 to 150U/mL. In addition, the assay could be used for the screening of the inhibitors of MTase. This PEC assay provides a promising platform for monitoring the activity and inhibition of DNA MTase, and thus shows a great potential in cancer diagnostics and anti-cancer drugs discovery. PMID:25282398

Shen, Qingming; Han, Li; Fan, Gaochao; Abdel-Halim, E S; Jiang, Liping; Zhu, Jun-Jie



High throughput miniature drug-screening platform using bioprinting technology  

Microsoft Academic Search

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

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



Screening and Brief Intervention for Unhealthy Drug Use: Little or No Efficacy  

PubMed Central

Unhealthy drug use ranges from use that risks health harms through severe drug use disorders. This narrative review addresses whether screening and brief intervention (SBI), efficacious for risky alcohol use, has efficacy for reducing other drug use and consequences. Brief intervention among those seeking help shows some promise. Screening tools have been validated though most are neither brief nor simple enough for use in general health settings. Several randomized trials have tested the efficacy of brief intervention for unhealthy drug use identified by screening in general health settings (i.e., in people not seeking help for their drug use). Substantial evidence now suggests that efficacy is limited or non-existent. Reasons likely include a range of actual and perceived severity (or lack of severity), concomitant unhealthy alcohol use and comorbid mental health conditions, and the wide range of types of unhealthy drug use (e.g., from marijuana, to prescription drugs, to heroin). Although brief intervention may have some efficacy for unhealthy drug users seeking help, the model of SBI that has effects in primary care settings on risky alcohol use may not be efficacious for other drug use. PMID:25228887

Saitz, Richard



Screening ToxCast? Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay  

EPA Science Inventory

An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...


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

Technology Transfer Automated Retrieval System (TEKTRAN)

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 ...


Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology  

PubMed Central

Abstract Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z?-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats. PMID:23679849

Watson, Venita Gresham; Drake, Katherine M.; Peng, Yu



1 Lessons from (patho)physiological tissue stiffness and their implications for drug 2 screening, drug delivery and regenerative medicine  

E-print Network

Received 23 October 2010 11 Accepted 5 January 2011 12 Available online xxxx 131415 16 Keywords: 17 and drug screening and aid in the rationale design of 35biomaterials for molecular therapy and tissue . . . . . . . . . . . . . . . . . . . . . . . . . . 0 49 3. Stiffness regulation of stem cell commitment and pathological differentiation

Simmons, Craig A.


Comparison of interferon-gamma release assay and tuberculin test for screening in healthcare workers.  


Healthcare workers (HCWs) have an increased risk of tuberculosis (TB). Screening for latent tuberculosis infection and active TB is therefore essential in infection control programs. Tuberculin skin test (TST) and Interferon -gamma Release Assay (IGRA) were used simultaneously in 1686 HCWs between May 2007 and April 2009. A chest X -ray was performed in order to exclude active TB when TST was >or=10mm or IGRA was positive and in HCWs with TB contact or symptoms. IGRA was positive in 33.1% and TST was >10mm in 78.3% of the HCWs. The proportion of positive IGRA results increased with the TST diameter. In those with a TST >15mm, 49.2% were IGRA positive. TST was more than twice as often positive than the IGRA. Therefore, TST+/IGRA- results were more often observed than concordant negative or positive results. In none of the HCWs with a TST+/IGRA- result active TB was diagnosed during the study period. Repeated BCG vaccination increased the number of TST+/IGRA- discordance. The smaller the interval after BCG vaccination, the higher was the TST+/IGRA- discordance. In the screened HCWs population, active TB was diagnosed in 9. At the time of diagnosis TST and IGRA were positive in all active TB cases. The study period covers 24 months, therefore the average annual incidence rate was 268/100 000. TB burden in HCWs in Portugal is high. Considering the limitations that TST and IGRA present, the best solution seems to be the use of both, using the IGRA higher specificity for confirming a positive TST, taking advantage of the best characteristics of each test. PMID:20437000

Costa, José Torres; Silva, Rui; Sá, Raul; Cardoso, Maria Joăo; Ribeiro, Carla; Nienhaus, Albert



Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens  

PubMed Central

Neonatal infection with Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis and meningitis in newborns. Recent guidelines have recommended universal screening of all pregnant women to identify those colonized with GBS and administration of peripartum prophylaxis to those identified as carriers to reduce the risk of early-onset GBS disease in neonates. Enriched culture methods are the current standard for prenatal GBS screening; however, the implementation of more sensitive molecular diagnostic tests may be able to further reduce the risk of early-onset GBS infection. We report a clinical evaluation of the Xpert GBS LB assay, a molecular diagnostic test for the identification of GBS from broth-enriched vaginal/rectal specimens obtained during routine prenatal screening. A total of 826 specimens were collected from women undergoing prenatal screening (35 to 37 weeks' gestation) and tested at one of three clinical centers. Each swab specimen was tested directly prior to enrichment using the Xpert GBS assay. Following 18 to 24 h of broth enrichment, each specimen was tested using the Xpert GBS LB assay and the FDA-cleared Smart GBS assay as a molecular diagnostic comparator. Results obtained using all three molecular tests were compared to those for broth-enriched culture as the gold standard. The sensitivity and specificity of the Xpert GBS LB assay were 99.0% and 92.4%, respectively, compared to those for the gold standard culture. The Smart GBS molecular test demonstrated sensitivity and specificity of 96.8% and 95.5%, respectively. The sensitivities of the two broth-enriched molecular methods were superior to those for direct testing of specimens using the Xpert GBS assay, which demonstrated sensitivity and specificity of 85.7% and 96.2%, respectively. PMID:25411176

Buchan, Blake W.; Faron, Matthew L.; Fuller, DeAnna; Davis, Thomas E.; Mayne, Donna



Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens.  


Neonatal infection with Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis and meningitis in newborns. Recent guidelines have recommended universal screening of all pregnant women to identify those colonized with GBS and administration of peripartum prophylaxis to those identified as carriers to reduce the risk of early-onset GBS disease in neonates. Enriched culture methods are the current standard for prenatal GBS screening; however, the implementation of more sensitive molecular diagnostic tests may be able to further reduce the risk of early-onset GBS infection. We report a clinical evaluation of the Xpert GBS LB assay, a molecular diagnostic test for the identification of GBS from broth-enriched vaginal/rectal specimens obtained during routine prenatal screening. A total of 826 specimens were collected from women undergoing prenatal screening (35 to 37 weeks' gestation) and tested at one of three clinical centers. Each swab specimen was tested directly prior to enrichment using the Xpert GBS assay. Following 18 to 24 h of broth enrichment, each specimen was tested using the Xpert GBS LB assay and the FDA-cleared Smart GBS assay as a molecular diagnostic comparator. Results obtained using all three molecular tests were compared to those for broth-enriched culture as the gold standard. The sensitivity and specificity of the Xpert GBS LB assay were 99.0% and 92.4%, respectively, compared to those for the gold standard culture. The Smart GBS molecular test demonstrated sensitivity and specificity of 96.8% and 95.5%, respectively. The sensitivities of the two broth-enriched molecular methods were superior to those for direct testing of specimens using the Xpert GBS assay, which demonstrated sensitivity and specificity of 85.7% and 96.2%, respectively. PMID:25411176

Buchan, Blake W; Faron, Matthew L; Fuller, DeAnna; Davis, Thomas E; Mayne, Donna; Ledeboer, Nathan A



Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.  


D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates. PMID:24949900

Barber, Janet E B; Damry, Adam M; Calderini, Guido F; Walton, Curtis J W; Chica, Roberto A



A validated, stability-indicating method for the assay of dexamethasone in drug substance and drug product analyses, and the assay of preservatives in drug product  

Microsoft Academic Search

Summary  A new high-performance liquid chromatographic (HPLC) procedure for the determination of dexamethasone, imprities, degradation\\u000a products and product preservatives is described. A three-stage, linear gradient with UV detection at 240 nm allows the alysis\\u000a of dexamethasone drug substance and dexamethasone in two formulated products, using the same chromatographic system. The Limit\\u000a of Quantitation (LOQ) of dexamethasone importies in drug substance is

M. Spangler; E. Mularz



Usefulness of a Rapid Real-time PCR Assay in Prenatal Screening for Group B Streptococcus Colonization  

PubMed Central

Background Group B streptococcus (GBS) infection is a leading cause of neonatal morbidity and mortality worldwide. Here, we present the analytical and diagnostic usefulness of a new real-time PCR-based assay (Xpert GBS; Cepheid, USA) for rapid and accurate prenatal GBS screening. Methods We enrolled 175 pregnant women who were between 35 and 39 weeks of gestation. The analytical performance of the Xpert GBS assay was first tested using a reference GBS strain. Next, to test diagnostic performance, rectovaginal swabs were obtained from pregnant women who visited the hospital for regular antenatal screening after 34 weeks of gestation. The results of the Xpert GBS assay were compared to those of standard culture for the detection of prenatal GBS colonization. Results When any positive result from Xpert GBS or culture was considered a true positive, the sensitivity of the Xpert GBS assay and culture were 91% (20/22; 95% CI [confidence interval], 72-98) and 68% (15/22; 95% CI, 47-84), respectively. The specificity of both methods was 100% (153/153; 95% CI, 97-100). The sensitivity and specificity of the Xpert GBS assay, using the culture results as a reference, were 86.7% and 95.6%, respectively. In the Xpert GBS assay, the median threshold cycle of vaginally colonized samples was significantly lower than rectally colonized samples (P<0.01). Conclusions The Xpert GBS assay is an accurate, rapid, easy-to-use test for the detection of maternal GBS colonization in prenatal screening that might be especially useful in clinical settings where standard culture is not feasible. PMID:23301221

Park, Jeong Su; Cho, Dong-Hee; Yang, Jae Hyug; Kim, Moon Young; Shin, Son Moon; Kim, Eui-Chong; Park, Sung Sup



Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres  

PubMed Central

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.



Identifying putative drug targets and potential drug leads: starting points for virtual screening and docking.  


The availability of 3D models of both drug leads (small molecule ligands) and drug targets (proteins) is essential to molecular docking and computational drug discovery. This chapter describes a simple approach that can be used to identify both drug leads and drug targets using two popular Web-accessible databases: (1) DrugBank and (2) The Human Metabolome Database. First, it is illustrated how putative drug targets and drug leads for exogenous diseases (i.e., infectious diseases) can be readily identified and their 3D structures selected using only the genomic sequences from pathogenic bacteria or viruses as input. The second part illustrates how putative drug targets and drug leads for endogenous diseases (i.e., noninfectious diseases or chronic conditions) can be identified using similar databases and similar sequence input. This chapter is intended to illustrate how bioinformatics and cheminformatics can work synergistically to help provide the necessary inputs for computer-aided drug design. PMID:25330974

Wishart, David S



Fluorescent biosensors for drug discovery new tools for old targets - Screening for inhibitors of cyclin-dependent kinases.  


Cyclin-dependent kinases play central roles in regulation of cell cycle progression, transcriptional regulation and other major biological processes such as neuronal differentiation and metabolism. These kinases are hyperactivated in most human cancers and constitute attractive pharmacological targets. A large number of ATP-competitive inhibitors of CDKs have been identified from natural substances, in high throughput screening assays, or through structure-guided approaches. Alternative strategies have been explored to target essential protein/protein interfaces and screen for allosteric inhibitors that trap inactive intermediates or prevent conformational activation. However this remains a major challenge given the highly conserved structural features of these kinases, and calls for new and alternative screening technologies. Fluorescent biosensors constitute powerful tools for the detection of biomolecules in complex biological samples, and are well suited to study dynamic processes and highlight molecular alterations associated with pathological disorders. They further constitute sensitive and selective tools which can be readily implemented to high throughput and high content screens in drug discovery programmes. Our group has developed fluorescent biosensors to probe cyclin-dependent kinases and gain insight into their molecular behaviour in vitro and in living cells. These tools provide a means of monitoring subtle alterations in the abundance and activity of CDK/Cyclins and can respond to compounds that interfere with the conformational dynamics of these kinases. In this review we discuss the different strategies which have been devised to target CDK/Cyclins, and describe the implementation of our CDK/Cyclin biosensors to develop HTS/HCS assays in view of identifying new classes of inhibitors for cancer therapeutics. PMID:25314935

Prével, Camille; Kurzawa, Laetitia; Van, Thi Nhu Ngoc; Morris, May C



Passive exposure to cocaine in medical personnel and its effect on urine drug screening tests.  


This report studies the importance of passive exposure of medical personnel to cocaine hydrochloride and its impact on urine screening testing. Eleven medical staff members were exposed to cocaine hydrochloride by means of aerosol and cutaneous application, similar to that which may occur in medical practice. Urine drug screening tests were negative for everyone tested. This finding is supported by known drug kinetics. It is unlikely that a single passive exposure of medical staff to cocaine hydrochloride will produce a positive urine screening test. In all cases of positive urine tests, contaminants should be tested for which may indicate a source of the drug. The routine use of gloves and masks--which is recommended to prevent HIV infection--should further decrease medical personnel's passive exposure to cocaine hydrochloride. PMID:1408218

Kavanagh, K T; Maijub, A G; Brown, J R



Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.  


Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-?-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase. PMID:24341595

Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni



Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration  

NASA Astrophysics Data System (ADS)

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (?103 M-1) ligands from moderate and high affinity (>105 M-1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

Kitova, Elena N.; El-Hawiet, Amr; Klassen, John S.



Assessment of DNA damage in Japanese nurses handling antineoplastic drugs by the comet assay.  


To clarify genotoxic effects of occupational exposure to antineoplastic drugs in Japan, we examined DNA damage, assessed by the comet assay, in 121 female nurses and 46 female clerks working at three hospitals in the northeast of Japan. The comet assay is considered to be a sensitive and rapid method for DNA strand break detection in individual cells, and tail length and tail moment are used as the comet parameters. Concerning the basal characteristics, the 46 control subjects had higher rates of smoking and coffee-drinking habits and lower hemoglobin than the 121 nurses (p<0.05). The log-transformed tail length in the nurses was significantly longer than that in the control subjects after adjusting for possible covariates such as age and smoking habit (p<0.05). Also, the log-transformed tail length was significantly longer, in the 57 nurses who had handled antineoplastic drugs in the last six months, than that in the 46 control subjects (p<0.05); but, no significant difference in tail length or tail moment was seen between the two nurse groups with and without experience of handling hazardous drugs (p>0.05). These results suggest that Japanese nurses who have worked at hospitals using antineoplastic drugs may have a potential risk of DNA damage. To minimize this risk in Japan, use of biological safety cabinet and appropriate protective equipment, in addition to staff education and training, should be implemented in the healthcare environment. PMID:18285639

Sasaki, Makiko; Dakeishi, Miwako; Hoshi, Shigeko; Ishii, Noriko; Murata, Katsuyuki



Preventing drug interactions by online prescription screening in community pharmacies and medical practices  

Microsoft Academic Search

Background: Drug interactions have been shown to be preventable by computerized prescription entry and screening only in hospitals and not in community-based practice.Methods: We retrospectively evaluated the effect of online prescription screening in community pharmacies and physician offices of one health maintenance organization, phased in during 3 consecutive 6-month periods in 1998 to 1999 (period I, system active only in

Hillel Halkin; Itzhak Katzir; Irena Kurman; Joseph Jan; Becky Ben-Oz Malkin



High-Content Screening: A New Approach to Easing Key Bottlenecks in the Drug Discovery Process  

Microsoft Academic Search

Recent improvements in target discovery and high throughput screening (HTS) have increased the pressure at key points along the drug discovery pipeline. High-content screening (HCS) was developed to ease bottlenecks that have formed at target validation and lead optimization points in the pipeline. HCS defines the role of targets in cell functions by combining fluorescence-based reagents with the ArrayScan™ System

Kenneth A. Giuliano; Robbin L. DeBiasio; R. Terry Dunlay; Albert Gough; Joanne M. Volosky; Joseph Zock; George N. Pavlakis; D. Lansing Taylor



Finding a better drug for epilepsy: Preclinical screening strategies and experimental trial design  

PubMed Central

SUMMARY The antiepileptic drugs (AEDs) introduced during the past two decades have provided several benefits: they offered new treatment options for symptomatic treatment of seizures, improved ease of use and tolerability, and lowered risk for hypersensitivity reactions and detrimental drugdrug interactions. These drugs, however, neither attenuated the problem of drug-refractory epilepsy nor proved capable of preventing or curing the disease. Therefore, new preclinical screening strategies are needed to identify AEDs that target these unmet medical needs. New therapies may derive from novel targets identified on the basis of existing hypotheses for drug-refractory epilepsy and the biology of epileptogenesis; from research on genetics, transcriptomics, and epigenetics; and from mechanisms relevant for other therapy areas. Novel targets should be explored using new preclinical screening strategies, and new technologies should be used to develop medium- to high-throughput screening models. In vivo testing of novel drugs should be performed in models mimicking relevant aspects of drug refractory epilepsy and/or epileptogenesis. To minimize the high attrition rate associated with drug development, which arises mainly from a failure to demonstrate sufficient clinical efficacy of new treatments, it is important to define integrated strategies for preclinical screening and experimental trial design. An important tool will be the discovery and implementation of relevant biomarkers that will facilitate a continuum of proof-of-concept approaches during early clinical testing to rapidly confirm or reject preclinical findings, and thereby lower the risk of the overall development effort. In this review, we overview some of the issues related to these topics and provide examples of new approaches that we hope will be more successful than those used in the past. PMID:22708847

Simonato, Michele; Löscher, Wolfgang; Cole, Andrew J.; Dudek, F. Edward; Engel, Jerome; Kaminski, Rafal M.; Loeb, Jeffrey A.; Scharfman, Helen; Staley, Kevin J.; Velíšek, Libor; Klitgaard, Henrik



Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.  


A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption. PMID:24490924

Jones, Shuna A; Salter, Robert S; Goldsmith, Tim; Quintana, Julio; Rapnicki, Paul; Shuck, Karen; Wells, Jim E; Schneider, Marilyn J; Griffin, Dee



Indirect Competitive Assays on DVD for Direct Multiplex Detection of Drugs of Abuse in Oral Fluids.  


On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods. PMID:25540088

Zhang, Lingling; Li, Xiaochun; Li, Yunchao; Shi, Xiaoli; Yu, Hua-Zhong



Evaluation of California’s Alcohol and Drug Screening and Brief Intervention Project for Emergency Department Patients  

E-print Network

drug and alcohol use at multiple instrument for substancefor alcohol or drugs (or both) with the ASSIST instrument. AAlcohol, Smoking, and Substance Involvement Screening Test (ASSIST), a 9-item instrument

Woodruff, Susan I; Eisenberg, Kimberly; McCabe, Cameron T.; Clapp, John D.; Hohman, Melinda



Second-line anti-tuberculosis drug concentrations for susceptibility testing in the MODS assay.  


Multidrug-resistant tuberculosis (TB) threatens TB control worldwide. The microscopic observation drug susceptibility (MODS) assay is a low-cost, high-performance TB diagnostic tool for rapid liquid culture and direct isoniazid and rifampicin drug susceptibility testing (DST). The objective of this study was to explore the potential for extending the MODS assay to rapid second-line DST and to identify critical concentrations of candidate drugs for prospective testing. Sputum samples from 94 TB culture-positive patients receiving second-line TB agents were cultured following standardised MODS protocols, with a range of titrations of antimicrobial drugs added. Critical concentrations were determined using a modified Kaplan-Meier survival curve analysis. Candidate critical concentrations were determined for capreomycin (10 ?g·mL(-1)), ciprofloxacin (1.25 ?g·mL(-1)), cycloserine (40 ?g·mL(-1)), ethambutol (10 ?g·mL(-1)), ethionamide (5 ?g·mL(-1)), kanamycin (5 ?g·mL(-1)), para-aminosalicylic acid (10 ?g·mL(-1)) and streptomycin (10 ?g·mL(-1)). No cut-off point was identified for the other second-line drugs or for pyrazinamide. At particular concentrations of some second-line TB drugs this novel Kaplan-Meier analysis clearly differentiated populations that were susceptible or resistant. These candidate critical concentrations should now be tested in a range of epidemiological settings to define the performance of direct, second-line TB DST with MODS, offering potential low-cost second-line TB DST capacity. PMID:22903960

Fitzwater, Sean Patrick; Sechler, G Andrew; Jave, Oswaldo; Coronel, Jorge; Mendoza, Alberto; Gilman, Robert H; Friedland, Jon S; Moore, David A J



iScreen: world's first cloud-computing web server for virtual screening and de novo drug design based on TCM database@Taiwan.  


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 PMID:21647737

Tsai, Tsung-Ying; Chang, Kai-Wei; Chen, Calvin Yu-Chian



Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay.  


The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development. PMID:19592503

Hastwell, Paul W; Webster, Thomas W; Tate, Matthew; Billinton, Nicholas; Lynch, Anthony M; Harvey, James S; Rees, Robert W; Walmsley, Richard M



Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay  

PubMed Central

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (Ki) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol?1 atom?1 (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization. PMID:23991231

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, Joăo S.; McKerrow, James H.; Montanari, Carlos A.




EPA Science Inventory

In response to the 1996 legislative mandate for an endocrine screening and testing program, we are helping develop, standardize and validate relatively sensitive, robust and relatively simple methods for in vitro screening of chemicals that affect estrogen, and androgen function ...


Role for interferon-gamma release assays in latent tuberculosis screening before TNF-? antagonist therapy.  


TNF-? antagonist therapy is associated with a risk of severe, extrapulmonary, disseminated tuberculosis, which is fatal in 10% of cases. The risk of tuberculosis is increased four-fold in patients on TNF-? antagonist therapy. The main risk factors are a history of untreated or inadequately treated primary tuberculosis, recent contact with a tuberculosis patient, and residence in or travel to a high-endemicity region. Infection surveillance agencies throughout the world have issued recommendations to ensure the detection and treatment of latent tuberculosis before TNF-? antagonist initiation. These recommendations have returned the incidence of tuberculosis to the level seen before the introduction of TNF-? antagonists. Nevertheless, there is still room for improvement. Recommendations about latent tuberculosis screening include the use of tuberculin skin tests. However, these tests are positive in individuals vaccinated with the BCG vaccine, which leads to overuse of tuberculosis chemoprophylaxis and, therefore, to unnecessary patient exposure to hepatotoxic effects. Furthermore, tuberculin skin tests may be falsely negative in immunosuppressed patients, leading to underuse of tuberculosis prophylaxis. These shortcomings of tuberculin skin tests have generated interest in interferon-gamma release assays (IGRAs). In patients with overt tuberculosis, IGRAs are more sensitive and more specific than tuberculin skin tests. However, the accuracy of IGRAs for diagnosing latent tuberculosis remains unknown, because no reference standard is available. In addition, patients taking immunosuppressant agents to treat systemic disease may exhibit anergia, which complicates the interpretation of IGRAs. Until additional data become available, caution requires that IGRAs be used only when a positive or negative result, as assessed on a case-by-case basis, will help to decide whether tuberculosis chemoprophylaxis is in order. PMID:21251863

Lioté, Huguette; Lioté, Frédéric



A microfluidic digital single-cell assay for the evaluation of anticancer drugs.  


Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ?90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC50) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. PMID:25433683

Wang, Yao; Tang, Xiaolong; Feng, Xiaojun; Liu, Chao; Chen, Peng; Chen, Dongjuan; Liu, Bi-Feng



Diagnostic accuracy of a new point-of-care screening assay for celiac disease  

PubMed Central

AIM: To determine the diagnostic accuracy of a new point-of-care assay detecting anti-deamidated gliadin peptides in celiac disease (CD) patients. METHODS: One-hundred-and-twelve patients (age range: 1.8-79.2 years old) with clinical symptoms suggestive of CD and/or first-degree relatives (FDR) of CD patients (n = 66), and confirmed CD on a gluten-free diet (GFD) (n = 46), were prospectively enrolled in the study at Gastroenterology outpatient clinics for adult patients and from the Gastroenterology Consultation Ward at the Pediatric Department of the University Hospital of Geneva. Written informed consent was obtained from all subjects enrolled. The study received approval from the local ethics committee. The original CD diagnosis had been based on serum-positive IgA anti-tissue transglutaminase enzyme-linked immunosorbent assay (ELISA) (QuantaLite™, Inova Diagnostics, San Diego, CA, United States) and on biopsy results. Serum samples from all study participants were tested by the new CD lateral flow immunochromatographic assay (CD-LFIA) device, Simtomax® Blood Drop (Augurix SA, BioArk, Monthey, Switzerland) to detect immunoglobulin (Ig)A and IgG antibodies against deamidated gliadin peptides. The diagnostic performance was evaluated using receiver operating characteristic curves with 95%CIs. A cut-off of 2 on the Rann colorimetric scale was used to calculate the device’s sensitivity and specificity. RESULTS: CD-LFIA was highly accurate in detecting untreated celiac patients. In the group of patients with CD symptoms and/or FDR, eight new cases of CD were detected by ELISA and biopsy. All of these new cases were also correctly identified by CD-LFIA. The test yielded four false positive and four false negative results. The false positive results were all within the groups with clinical symptoms suggestive of CD and/or FDR, whereas the false negative results were all within the GFD group. The test yeld a sensitivity of 78.9% (95%CI: 54.4-93.9) and specificity of 95.7% (95%CI: 89.4-98.8), and the area under the curve reached 0.893 (95%CI: 0.798-0.988). The Kappa coefficient, calculated according to the values obtained by two readers from the same device, was of 0.96 (SE: 0.06). When the GFD patients were excluded from the analysis, the area under the curve reached 0.989 (95%CI: 0.971-1.000) and the Kappa coefficient, calculated according to the values obtained by two readers from the same device, became 0.96 (SE: 0.07). Furthermore, using the Rann scale cut-off of 2 without the GFD patients, sensitivity was 100% and specificity was 93.1% (95%CI: 83.3-98.1). CONCLUSION: The new CD-LFIA rapid screening test shows good diagnostic accuracy, sensitivity and specificity, and may rule out CD in patients with CD-related symptoms. PMID:23964145

Benkebil, Faiza; Combescure, Christophe; Anghel, Silvia I; Besson Duvanel, Cécile; Schäppi, Michela G



Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay  

Microsoft Academic Search

BACKGROUND: There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. RESULTS: A natural

Mark E Shaneyfelt; Anna D Burke; Joel W Graff; Mark A Jutila; Michele E Hardy



Investigation of a Broad-Spectrum PCR Assay for Human Papillomaviruses in Screening Benign Lesions of the Upper Aerodigestive Tract  

Microsoft Academic Search

Background: A variety of different human papillomavirus (HPV) types can be found in benign and malignant lesions of the upper aerodigestive tract. Therefore a broad-spectrum assay is needed for screening reasons. Methods: A PCR system with degenerate consensus primers originating from a very conserved region (e.g. L1) of the HPV genome was applied. The sensitivity level was improved by combining

Markus Fischer



Mass spectrometric techniques for label-free high-throughput screening in drug discovery.  


High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens. PMID:17902631

Roddy, Thomas P; Horvath, Christopher R; Stout, Steven J; Kenney, Kristin L; Ho, Pei-I; Zhang, Ji-Hu; Vickers, Chad; Kaushik, Virendar; Hubbard, Brian; Wang, Y Karen



Comparing rapid-screening and standard toxicity assays to assess known chemical contamination at a hazardous waste site  

SciTech Connect

The thrust to streamline the Superfund site investigation/remediation program makes it critical for site investigators to utilize rapid screening methodologies to facilitate decision-making. However, screening methodologies providing information upon which decision-making is based must not only be rapid but also scientifically valid. This presentation compares and contrasts two rapid screening toxicity assessments, the Daphnia magna IQ Toxicity Test {trademark} and Microtox{trademark}, to a battery of standard aquatic toxicity tests using Lemna, Rana, Pimephales, Selenastruni and Ceriodaphnia. Chemical analysis of test water samples provided evidence of potential toxicological risk associated with the test samples. The study site was J-Field, Aberdeen Proving Ground, Maryland, a federal facility listed on the National Priority List that used to test and/or dispose of high explosives and chemical warfare agents in open pits or fields. Surface water samples from 20 sites were collected and used in the toxicity assessments. Water samples also were analyzed for explosives, chemical surety degradation compounds, Target Analyte List (inorganics), Target Compound List (organics) and selected pesticides and PCBs. The Microtox{trademark} assay did not reveal any toxicity present in the samples analyzed. Correlation analyses showed only slight correlation between the Daphnia magna IQ{trademark} assay and the standard 48-hour toxicity test. No correlation existed between the Microtox{trademark} assay and the aquatic toxicity tests. Results are discussed in light of the expected risk of the chemicals known to be present and the outcome of the toxicity tests.

Martino, L. [Argonne National Labs., Washington, DC (United States); Swigert, J.; Roberts, C. [Wildlife International Ltd., Easton, MD (United States)



Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia  

PubMed Central

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol. PMID:23418481

Li, Lingli; Voullaire, Lucille; Sandi, Chiranjeevi; Pook, Mark A.



Bridging the Gap From Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes  

PubMed Central

This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hER?. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hER? and hER?, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ER? subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ER? subtypes are critically involved in the teleost estrogenic response, with the ER?:ER? ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures. PMID:24422420



The response of tumour cells to radiation and cytotoxic drugs--a comparison of clonogenic and isotope uptake assays.  

PubMed Central

We have carried out a series of experiments to compare the response to radiation and drugs of cells disaggregated from solid tumours as assayed by clonogenic survival and by an isotope incorporation method. This latter assay consisted of measuring the 24 h uptake of tritium labelled thymidine into cells plated in liquid medium upon a layer of semi-solid agar. The isotope was administered 4 days after plating. For cells from the RIF-1 mouse tumour, good agreement was seen between response to radiation, adriamycin, vincristine and CCNU as measured by the two assays. The two curves for radiation response, for example, showed similar shoulders and subsequent exponential regions. For cells from xenografts of the NCI-H69 human small cell lung cancer line, the response to radiation was dose-related for both assays, but the curve for clonogenic assay was about twice as steep as that for isotope uptake. For a range of five cytotoxic drugs, good agreement was seen between the two assays over the first 1 1/2 decades of response but with a tendency for the isotope uptake curve to plateau with further increasing drug dose. It appears that, at least for these two well-defined experimental tumour systems, the isotope uptake assay can provide a rapid quantitative assessment of cellular drug and radiation sensitivity comparable to that provided by clonogenic assay but in a much shorter period of time. PMID:6093839

Twentyman, P. R.; Walls, G. A.; Wright, K. A.



The US EPA's Endocrine Disruptor Screening Program: In VItro and In Vivo Mammalian Tier 1 Screening Assays  

EPA Science Inventory

In response to emerging concerns that environmental chemicals may have adverse effects on human health by altering the function of the endocrine system, the Food Quality Protection Act mandated that the U.S. EPA develop and implement an endocrine disruptor screening program (EDSP...


Validity of the Drug Use Screening Inventory for Predicting DSM-III-R Substance Use Disorder.  

ERIC Educational Resources Information Center

Investigates the Drug Use Screening Inventory's (DUSI) utility for identifying youth who will develop a disorder of abuse/dependence. Findings indicate that the DUSI, taking only 15-20 minutes to complete, is a useful practical instrument to identify youth who could benefit most from prevention interventions. (Contains 12 references and 4 tables.)…

Tarter, Ralph E.; Kirisci, Levent



Micellar electrokinetic chromatographic screening method for common sexual assault drugs administered in beverages  

Microsoft Academic Search

Recently, much attention has been given to benzodiazepines and ?-hydroxybutyric acid (GHB) related compounds owing to their alleged widespread use as date-rape drugs. Toxicologists would greatly benefit from a screening method that allows for the simultaneous detection of both groups of substances. A new capillary electrophoresis (CE) method has been developed in the micellar mode to accomplish this separation in

Sandra C Bishop; Margaret Lerch; Bruce R McCord



Modified local straight-line screening to detect synthetic drugs in adulterated herbal medicines.  


A local straight-line screening (LSLS) algorithm was recently designed as a method to detect synthetic drug(s) in adulterated herbal medicines based on infrared spectroscopy. Some modifications are made in this paper to improve the existing LSLS algorithm, including interpolation, second derivation, and change of calculation regions from 3 to 7 data points. These modifications have decreased the effect of unpredicted noises and baseline shift on infrared spectroscopy, resulting in outstanding detailed spectral characteristics of the suspected synthetic drugs. The algorithm has been tested using five kinds of synthetic drugs (sibutramine, fenfluramine, lovastatin, sildenafil, and methyldopa) in 40 herbal medicine samples. The concentration of the synthetic drug(s) predicted by the modified LSLS algorithm is closer to those determined by high-performance liquid chromatography. Consequently, the correct results rise from 30 obtained using the original LSLS to 36 obtained using the modified LSLS in 40 samples, the false negative responses drop from 5 to 1, and the false positive responses drop from 5 to 3. The results obtained using the M-LSLS algorithm based on the sibutramine spectrum collected at different times and on different instruments also vary within acceptable ranges. These allow the method to be more appropriate for the preliminary screening of herbal medicines suspected of adulteration with synthetic drugs, with high rapidity, accuracy, and cost effectiveness. PMID:19366515

Xueyi, Zhu; Zhonghu, Zhang; Feng, Lu; Yutian, Wu; Yunpeng, Qi



An Algorithm for the Preclinical Screening of Anticancer Drugs Effective against Brain Tumors  

PubMed Central

The anticancer drugs screening program is a long and expensive process. It is estimated that only 5% of drugs entering clinical trials are approved by the FDA. Moreover, many of the drugs that enter clinical trials are often of limited use in clinical practice, and most cancers remain untreatable. Brain tumors are particularly difficult to treat due to the presence of the blood brain barrier that limits the penetration of anticancer drugs. Additionally the isolation from most brain tumors of putative cancer stem cells and novel models of cancer stem cell biology suggest that anticancer drugs should be delivered for prolonged time and at higher concentrations to deplete any potential tumorigenic cell. In this paper, current concepts of cancer stem cell biology and novel concepts of anticancer drugs screening are integrated to develop a seven-steps algorithm as a guideline for the preclinical evaluation of active compounds for the treatment of brain tumors. The flexibility of the algorithm allows the inclusion of alternative studies to exhaustively investigate anticancer drugs and creates multiple opportunities where decisions to engage or not in early clinical trials can be made providing a useful tool for translational research in neurooncology. PMID:22830045

Yakisich, Juan Sebastian



Drug screening in medical examiner casework by high-resolution mass spectrometry (UPLC-MSE-TOF).  


Postmortem drug findings yield important analytical evidence in medical examiner casework, and chromatography coupled with nominal mass spectrometry (MS) serves as the predominant general unknown screening approach. We report screening by ultra performance liquid chromatography (UPLC) coupled with hybrid quadrupole time-of-flight mass spectrometer (MS(E)-TOF), with comparison to previously validated nominal mass UPLC-MS and UPLC-MS-MS methods. UPLC-MS(E)-TOF screening for over 950 toxicologically relevant drugs and metabolites was performed in a full-spectrum (m/z 50-1,000) mode using an MS(E) acquisition of both molecular and fragment ion data at low (6 eV) and ramped (10-40 eV) collision energies. Mass error averaged 1.27 ppm for a large panel of reference drugs and metabolites. The limit of detection by UPLC-MS(E)-TOF ranges from 0.5 to 100 ng/mL and compares closely with UPLC-MS-MS. The influence of column recovery and matrix effect on the limit of detection was demonstrated with ion suppression by matrix components correlating closely with early and late eluting reference analytes. Drug and metabolite findings by UPLC-MS(E)-TOF were compared with UPLC-MS and UPLC-MS-MS analyses of postmortem blood in 300 medical examiner cases. Positive findings by all methods totaled 1,528, with a detection rate of 57% by UPLC-MS, 72% by UPLC-MS-MS and 80% by combined UPLC-MS and UPLC-MS-MS screening. Compared with nominal mass screening methods, UPLC-MS(E)-TOF screening resulted in a 99% detection rate and, in addition, offered the potential for the detection of nontargeted analytes via high-resolution acquisition of molecular and fragment ion data. PMID:23999055

Rosano, Thomas G; Wood, Michelle; Ihenetu, Kenneth; Swift, Thomas A



Corifungin, a New Drug Lead against Naegleria, Identified from a High-Throughput Screen  

PubMed Central

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM. PMID:22869574

Debnath, Anjan; Tunac, Josefino B.; Galindo-Gómez, Silvia; Silva-Olivares, Angélica; Shibayama, Mineko



Validation of the direct analysis in real time source for use in forensic drug screening.  


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. PMID:19302399

Steiner, Robert R; Larson, Robyn L



Screening for Oxidative Stress Elicited by Engineered Nanomaterials: Evaluation of Acellular DCFH Assay  

PubMed Central

The DCFH assay is commonly used for measuring free radicals generated by engineered nanomaterials (ENM), a well-established mechanism of ENM toxicity. Concerns exist over susceptibility of the DCFH assay to: assay conditions, adsorption of DCFH onto ENM, fluorescence quenching and light scattering. These effects vary in magnitude depending on ENM physiochemical properties and concentration. A rigorous evaluation of this method is still lacking. The objective was to evaluate performance of the DCFH assay for measuring ENM-induced free radicals. A series of diverse and well-characterized ENM were tested in the acellular DCFH assay. We investigated the effect of sonication conditions, dispersion media, ENM concentration, and the use of horseradish peroxidase (HRP) on the DCFH results. The acellular DCFH assay suffers from high background signals resulting from dye auto-oxidation and lacks sensitivity and robustness. DCFH oxidation is further enhanced by HRP. The number of positive ENM in the assay and their relative ranking changed as a function of experimental conditions. An inverse dose relationship was observed for several Carbon-based ENM. Overall, these findings indicate the importance of having standardized assays for evaluating ENM toxicity and highlights limitations of the DCFH assay for measuring ENM-induced free radicals. PMID:22942866

Pal, Anoop K.; Bello, Dhimiter; Budhlall, Bridgette; Rogers, Eugene; Milton, Donald K.



The development and validation of the Indigenous Risk Impact Screen (IRIS): a 13-item screening instrument for alcohol and drug and mental health risk  

Microsoft Academic Search

The study aimed to assess the psychometric properties of the Indigenous Risk Impact Screen (IRIS) as a screening instrument for determining (i) the presence of alcohol and drug and mental health risk in Indigenous adult Australians and (ii) the cut-off scores that discriminate most effectively between the presence and absence of risk. A cross-sectional survey was used in clinical and




A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine  

PubMed Central

Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called ‘caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535?nm (green) to 486?nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery. PMID:17179946

Tian, H; Ip, L; Luo, H; Chang, D C; Luo, K Q



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

PubMed Central

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

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



Screening of drug target proteins by 2D ligand matching approach.  


Drugs interacting with off-target proteins would bring about side-effects. The identification of the proteins that a drug can bind is thus valuable for evaluating its side-effects. We established a system based on PDB database for screening for proteins a drug could bind. Firstly, all complexes in the PDB database were sorted by species; then, a ligand database was established by extracting ligands from the structure data files. Secondly, all proteins were clustered according to their sequence similarity with the protein originally bound with the ligand in PDB. To search the potential target proteins of a drug, the query drug structure is compared with all ligands in the database to obtain similar scores. Ligands with similar sores greater than a certain threshold were flagged. Protein clusters associating with these ligands would be considered as potential targets of the query drug. To test the reliability of this approach, three drugs from DrugBank were used to search for their binding proteins by our method. The results showed that all the corresponding target proteins were found. The method presented here was rapid, scalable and could be used for high efficient drug side-effects analysis. PMID:24034065

Feng, Jianyu; Guo, Hong; Wang, Jian; Lu, Tun



Chemical microarray: a new tool for drug screening and discovery  

PubMed Central

HTS with microtiter plates has been the major tool used in the pharmaceutical industry to explore chemical diversity space and to identify active compounds and pharmacophores for specific biological targets. However, HTS faces a daunting challenge regarding the fast-growing numbers of drug targets arising from genomic and proteomic research, and large chemical libraries generated from high-throughput synthesis. There is an urgent need to find new ways to profile the activity of large numbers of chemicals against hundreds of biological targets in a fast, low-cost fashion. Chemical microarray can rise to this challenge because it has the capability of identifying and evaluating small molecules as potential therapeutic reagents. During the past few years, chemical microarray technology, with different surface chemistries and activation strategies, has generated many successes in the evaluation of chemical–protein interactions, enzyme activity inhibition, target identification, signal pathway elucidation and cell-based functional analysis. The success of chemical microarray technology will provide unprecedented possibilities and capabilities for parallel functional analysis of tremendous amounts of chemical compounds. PMID:16793536

Ma, Haiching; Horiuchi, Kurumi Y.



Toxicity Screening of the ToxCast Chemical Library Using a Zebrafish Developmental Assay  

EPA Science Inventory

As part of the chemical screening and prioritization research program of the U.S. Environmental Protection Agency, the toxicity of the 320 ToxCast? Phase I chemicals were assessed using a vertebrate screen of developmental toxicity. Zebrafish embryos/larvae (Danio rerio) were exp...


Dose response screening of the Toxcast chemical library using a Zebrafish developmental assay  

EPA Science Inventory

As part of the chemical screening and prioritization research program of the U.S. Environmental Protection Agency, the toxicity of the 320 ToxCaspM Phase I chemicals was assessed using a vertebrate screen of developmental toxicity. Zebrafish embryos/larvae (Danio rerio) were expo...


Screening, isolation and optimization of anti–white spot syndrome virus drug derived from marine plants  

PubMed Central

Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1?000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug. PMID:25183065

Chakraborty, Somnath; Ghosh, Upasana; Balasubramanian, Thangavel; Das, Punyabrata



Screening, isolation and optimization of anti–white spot syndrome virus drug derived from terrestrial plants  

PubMed Central

Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug. PMID:25183066

Ghosh, Upasana; Chakraborty, Somnath; Balasubramanian, Thangavel; Das, Punyabrata



False-positive interferences of common urine drug screen immunoassays: a review.  


Urine drug screen (UDS) immunoassays are a quick and inexpensive method for determining the presence of drugs of abuse. Many cross-reactivities exist with other analytes, potentially causing a false-positive result in an initial drug screen. Knowledge of these potential interferents is important in determining a course of action for patient care. We present an inclusive review of analytes causing false-positive interferences with drugs-of-abuse UDS immunoassays, which covers the literature from the year 2000 to present. English language articles were searched via the SciFinder platform with the strings 'false positive [drug] urine' yielding 173 articles. These articles were then carefully analyzed and condensed to 62 that included data on causes of false-positive results. The discussion is separated into six sections by drug class with a corresponding table of cross-reacting compounds for quick reference. False-positive results were described for amphetamines, opiates, benzodiazepines, cannabinoids, tricyclic antidepressants, phencyclidine, lysergic acid diethylamide and barbiturates. These false-positive results support the generally accepted practice that immunoassay positive results are considered presumptive until confirmed by a second independent chemical technique. PMID:24986836

Saitman, Alec; Park, Hyung-Doo; Fitzgerald, Robert L



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

NASA Technical Reports Server (NTRS)

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.

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.



An Improved Whole-Seed Assay for Screening Wheat Germplasm for Polyphenol Oxidase Activity  

Microsoft Academic Search

portant for consumer acceptance (Moss, 1971; Miskelly, 1984, 1996), while low L* values indicate undesirable Polyphenol oxidase (PPO) causes darkening and discoloration of discoloration or darkening of noodles. wheat (Triticum aestivum L.) foods such as noodles. Consequently, a simple, nondestructive, quantitative assay for determining PPO on There is a need for a simple, quantitative assay for one to a few

James V. Anderson; Craig F. Morris



Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential  

Microsoft Academic Search

The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent

M. K. Johnson



Simultaneous Screening of 177 Drugs of Abuse in Urine Using Ultra-performance Liquid Chromatography with Tandem Mass Spectrometry in Drug-intoxicated Patients  

PubMed Central

Objective The demand for rapid and broad clinical toxicology screening methods to identify drugs of abuse and medicinal drugs is increasing steadily. Liquid chromatography-tandem mass spectrometry (LC-TMS) is increasingly used to screen for drugs of abuse and to identify a wide range of drugs and metabolites in clinical samples. We revised a high-throughput and rapid ultra-performance (UP) LC-TMS method for simultaneous screening of 177 of the most prevalent medicinal drugs and drugs of abuse in urine and validated the quality of performance using system suitability mixture (SSM) and quality control (QC) materials. Methods We assessed the limits of detection (LOD) using high concentrations of the test substances. The method was applied to 473 urine samples obtained from patients intoxicated with drugs who visited the emergency center. Results The retention time, peak area, and total ion chromatogram of the SSM and QC materials were within the acceptance criteria of the pre-defined acceptance interval. The LODs were <62 ng/ml for 12 commonly encountered drugs. In total, 418 patients (88.4%) tested positive for one or more medicinal drugs or drugs of abuse. Twenty-eight drugs were detected over ten times; the most commonly detected were zolpidem, ephedrine, paracetamol, and chlorpheniramine. Conclusion The UPLC-TMS method provided excellent performance for simultaneous screening of a large number of the drugs of abuse in urine samples. We conclude that this robust technique is useful for screening for a large number of drugs and for rapid screening of the most commonly encountered substances in emergency cases. PMID:24465253



Evaluating the Predictivity of Virtual Screening for Abl Kinase Inhibitors to Hinder Drug Resistance  

PubMed Central

Virtual screening methods are now widely used in early stages of drug discovery, aiming to rank potential inhibitors. However, any practical ligand set (of active or inactive compounds) chosen for deriving new virtual screening approaches cannot fully represent all relevant chemical space for potential new compounds. In this study, we have taken a retrospective approach to evaluate virtual screening methods for the leukemia target kinase ABL1 and its drug-resistant mutant ABL1-T315I. ‘Dual active’ inhibitors against both targets were grouped together with inactive ligands chosen from different decoy sets and tested with virtual screening approaches with and without explicit use of target structures (docking). We show how various scoring functions and choice of inactive ligand sets influence overall and early enrichment of the libraries. Although ligand-based methods, for example principal component analyses of chemical properties, can distinguish some decoy sets from active compounds, the addition of target structural information via docking improves enrichment, and explicit consideration of multiple target conformations (i.e. types I and II) achieves best enrichment of active versus inactive ligands, even without assuming knowledge of the binding mode. We believe that this study can be extended to other therapeutically important kinases in prospective virtual screening studies. PMID:23746052

Gani, Osman A B S M; Narayanan, Dilip; Engh, Richard A



A comparison of the analytical level of agreement of nine treponemal assays for syphilis and possible implications for screening algorithms  

PubMed Central

Objective The serological diagnosis of syphilis requires the detection of two distinct antibodies, the non-treponemal and trepomenal. Center for Disease Control and Prevention (CDC) recommends screening first with a non-treponemal test such as (Rapid Plasma Reagin/Venereal Disease Research Laboratory), and then confirming those results with one of the several treponemal tests (Fluorescent Treponemal Antibody-Absorption (FTA-ABS), Enzyme Immunoassay, chemiluminescence, treponema pallidum particle agglutination (TP-PA) or Point of Care). Owing to the high volume of samples processed by some laboratories using automated systems, the screening with treponemal assays and confirming with non-treponemal tests is becoming the established norm. The purpose of this study was to evaluate eight treponemal assays using TP-PA as the predicate assay. Methods 290 stored serum samples were tested qualitatively according to the manufacturer's directions. Results Concordance with specimens tested as reactive or non-reactive using TP-PA was: FTA-ABS 94.5–100%, Trep-Sure 100–98.9%, BioELISA 100–98.9%, INNO-LIA 99.1–99.4%, BIOLINE 100–98.9%, CAPTIA IgG 100–97.2%, Trep-ID 100–100% and LIAISON 100–99.4%. In order to properly evaluate the performance of these assays, the analytical sensitivity was determined by endpoint titration of serial dilutions of the reactive serum samples in normal sera. The median endpoint titre varied from 1:4 for FTA-ABS to 1:512 for Trep-Sure. Conclusions The performance of the treponemal serological assays was comparable while using medium and high-titre sera. However, the varying performance on specimen dilutions suggests that there may be differences in sensitivity with low-titre sera that are more prevalent in primary and late syphilis cases. PMID:24056483

Castro, Arnold; Jost, Heather; Cox, David; Fakile, Yetunde; Kikkert, Susan; Tun, Ye; Zaidi, Akbar; Park, Mahin



Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of TB  

PubMed Central

BACKGROUND New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth. METHODS In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein–Jensen medium with the proportion method. RESULTS Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein–Jensen culture (P<0.001); the median time to culture positivity was 7 days, 13 days, and 26 days, respectively (P<0.001), and the median time to the results of susceptibility tests was 7 days, 22 days, and 68 days, respectively. The incremental benefit of a second MODS culture was minimal, particularly in patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Agreement between MODS and the reference standard for susceptibility was 100% for rifampin, 97% for isoniazid, 99% for rifampin and isoniazid (combined results for multidrug resistance), 95% for ethambutol, and 92% for streptomycin (kappa values, 1.0, 0.89, 0.93, 0.71, and 0.72, respectively). CONCLUSIONS A single MODS culture of a sputum sample offers more rapid and sensitive detection of tuberculosis and multidrug-resistant tuberculosis than the existing gold-standard methods used. PMID:17035648

Moore, David A.J.; Evans, Carlton A.W.; Gilman, Robert H.; Caviedes, Luz; Coronel, Jorge; Vivar, Aldo; Sanchez, Eduardo; Pińedo, Yvette; Saravia, Juan Carlos; Salazar, Cayo; Oberhelman, Richard; Hollm-Delgado, Maria-Graciela; LaChira, Doris; Escombe, A. Roderick; Friedland, Jon S.



Transgenic Plants as Low-Cost Platform for Chemotherapeutic Drugs Screening  

PubMed Central

In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects. PMID:25608652

Vergara, Daniele; de Domenico, Stefania; Maffia, Michele; Piro, Gabriella; Di Sansebastiano, Gian-Pietro



Transgenic plants as low-cost platform for chemotherapeutic drugs screening.  


In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects. PMID:25608652

Vergara, Daniele; de Domenico, Stefania; Maffia, Michele; Piro, Gabriella; Sansebastiano, Gian-Pietro Di



Image-Based Chemical Screening Identifies Drug Efflux Inhibitors In Lung Cancer Cells  

PubMed Central

Cancer cells with active drug-efflux capability are multidrug resistant and pose a significant obstacle for the efficacy of chemotherapy. Moreover, recent evidence suggests that high drug-efflux cancer cells (HDECCs) may be selectively enriched with stem-like cancer cells, which are believed to be the cause for tumor initiation and recurrence. There is a great need for therapeutic reagents that are capable of eliminating HDECCs. We developed an image-based high-content screening (HCS) system to specifically identify and analyze the HDECC population in lung cancer cells. Using the system, we screened 1,280 pharmacologically active compounds which identified twelve potent HDECC inhibitors. It is shown that these inhibitors are able to overcome MDR and sensitize HDECCs to chemotherapeutic drugs, or directly reduce the tumorigenicity of lung cancer cells possibly by affecting stem-like cancer cells. The HCS system we established provides a new approach for identifying therapeutic reagents overcoming MDR. The compounds identified by the screening may potentially be used as potential adjuvant to improve the efficacy of chemotherapeutic drugs. PMID:20841476

Xia, Xiaofeng; Yang, Jian; Li, Fuhai; Li, Ying; Zhou, Xiaobo; Dai, Yue; Wong, Stephen T C



Combinatorial drug screening identifies compensatory pathway interactions and adaptive resistance mechanisms  

PubMed Central

Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment. PMID:23599172

Axelrod, Mark; Gordon, Vicki L.; Conaway, Mark; Tarcsafalvi, Adel; Neitzke, Daniel J.; Gioeli, Daniel; Weber, Michael J.



A High-Throughput Assay for Arylamine Halogenation Based on a Peroxidase-Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations  

PubMed Central

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one-pot” workflow. PMID:25319801

Hosford, Joseph; Shepherd, Sarah A; Micklefield, Jason; Wong, Lu Shin



Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression  

NASA Astrophysics Data System (ADS)

Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda



Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals.  


Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelope proteins and their mRNAs are produced in the liver of oviparous species by stimulation of endogenous estrogen and exogenous estrogenic chemicals. The VTG assay based on enzyme-linked immunosorbent assay (ELISA) has been widely used for many fish species to screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents using immature fish and/or male fish. In order to reduce the number of fish for screening of estrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can be used. In fact, primary cultured hepatocytes have been successfully used for the detection of estrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fish species, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream (Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish (Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these include frogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallus domesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantitative reverse transcription-polymerase chain reaction has also been successfully applied in the primary cultured hepatocytes of various species. PMID:16883298

Iguchi, Taisen; Irie, Fumi; Urushitani, Hiroshi; Tooi, Osamu; Kawashima, Yukio; Roberts, Mike; Norrgren, Leif; Hutchinson, Thomas



Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni.  


Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ?-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2. PMID:25325257

Schiedel, Matthias; Marek, Martin; Lancelot, Julien; Karaman, Berin; Almlöf, Ingrid; Schultz, Johan; Sippl, Wolfgang; Pierce, Raymond J; Romier, Christophe; Jung, Manfred



The use of screening instruments for detecting alcohol and other drug use disorders in first-episode psychosis  

Microsoft Academic Search

The high rate of drug abuse among patients with psychosis represents a challenge to clinicians in their treatment of the patients. Powerful screening tools to detect problematic drug use in an early phase of psychotic illness are needed. The aim of the present study was to investigate prevalence of drug use disorders and psychometric properties of the Alcohol Use Disorder

Ragnar Nesvĺg; Elisabeth H. Lange; Ann Fćrden; Elizabeth Ann Barrett; Björn Emilsson; Petter Andreas Ringen; Ole A. Andreassen; Ingrid Melle; Ingrid Agartz



Rapid Identification and Drug Susceptibility Testing of Mycobacterium tuberculosis: Standard Operating Procedure for Non-Commercial Assays: Part 2: Nitrate Reductase Assay v1.3.12  

PubMed Central

In the previous part, we presented the standard operating procedure (SOP) of the microscopic observation drug susceptibility assay drug susceptibility testing (DST) for Mycobacterium tuberculosis. The present SOP is devoted to another non-commercial culture and DST method known as nitrate reductase assay (NRA). As the name implies, the NRA detects the ability of M. tuberculosis to reduce nitrate to nitrite. In the assay, the presence of nitrite is detected by the addition of p-nitrobenzoate into the growth yield. The reaction is detected by the naked eye. The incorporation of drugs in the medium allows to use the test for DST, which can be interpreted with naked eyes. The identification and drug susceptibility results can be obtained in 2-3 weeks. This SOP document has been developed through the culture and DST subgroup of the STOP tuberculosis (TB) Partnership New Diagnostic Working Group. It is intended for laboratories that would want to use or already using this rapid non-commercial method for culture identification and DST of M. tuberculosis, notably in resource-constraint settings in Asia and Africa. PMID:23440455

Singh, Sarman; Kumar, Parveen; Sharma, Shreya; Mumbowa, Francis; Martin, Anandi; Durier, Nicolas



Spectrofluorimetric methods of stability-indicating assay of certain drugs affecting the cardiovascular system  

NASA Astrophysics Data System (ADS)

Two stability-indicating spectrofluorimetric methods have been developed for the determination of ezetimibe and olmesartan medoxomil, drugs affecting the cardiovascular system, and validated in the presence of their degradation products. The first method, for ezetimibe, is based on an oxidative coupling reaction of ezetimibe with 3-methylbenzothiazolin-2-one hydrazone hydrochloride in the presence of cerium (IV) ammonium sulfate in an acidic medium. The quenching effect of ezetimibe on the fluorescence of excess cerous ions is measured at the emission wavelength, ?em, of 345 nm with the excitation wavelength, ?ex, of 296 nm. Factors affecting the reaction were carefully studied and optimized. The second method, for olmesartan medoxomil, is based on measuring the native fluorescence intensity of olmesartan medoxomil in methanol at ?em = 360 nm with ?ex = 286 nm. Regression plots revealed good linear relationships in the assay limits of 10-120 and 8-112 g/ml for ezetimibe and olmesartan medoxomil, respectively. The validity of the methods was assessed according to the United States Pharmacopeya guidelines. Statistical analysis of the results exposed good Student's t-test and F-ratio values. The introduced methods were successfully applied to the analysis of ezetimibe and olmesartan medoxomil in drug substances and drug products as well as in the presence of their degradation products.

Moussa, B. A.; Mohamed, M. F.; Youssef, N. F.



A rapid and sensitive HPLC assay of some concomitant anti-migraine drugs.  


This work describes a simple and sensitive method for simultaneous determination of zolmitriptan, naproxen and propranolol in their dosage forms using HPLC. The drugs were separated isocratically on a Zorbax C8 (4.6 × 250 mm with 5 µm particle size) column using a mobile phase composed of 20 mM phosphate citrate buffer [0.1% TEA (pH 3.1)]:methanol:THF (5:3:2, by volumes). The detection was accomplished fluorometrically setting the excitation wavelength at 280 nm and emission wavelength at 360 nm. The method was validated over a linearity range of 100-900 ng/mL for zolmitriptan, 50-300 ng/mL for naproxen and 100-800 ng/mL for propranolol. The assay was successfully applied to the determination of the studied drugs in pharmaceutical dosage forms without interference from tablet excipients with high specificity. The method can be applied successfully in the future for the pharmacokinetic study of these drugs in the human plasma with high accuracy especially that LOQs of zolmitriptan and propranolol in the proposed method cover their Cmax. PMID:23845885

Rezk, Mamdouh R; Michael, Adel M; Lotfy, Hayam M; El-Kadi, Ayman O; Shehata, Mostafa A



Application of simulated intestinal fluid on the phospholipid vesicle-based drug permeation assay.  


The compatibility of fasted state simulated intestinal fluid (FaSSIF) in drug permeation studies employing the phospholipid vesicle-based permeation assay (PVPA) model was confirmed by a set of different integrity indicators. Neither calcein permeability nor electrical resistance were found significantly changed indicating unaffected barrier tightness. Furthermore, the release of phospholipid from the barriers in contact with FaSSIF was negligible, although sodium taurocholate disappeared from the donor - possibly due to transfer into the barrier. Visual examination of the barrier structure by confocal laser scanning microscopy (CLSM) revealed no changes. The model drugs, cimetidine, nadolol, ketoprofen and griseofulvin showed either slightly enhanced or unchanged permeability values in the presence of FaSSIF. This may be attributed to micellar encapsulation and/or slight changes in barrier characteristics. Particularly for poorly soluble drugs, FaSSIF appeared favourable in terms of markedly improved recovery. Moreover, utilisation of BSA in the receiver compartment seems to augment this beneficial effect on recovery rate. It is likely that this experimental set-up affords better sink conditions in the receiver phase, which results in higher fluxes. Overall, a combination of FaSSIF in the donor phase and BSA in the receiver phase facilitates improved experimental output. PMID:22027395

Fischer, Sarah Maud; Buckley, Stephen Timothy; Kirchmeyer, Wiebke; Fricker, Gert; Brandl, Martin



Rapid screening technique for detecting and assaying endopeptidase mutants of Aeromonas proteolytica  

E-print Network

T to Hemoglobin Assay Values Plot of Halo Diameter vs. log of Endopeptidase Units (Hemoglobin Assay) Assay Plates for Determination of' Optimal Broth Medium for Endo- peptidase Production. Top: Heart Infusion Broth, Bottom: Casamino Acids-SW Page 5C 54... cultures, it consisted of a 20 1. $$/a solution of SSMN enriched with 0. $$ mg/1 of K&IB'04. The media tested were heart infusion made with distilled water, heart infusion made with SW, 4/o casamino acids-SW, 2/a casamino acids-SW, 4~/o casitone- SW...

Soli, Teri Cullen



Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds  

E-print Network

), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 2 Screening Technology & Pharmacology Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 3 Active Compound Space Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 4 Image Mining Group, Institut Pasteur Korea

Paris-Sud XI, Université de


Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells  

PubMed Central

Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed “interactome.” Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET) technique was primarily developed to allow the dynamic monitoring of protein/protein interactions (PPI) in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of PPI and here is described why and how to set up and optimize a high throughput screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence of substrate concentration, number of cells and medium composition used on the Z? factor, and expected interferences from colored or fluorescent compounds. PMID:22973258

Couturier, Cyril; Deprez, Benoit



Large-scale virtual screening  

E-print Network

. Perkins R. A. Sykes J. Li Virtual screening, or in silico screening, is a new approach attracting the impact of automated laboratory systems in the fields of chemical synthesis, biological assay, drug payment of royalty provided that (1) each reproduction is done without alteration and (2) the Journal

Ferreira, Márcia M. C.


Synergistic Drug Combinations for Tuberculosis Therapy Identified by a Novel High-Throughput Screen?†  

PubMed Central

Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies. PMID:21576426

Ramón-García, Santiago; Ng, Carol; Anderson, Hilary; Chao, Joseph D.; Zheng, Xingji; Pfeifer, Tom; Av-Gay, Yossef; Roberge, Michel; Thompson, Charles J.



Patterned Cardiomyocytes on Microelectrode Arrays as a Functional, High Information Content Drug Screening Platform  

PubMed Central

Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high-information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes. PMID:21453966

Natarajan, Anupama; Stancescu, Maria; Dhir, Vipra; Armstrong, Christopher; Sommerhage, Frank; Hickman, James J.; Molnar, Peter



Influenza virus-host interactome screen as a platform for antiviral drug development.  


Host factors required for viral replication are ideal drug targets because they are less likely than viral proteins to mutate under drug-mediated selective pressure. Although genome-wide screens have identified host proteins involved in influenza virus replication, limited mechanistic understanding of how these factors affect influenza has hindered potential drug development. We conducted a systematic analysis to identify and validate host factors that associate with influenza virus proteins and affect viral replication. After identifying over 1,000 host factors that coimmunoprecipitate with specific viral proteins, we generated a network of virus-host protein interactions based on the stage of the viral life cycle affected upon host factor downregulation. Using compounds that inhibit these host factors, we validated several proteins, notably Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) and JAK1, as potential antiviral drug targets. Thus, virus-host interactome screens are powerful strategies to identify targetable host factors and guide antiviral drug development. PMID:25464832

Watanabe, Tokiko; Kawakami, Eiryo; Shoemaker, Jason E; Lopes, Tiago J S; Matsuoka, Yukiko; Tomita, Yuriko; Kozuka-Hata, Hiroko; Gorai, Takeo; Kuwahara, Tomoko; Takeda, Eiji; Nagata, Atsushi; Takano, Ryo; Kiso, Maki; Yamashita, Makoto; Sakai-Tagawa, Yuko; Katsura, Hiroaki; Nonaka, Naoki; Fujii, Hiroko; Fujii, Ken; Sugita, Yukihiko; Noda, Takeshi; Goto, Hideo; Fukuyama, Satoshi; Watanabe, Shinji; Neumann, Gabriele; Oyama, Masaaki; Kitano, Hiroaki; Kawaoka, Yoshihiro




EPA Science Inventory

In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...



EPA Science Inventory

A simple, inexpensive and sensitive method for detecting organophosphate and carbamate insecticides is reported. Acetylcholinesterase was immobilized to PorexR Lateral-FloTM membrane material and remained active for several months at room temperature. The assay was sensitive ...


Nanotechnology in drug delivery: the need for more cell culture based studies in screening  

PubMed Central

Advances in biomedical science are leading to upsurge synthesis of nanodelivery systems for drug delivery. The systems were characterized by controlled, targeted and sustained drug delivery ability. Humans are the target of these systems, hence, animals whose systems resembles humans were used to predict outcome. Thus, increasing costs in money and time, plus ethical concerns over animal usage. However, with consideration and planning in experimental conditions, in vitro pharmacological studies of the nanodelivery can mimic the in vivo system. This can function as a simple method to investigate the effect of such materials without endangering animals especially at screening phase. PMID:25057288



Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays  

PubMed Central

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. PMID:22470516

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.



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

SciTech Connect

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.

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



A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay.  


The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Iad-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes. PMID:7740543

Hatao, M; Hariya, T; Katsumura, Y; Kato, S



Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei  

SciTech Connect

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.

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)



Inter- and Intra-Assay Reproducibility of Microplate Alamar Blue Assay Results for Isoniazid, Rifampicin, Ethambutol, Streptomycin, Ciprofloxacin, and Capreomycin Drug Susceptibility Testing of Mycobacterium tuberculosis? †  

PubMed Central

The intersample and intrasample variability of the results obtained with the microplate Alamar blue assay for the indirect drug susceptibility testing of Mycobacterium tuberculosis was investigated. Between 1.2 and 8.5% of paired MICs differed by more than one twofold dilution, resulting in discordant susceptible-resistant designations at frequencies between 0.6% (rifampin) and 18.9% (ethambutol). PMID:18701659

Leonard, Brian; Coronel, Jorge; Siedner, Mark; Grandjean, Louis; Caviedes, Luz; Navarro, Pilar; Gilman, Robert H.; Moore, David A. J.



Sediment toxicity screening with cost-effective microbiotests and conventional assays: A comparative study  

SciTech Connect

A large monitoring study of freshwater sediments, using the TRIAD approach, was conducted in Flanders (Belgium). This paper reports on the results of the toxicity assessment of 80 sediment samples evaluated with a battery of microbiotests and conventional assays. Sediment pore waters, extracted by squeezing, were tested with the Microtox{reg_sign} (Vibrio fischerii) and Thamnotoxkit{trademark} F (Thamnocephalus platyurus) microbiotests and the conventional (acute) assays with algae (Selenastrum capricornutum) and daphnids (Daphnia magna). A newly developed 5 day ELS test with the catfish Clarias gariepinus was also applied to the pore waters. Solid-phase testing was performed with the Microtox Sp{reg_sign} assay and the 10 day tests with Chironomus riparius and Hyalella azteca. Uni- and multivariate statistical techniques were applied to the data matrix to select a minimal test battery from the water phase and solid phase assays and from all tests combined. The influence of sediment associated confounding factors on the validity of the test results obtained with the various assays will be discussed. Finally a comparison of the predictive power of the selected battery of signal tests and that of the complete battery will be made and the potential use of the minimal battery for the initial hazard assessment of contaminated sediments will be reviewed.

Vanciheluwe, M.L.; Janssen, C.R.; Persoone, G. [Univ. of Ghent (Belgium). Lab. for Biological Research in Aquatic Pollution



Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY  

PubMed Central

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N?-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors. PMID:14982781

Stachyra, Thérčse; Dini, Christophe; Ferrari, Paul; Bouhss, Ahmed; van Heijenoort, Jean; Mengin-Lecreulx, Dominique; Blanot, Didier; Biton, Jacques; Le Beller, Dominique



Assessment of the TLC/Salmonella assay for screening hazardous wastes  

SciTech Connect

Using a modified version of the TLC/Salmonella assay developed by Bjorseth et al. (1982), 10 complex hazardous wastes were tested for mutagenic activity. The method couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude hazardous wastes and selected hazardous-waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed by applying a single overlay of minimal growth agar containing a tester strain of Salmonella and the optional metabolic activation system directly onto the developed chromatogram. Seven of 10 hazardous wastes demonstrated mutagenic activity when tested by the method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Eleven of the 14 mutagens were positive in the test system.

Houk, V.S.; Claxton, L.D.



Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).  


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. PMID:23123550

Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G



Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay.  


The risk of contracting hepatitis from blood transfusions is estimated to be 1 in 63000 in the case of hepatitis B virus (HBV) and 1 in 103000 in the case of hepatitis C virus (HCV). In some countries (Germany, USA and England, for example), molecular protocols are evaluated to detect viral genomes in blood donations in order to reduce the seroconversion period. However, no such method is available currently to screen large series samples for HBV and HCV. While strategies involving the pooling of plasma samples have been proposed and tested in Germany, there is the question of sensitivity. We developed a novel approach to screen for HBV and HCV based on the TaqMan technology that allows for the quantification of an amplified fragment during PCR analysis (Lee et al., 1993). This approach is more sensitive than other quantification methods. As a first step primers and probes were designed to detect the different sub-types of HBV and HCV genomes. We then optimized the reaction conditions in order to screen for the two viruses at the same time. The observed sensitivity is less than 50 molecules per ml for HBV and less than 50 molecules per ml for HCV. This assay is, to our knowledge, the first that allows the simultaneous detection of DNA and RNA viral genomes. In conclusion, this TaqMan approach could be used as a single test to screen for HBV and HCV genomes in a series of 96 samples in less than 5 h. Such an approach is a first step for development of automation allowing a systematic screening of blood donations. PMID:10029319

Mercier, B; Burlot, L; Férec, C



A multilayer microdevice for cell-based high-throughput drug screening  

NASA Astrophysics Data System (ADS)

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.

Liu, Chong; Wang, Lei; Xu, Zheng; Li, Jingmin; Ding, Xiping; Wang, Qi; Chunyu, Li



Zebrafish as a genetic model in pre-clinical drug testing and screening.  


The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. However, the zebrafish is increasingly providing alternative strategies for various components of this pipeline. Zebrafish and their embryos are small, easily accessible and relatively low cost, making them applicable to high-throughput, small molecule screening. Zebrafish can also be manipulated by a range of forward and reverse genetics techniques to facilitate gene discovery and functional studies. Moreover, their physiological and developmental complexity provides accurate models of human disease to underpin mechanism of action and in vivo validation studies. Finally, several of these biological characteristics make zebrafish eminently suitable for toxicity testing, including eco-toxicology. Here we review the application of zebrafish to preclinical drug development and toxicity testing, including recent advances in mutant generation, drug screening and toxicology that serve to further enhance the capabilities of this valuable model organism in drug discovery. PMID:23521675

Gibert, Y; Trengove, M C; Ward, A C



The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?  


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. PMID:16650091

Kimber, Ian; Dearman, Rebecca J; Betts, Catherine J; Gerberick, G Frank; Ryan, Cindy A; Kern, Petra S; Patlewicz, Grace Y; Basketter, David A



An electrochemical assay for DNA methylation, methyltransferase activity and inhibitor screening based on methyl binding domain protein.  


DNA methylation is one of important epigenetics events, and responsible to transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Methyl binding domain (MBD) proteins can specifically bind to the methylated CpG dinucleotides. Conventional assay for DNA methylation normally need bisulfide treatment, methylated nucleotide labeling or PCR amplification. Here, we fabricated a novel electrochemical biosensor for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor based on MBD protein and coomassie brilliant blue G250 (CBB-G250), where the electrochemical signal of CBB-G250 was used to monitor the methylation event. After the hybrids of DNA S1 and DNA S2 were treated with M. SssI MTase in the presence of S-adenosylmethionine, the MBD proteins were specifically conjugated to the methylation site of CpG dinucleotides, and then, the MBD proteins were stained with CBB-G250. The electrochemical signal of CBB-G250 increased linearly with increasing M. SssI MTase concentration in the range from 0.1 to 40 unit/mL. Furthermore, the inhibition investigation demonstrates that fisetin and chlorogenic acid can inhibit the M. SssI MTase activity with the IC(50) value of 153.12 and 137.07 ?M, respectively. Therefore, we think that this study may provide a sensitive platform for screening of DNA MTase inhibitors. PMID:23021851

Yin, Huanshun; Zhou, Yunlei; Xu, Zhenning; Chen, Lijian; Zhang, Di; Ai, Shiyun



Using pathway modules as targets for assay development in xenobiotic screening  

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...


Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals  

EPA Science Inventory

Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...


Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening**  

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. As such, we are exploring a behavioral testing paradigm, which can assess the effect of sublethal and subteratogenic concentrations of de...


High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast  

EPA Science Inventory

High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...


Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening  

EPA Science Inventory

The U.S. Environmental Protection Agency is developing and evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. Towards this goal, we are exploring methods to detect developmental neurotoxicants in very young larval zebrafish. We have...



EPA Science Inventory

A time-integrated sampling system interfaced with a toxicity-based assay is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethyl sulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...



EPA Science Inventory

A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...



E-print Network

, image processing software is necessary to have automatic algorithms for segmenting the cells individ, watershed transformation and granulometries for seg- menting cells of different size, contrast, etc. In particular, the performance of the algorithms is illustrated with cell im- ages from a toxicity assay



A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways  

EPA Science Inventory

The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. ?Toxicity Testin...


A novel microfluidic platform with stable concentration gradient for on chip cell culture and screening assays.  


In this work a novel microfluidic platform for cell culture and assay is developed. On the chip a static cell culture region is coupled with dynamic fluidic nutrition supply structures. The cell culture unit has a sandwich structure with liquid channels on the top, the cell culture reservoir in the middle and gas channels on the bottom. Samples can be easily loaded into the reservoir and exchange constantly with the external liquid environment by diffusion. Since the flow direction is perpendicular to the liquid channel on the top of the reservoir, the cells in the reservoir are shielded from shear-force. By assembling the basic units into an array, a steady concentration gradient can be generated. Cell culture models both for continuous perfusion and one-off perfusion were established on the chip. Both adherent and suspended cells were successfully cultured on the chip in 2D and 3D culture modes. After culturing, the trapped cells were recovered for use in a later assay. As a competitive candidate for a standard cell culture and assay platform, this chip is also adaptable for cytotoxicity and cell growth assays. PMID:23884407

Xu, Bi-Yi; Hu, Shan-Wen; Qian, Guang-Sheng; Xu, Jing-Juan; Chen, Hong-Yuan



Comparison of LUCIO®-direct ELISA with CEDIA immunoassay for 'zero tolerance' drug screening in urine as required by the German re-licensing guidelines.  


The performance of the previously validated LUCIO(®)-Direct-enzyme linked immunosorbent assay (direct ELISA) screening tests according to forensic guidelines is compared to that of cloned enzyme donor immunoassays (CEDIA) test for drugs of abuse in urine as defined in the new re-licensing German medical and psychological assessment (MPA) guidelines. The MPA screening cut-offs correspond to 10?ng/ml 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 50?ng/ml amphetamine and designer amphetamines, 25?ng/ml morphine, codeine and dihydrocodeine, 30?ng/ml benzoylecgonine, 50?ng/ml methadone metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and metabolites of diazepam, oxazepam, bromazepam, alprazolam, flunitrazepam and lorazepam at 50?ng/ml. Average relative sensitivities and relative specificities were 99.7 % and 98.4 % for direct ELISA and 66 % and 91.4 % for CEDIA, respectively. PMID:23349145

Agius, Ronald; Nadulski, Thomas; Kahl, Hans-Gerhard; Dufaux, Bertin



Evaluation of a high-throughput screening method for the detection of the excipient-mediated precipitation inhibition of poorly soluble drugs.  


The objective of the present study was to develop and evaluate a high-throughput (HT) precipitation inhibitor screening method that can be used for the identification of the excipient-mediated precipitation inhibition of poorly soluble drugs. The impact of incubation temperature, shaking intensity, phase separation, inter- and intraday variability, cosolvent, and plate selection on the HT screening method performance was investigated. Additionally, the pipetting quality of the automated workstation, the correlation with the classical laboratory approach, and the practical implementation of the developed HT screening method using two model compounds are disclosed. Investigation of the HT method resulted in optimized experimental conditions, which showed low inter- and intraday variability (relative standard deviation [RSD]<5.88%). Higher shaking intensity (7 Hz) and incubation temperature (37°C) resulted in a lower likelihood of obtaining false-negative results. The acceptable dimethyl sulfoxide concentration in the precipitation inhibitor screening assay was set to ?1% (v/v). All liquid dispensing steps resulted in an RSD of <3.4%, and an excellent correlation (R(2)=0.96, P<0.01) with the classical laboratory method was obtained. The practical implementation of the developed HT method was demonstrated by investigating the impact of 23 diverse excipients on the precipitation inhibition of two poorly soluble drugs (fenofibrate and carbamazepine). The screen resulted in the identification of hit excipients, which were not identical for fenofibrate and carbamazepine. This outcome emphasized that the HT screening approach is a reasonable starting point for searching for effective precipitation inhibitors, especially because the excipient-mediated precipitation inhibition effect is case specific and cannot be predicted in a straightforward manner. PMID:23116459

Petruševska, Marija; Urleb, Uroš; Peternel, Luka



Screening diabetic cats for hypersomatotropism: performance of an enzyme-linked immunosorbent assay for insulin-like growth factor 1.  


Screening diabetic cats for feline hypersomatotropism (HS) is currently dependent on using a radioimmunoassay (RIA) for measurement of growth hormone or insulin-like growth factor 1 (IGF-1), both of which require radioactivity, are costly and have limited availability. Performance of an enzyme-linked immunosorbent assay (ELISA) using anti-human IGF-1 antibodies was assessed. Total IGF-1 was determined in diabetic cat samples across a wide range of IGF-concentrations using a previously validated RIA (serum: 92 cats; plasma: 31 cats). Repeat IGF-1 measurement was then performed using the ELISA-system. Mean IGF-1 recovery after serial dilution proved satisfactory with a correlation coefficient of 0.96 (serum) and 0.97 (plasma). Appropriate precision was established [intra-assay coefficient of variation (CV) 9.5 ± 2% (serum) and 13.6 ± 7% (plasma); inter-assay CV 11.4 ± 4% (serum) and 7.6 ± 6% (plasma)] and significant effect of hyperlipidaemia, haemoglobinaemia, bilirubinaemia and storage was excluded, with the exception of an increase in serum IGF-1 when left at room temperature for more than 24 h. ELISA concentrations correlated significantly with RIA concentrations (serum Pearson r(2): 0.75; plasma: 0.83, P <0.001). Receiver operating characteristics analysis showed an area under the curve of 0.99 (serum) and 0.96 (plasma), and indicated high diagnostic accuracy for categorising a diabetic cat correctly as suspicious for HS at a serum IGF-1 cut-off of 997 ng/ml (sensitivity, 100%; specificity, 88.1%). The current study is the first to validate an easy-to-use and economical IGF-1 ELISA for the screening for HS among diabetic cats, which is important given the suspected significant prevalence of HS-induced diabetes mellitus. PMID:23828811

Rosca, Madalina; Forcada, Yaiza; Solcan, Gheorghe; Church, David B; Niessen, Stijn J M



Validation of an enzyme-linked immunosorbent assay for qualitative screening of neomycin in muscle, liver, kidney, eggs and milk.  


A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was used for the qualitative screening analysis of neomycin in food of animal origin (muscle, liver, kidney, eggs and milk) at levels corresponding to the European Union maximum residue limit (MRL) set for this substance. The method validation was performed according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CC?), specificity, detection limit (LOD), quantification limit (LOQ), recovery, precision, linearity and ruggedness. LODs ranged from 5.7 microg kg(-1) in kidney to 29.3 microg kg(-1) in milk; LOQs ranged from 11.4 microg kg(-1) in kidney to 59.7 microkg(-1) in eggs. The recoveries from spiked samples at the MRL, half the MRL and double the MRL levels ranged from 65.8% to 122.8%, with a coefficient of variation (CV) between 5.9% and 28.6%. The CC? value was less than the MRL for all examined matrices. Moderate variations of some critical factors in the sample pretreatment for muscle, milk and eggs were deliberately introduced for ruggedness evaluation and had a slight but not statistically significant effect on method performance. The proposed method is suitable for qualitative screening analysis of neomycin in the above-mentioned food in conformity with current European Union performance requirements. PMID:21082465

Solomun, B; Bilandzic, N; Varenina, I; Scortichini, G



Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells  

PubMed Central

Background Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. Methods and Findings In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. Conclusions Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell growth. Disulfiram-sunitinib combination was identified as one of the potent synergistic approaches. Since sunitinib alone has been reported to lack efficacy in prostate cancer clinical trials, our results provide a rationale for novel combinatorial approach to target prostate cancer more efficiently. PMID:23251544

Ketola, Kirsi; Kallioniemi, Olli; Iljin, Kristiina



Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells  

SciTech Connect

Apoptosis, originally defined by specific morphological changes, is characterized biochemically by non-random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50 kbp fragments prior to, concomitantly with, or in the absence of 180 bp integer fragmentation. Incorporation into fragmented DNA of biotin-labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorportion is visualized using avidin-tagged fluorescein isothiocyanate (FITC). Here, we characterize this assay further in three different hemopoietic cell lines. Drug-induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson-encoded protein tyrosine kinase suppressed drug-induced apoptosis, but did not inhibit drug-induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N-methylformamide in HL60 and MOLT-4 cells, including those high molecular weight DNA fragments formed in MOLT-4 cells which were not further cleaved to 180-200 bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples. 55 refs., 7 figs., 2 tabs.

Chapman, R.S.; Chresta, C.M.; Herberg, A.A. [Manchester Univ. (United Kingdom)] [and others



In Silico Assay Development for Screening of Tetracyclic Triterpenoids as Anticancer Agents against Human Breast Cancer Cell Line MCF7.  


Experimental activity of a compound on cancer cell line/target is mostly analyzed in the form of percentage inhibition at different concentration gradient and time of incubation. In this study a statistical model has been developed referred as in silico assay using support vector regression model, which can act with change in concentration gradient and time of incubation. This model is a function of concentration gradient, treatment hour and independent components; which calculate the percentage inhibition in combination of above three components. This model is designed to screen tetracyclic triterpenoids active against human breast cancer cell line MCF7. The model has been statistically validated, checked for applicability domain and predicted results were reconfirmed by MTT assay, for example Oenotheranstrol derivatives, OenA & B. Computational SAR, target and docking studies were performed to understand the cytotoxic mechanism of action of Oenotheranstrol compounds. The proposed in silico assay model will work for specific chemical family for which it will be optimized. This model can be used to analyze growth kinetics pattern on different human cancer cell lines for designed compounds. PMID:25365399

Prakash, Om; Ahmad, Ateeque; Tripathi, Vinay Kumar; Tandon, Sudeep; Pant, Aditya Bhusan; Khan, Feroz



Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.  


To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 ?g/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 ?g/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process. PMID:24147865

Jiang, Wenxiao; Zhang, Huiyan; Li, Xiangmei; Liu, Xinxin; Zhang, Suxia; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui



Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay.  


Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified. PMID:23740574

Sharma, Pooja; Jindal, Swati; Bala, Kiran; Kumari, Kirti; Niharika, Neha; Kaur, Jasvinder; Pandey, Gunjan; Pandey, Rinku; Russell, Robyn J; Oakeshott, John G; Lal, Rup



In Silico Assay Development for Screening of Tetracyclic Triterpenoids as Anticancer Agents against Human Breast Cancer Cell Line MCF7  

PubMed Central

Experimental activity of a compound on cancer cell line/target is mostly analyzed in the form of percentage inhibition at different concentration gradient and time of incubation. In this study a statistical model has been developed referred as in silico assay using support vector regression model, which can act with change in concentration gradient and time of incubation. This model is a function of concentration gradient, treatment hour and independent components; which calculate the percentage inhibition in combination of above three components. This model is designed to screen tetracyclic triterpenoids active against human breast cancer cell line MCF7. The model has been statistically validated, checked for applicability domain and predicted results were reconfirmed by MTT assay, for example Oenotheranstrol derivatives, OenA & B. Computational SAR, target and docking studies were performed to understand the cytotoxic mechanism of action of Oenotheranstrol compounds. The proposed in silico assay model will work for specific chemical family for which it will be optimized. This model can be used to analyze growth kinetics pattern on different human cancer cell lines for designed compounds. PMID:25365399

Prakash, Om; Ahmad, Ateeque; Tripathi, Vinay Kumar; Tandon, Sudeep; Pant, Aditya Bhusan; Khan, Feroz



Validation of the performance of a GMO multiplex screening assay based on microarray detection  

Microsoft Academic Search

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray,\\u000a has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target\\u000a elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip® GMO, Eppendorf, Germany).

Serge Leimanis; Sandrine Hamels; Florence Naze ´; Guillaume Mbongolo Mbella; Myriam Sneyers; Rupert Hochegger; Hermann Broll; Lillian Roth; Klára Dallmann; Adrienn Micsinai; Maria Pla; Claudia Brünen-Nieweler; Nina Papazova; Isabel Taverniers; Norbert Hess; Britta Kirschneit; Yves Bertheau; Colette Audeon; Valérie Laval; Ulrich Busch; Sven Pecoraro; Katrin Neumann; Sibylle Rösel; Jeroen van Dijk; Esther Kok; Gianni Bellocchi; Nicoletta Foti; Marco Mazzara; William Moens; José Remacle; Guy Van Den Eede



Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets  

PubMed Central

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depend on activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies. PMID:24065146

Miller, Martin L.; Molinelli, Evan J.; Nair, Jayasree S.; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M.; Singer, Samuel; Schwartz, Gary K.; Sander, Chris



Screening for phenotype selective activity in multidrug resistant cells identifies a novel tubulin active agent insensitive to common forms of cancer drug resistance  

PubMed Central

Background Drug resistance is a common cause of treatment failure in cancer patients and encompasses a multitude of different mechanisms. The aim of the present study was to identify drugs effective on multidrug resistant cells. Methods The RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 was screened for cytotoxicity in response to 3,000 chemically diverse compounds using a fluorometric cytotoxicity assay (FMCA). Follow-up profiling was subsequently performed using various cellular and biochemical assays. Results One compound, designated VLX40, demonstrated a higher activity against 8226/Dox40 cells compared to its parental counterpart. VLX40 induced delayed cell death with apoptotic features. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. Strong connections to tubulin inhibitors and microtubule cytoskeleton were retrieved. The mechanistic hypothesis of VLX40 acting as a tubulin inhibitor was confirmed by direct measurements of interaction with tubulin polymerization using a biochemical assay and supported by demonstration of G2/M cell cycle arrest. When tested against a broad panel of primary cultures of patient tumor cells (PCPTC) representing different forms of leukemia and solid tumors, VLX40 displayed high activity against both myeloid and lymphoid leukemias in contrast to the reference compound vincristine to which myeloid blast cells are often insensitive. Significant in vivo activity was confirmed in myeloid U-937 cells implanted subcutaneously in mice using the hollow fiber model. Conclusions The results indicate that VLX40 may be a useful prototype for development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance. PMID:23919498



Decolorization Screening of Synthetic Dyes by Anaerobic Methanogenic Sludge Using a Batch Decolorization Assay  

Microsoft Academic Search

The nonspecific ability of anaerobic sludge bacteria obtained from cattle dung slurry was investigated for 17 different dyes\\u000a in a batch assay system using sealed serum vials. Experiments using Reactive Violet 5 (RV 5) showed that sludge bacteria could\\u000a effectively decolorize solutions having dye concentrations up to 1000 mg l?1 with a decolorization efficiency of above 75% during 48 h

Haresh Keharia; Hardik Patel; Datta Madamwar



Screening of antibiotic residues in ewes' milk destined to cheese by a commercial microbiological inhibition assay  

Microsoft Academic Search

Bulk ewes’ milk from Spanish dairy farms in the Castilla-La Mancha region and destined for production of protected denomination of origin (PDO) Manchego cheese were analysed each month for antimicrobial residues during the course of 1 year. A microbiological assay specific for ewes’ milk (Eclipse ‘100ov’®) was used. The number of positive samples by the Eclipse ‘100ov’ test was 2.6%.

M. Yamaki; M. I. Berruga; R. L. Althaus; M. P. Molina; A. Molina



Retinal and Ocular Toxicity in Ocular Application of Drugs and Chemicals – Part I: Animal Models and Toxicity Assays  

Microsoft Academic Search

Aims: Experimental retinal research has gained great importance due to the ophthalmic pharmacotherapy era. An increasing number of drugs are constantly released into the market for the treatment of retinal diseases. In this review, animal species, animal models and toxicity assays in retinal research are discussed. Methods: An extensive search of the literature was performed to review various aspects of

Fernando Marcondes Penha; Eduardo B. Rodrigues; Maurício Maia; Eduardo Dib; Elaine Fiod Costa; Bruno A. Furlani; Milton Nunes Moraes Filho; Juliana L. Dreyfuss; Juliana Bottós; Michel E. Farah



A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs.  


With the recent emergence of Middle East Respiratory Syndrome coronavirus in humans and the outbreak of devastating porcine epidemic diarrhea coronavirus in swine, therapeutic intervention is urgently needed. However, anti-coronavirus drugs currently are not available. In an effort to assist rapid development of anti-coronavirus drugs, here we screened the NIH Clinical Collection in cell culture using a luciferase reporter-expressing recombinant murine coronavirus. Of the 727 compounds screened, 84 were found to have a significant anti-coronavirus effect. Further experiments revealed that 51 compounds blocked virus entry while 19 others inhibited viral replication. Additional validation studies with the top 3 inhibitors (hexachlorophene, nitazoxanide and homoharringtonine) demonstrated robust anti-coronavirus activities (a reduction of 6 to 8log10 in virus titer) with an IC50 ranging from 11nM to 1.2?M. Furthermore, homoharringtonine and hexachlorophene exhibited broad antiviral activity against diverse species of human and animal coronaviruses. Since the NIH Clinical Collection consists of compounds that have already been through clinical trials, these small molecule inhibitors have a great potential for rapid development as anti-coronavirus drugs. PMID:25451075

Cao, Jianzhong; Forrest, J Craig; Zhang, Xuming



Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet Syndrome treatment  

PubMed Central

Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterise zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviors. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviors and electrographic seizures. This approach represents a new direction in modeling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder. PMID:24002024

Baraban, Scott C.; Dinday, Matthew T.; Hortopan, Gabriela A.



Active Screening of Multi-Drug Resistant Bacteria Effectively Prevent and Control the Potential Infections.  


Our objective is to determine if actively screen the multi-drug resistant bacteria (MDRB) infection in intensive care unit (ICU) to prevent, control, and decrease the infection rate and transmission of MDRB. The patients admitted in ICU of one hospital in 2013 were analyzed. The throat swab, blood, defecation, and urine of patients were actively collected for bacteria cultures to screen Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii in patients. All patients received screening of MDRB infection and colonization within 2 days and after 2 days of admission, the results showed that there were 418 infectious bacterial strains in total and P. aeruginosa was the main bacterium. The asymptomatic infection rates of P. aeruginosa, K. pneumonia, E. coli, S. aureus, and A. baumannii were 39.02, 24.74, 44.00, 29.17, and 33.33 %, respectively; the symptomatic infection rates were 60.98, 75.26, 56.00, 70.83, and 66.67 %. 59.70 % patients received antibiotics treatment, 27.45 % patients received trachea cannula, 32.95 % patients received mechanism ventilation, 2.27 % patients received arterial cannula or venous cannula and 4.00 % patients received indwelling urinary catheters. The main MDRB in ICU is P. aeruginosa. The active screening of MDRB infection and colonization can provide the opportunity to take the life-saving measure against MDRB and treat patients. This can decrease the infection risk and the nosocomial transmission of MDRB. PMID:25416582

Ren, Yuguo; Ma, Guoliang; Peng, Lin; Ren, Yufeng; Zhang, Fengmei



[Local straight line screening method for the detection of Chinese proprietary medicines containing undeclared prescription drugs].  


The present paper constructs a new approach named local straight-line screening (LSLS) to detect Chinese proprietary medicines (CPM) containing undeclared prescription drugs (UPD). Different from traditional methods used in analysis of multi-component spectrum, LSLS is proposed according to the characteristics of original infrared spectra of the UPD and suspected CPM, without any pattern recognition or concentration model establishment. Spectrum-subtraction leads to the variance in local straight line, which serves as a key in discrimination of whether suspected CPD is adulterated or not. Sibutramine hydrochloride, fenfluramine hydrochloride, sildenafil citrate and lovastatin were used as reference substances of UPD to analyze 16 suspected CPM samples. The results show that LSLS can obtain an accurate quantitative and qualitative analysis of suspected CPM. It is possible for the method to be potentially used in the preliminary screening of CPM containing possible UPD. PMID:19445196

Li, Shu; Cao, Yan; Le, Jian; Chen, Gui-Liang; Chai, Yi-Feng; Lu, Feng



Data-independent Proteomic Screen Identifies Novel Tamoxifen Agonist that Mediates Drug Resistance  

PubMed Central

A label-free quantitative variation of the recently developed data-independent shotgun proteomic method Precursor Acquisition Independent From Ion Count (PAcIFIC) was used to identify novel proteins implicated in cancer progression and resistance. Specifically, this screen identified the pro-metastatic protein anterior gradient 2 (AGR2) as significantly up-regulated in tamoxifen treated cells. Highlighting the need for direct proteome profiling methods like PAcIFIC, neither data-dependent gas-phase fractionation nor a transcriptomic screen detected AGR2 protein/transcript at significantly up-regulated levels. Further cell-based experiments using human cancer cell lines and in vivo xenografts confirmed the PAcIFIC hypothesis that AGR2 is up-regulated in MCF-7 cells post tamoxifen treatment, and that it is implicated in drug resistance mediation. PMID:21936522

Hengel, Shawna Mae; Murray, Euan; Langdon, Simon; Hayward, Larry; O’Donoghue, Jean; Panchaud, Alexandre; Hupp, Ted; Goodlett, David R.



Design or screening of drugs for the treatment of Chagas disease: what shows the most promise?  

PubMed Central

Introduction Endemic in Latin America, Chagas disease is now becoming a serious global health problem, and yet has no financial viability for the pharmaceutical industry and remains incurable. In 2012, two antimycotic drugs inhibitors of fungal sterol 14?-demethylase (CYP51) – posaconazole and ravuconazole – entered clinical trials. Availability of the X-ray structure of the orthologous enzyme from the causative agent of the disease, protozoan parasite Trypanosoma cruzi, determined in complexes with posaconazole as well as with several experimental protozoa-specific CYP51 inhibitors opens an excellent opportunity to improve the situation. Areas covered This article summarizes the information available in PubMed and Google on the outcomes of treatment of the chronic Chagas disease. It also outlines the major features of the T. cruzi CYP51 structure and the possible structure-based strategies for rational design of novel T. cruzi specific drugs. Expert opinion There is no doubt that screenings for alternative drug-like molecules as well as mining the T. cruzi genome for novel drug targets are of great value and might eventually lead to groundbreaking discoveries. However, all newly identified molecules must proceed through the long, expensive and low-yielding drug optimization process, and all novel potential drug targets must be validated in terms of their essentiality and druggability. CYP51 is already a well-validated and highly successful target for clinical and agricultural antifungals. With minimal investments into the final stages of their development/trials, T. cruzi-specific CYP51 inhibitors can provide an immediate treatment for Chagas disease, either on their own or in combination with the currently available drugs. PMID:24079515

Lepesheva, Galina I.



Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.  

PubMed Central

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

Ginouves, Marine; Carme, Bernard; Couppie, Pierre



An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.  


Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

Rocha, Marcele N; Corręa, Célia M; Melo, Maria N; Beverley, Stephen M; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P



Screening method for nonsteroidal antiinflammatory drugs based on the cyclooxygenase 2 pathway activated by serum-free stimulation in A549 cells.  


Cyclooxygenase 2 (COX-2) pathway inhibitors were regarded as promising nonsteroidal antiinflammatory drugs (NSAIDs). We discovered that the COX-2 pathway in A549 cells, a human lung cancer cell line, was activated by serum-free stimulation, and a drug screening model for NSAIDs was established based on this principle with simple performance and sufficient reliability. The COX-2 pathway was activated by treating with serum-free medium for 12 h. The activated cells were incubated with NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), acetyl salicylic acid (ASA) (nonselective COX inhibitor) at 37 degrees C for 15 min. Then the cells were incubated with 10 microM of arachidonic acid (AA) for another 30 min prostaglandin E2 and 6-keto-prostaglandin F(1alpha) were assayed in an enzyme immunoassay (EIA). The results showed that the COX-2 pathway was dominant in A549 cells whether activated by serum-free medium or not, and the COX-1 pathway could be ignored. The model accepted the positive inhibition threshold as NS398 2 microM; if a compound (10 microM) inhibited COX-2 pathway more than NS398 (2 microM), it was regarded as a hit. The COX-2 pathway inhibition experiment showed that the Z;-factor of the screening model was 0.62, which suggests that the model is suitable for COX-2 pathway inhibitor screening. PMID:17329938

Yao, Jin Cheng; Duan, Wei Gang; Yun, Yu; Liu, De Quan; Yan, Ming; Jiang, Zhen Zhou; Zhang, Lu Yong



Hematin Polymerization Assay as a High-Throughput Screen for Identification of New Antimalarial Pharmacophores  

Microsoft Academic Search

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of 14 C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The




Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.  


Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-?B and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the ?-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-?B/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z. PMID:24395226

Lewis, Russell E; Liao, Guangling; Young, Katherine; Douglas, Cameron; Kontoyiannis, Dimitrios P



Validation of direct assay of an aqueous formulation of a drug compound AZY by chiral supercritical fluid chromatography (SFC).  


Supercritical fluid chromatography (SFC) is increasingly being recognized as a powerful technique for analysis of pharmaceutical compounds in various dosage forms. Assay of aqueous formulations of research compounds by SFC is, however, a relatively unexplored area primarily due to the potential problems associated with it. This work describes the development of a direct assay of a chiral drug compound AZY in a 100% aqueous formulation by SFC, and its qualification following ICH and FDA validation guidelines on chromatographic methods. The results indicated that SFC has the potential for assaying aqueous formulations of research compounds with high degree of selectivity, accuracy, precision, robustness, sensitivity, and linearity over a wide range of concentrations. This work also confirmed a previous hypothesis that direct formulation assay by SFC approach is applicable to both acidic and basic pharmaceutical compounds with equal degree of success. PMID:16930907

Mukherjee, Partha S



Quantifying Intrinsic Specificity: A Potential Complement to Affinity in Drug Screening  

NASA Astrophysics Data System (ADS)

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

Wang, Jin; Zheng, Xiliang; Yang, Yongliang; Drueckhammer, Dale; Yang, Wei; Verkhivker, Gennardy; Wang, Erkang



Faculty buy-in to teach alcohol and drug use screening.  


Educating nursing faculty about the use of an evidence-based practice to screen and intervene earlier along the continuum of alcohol and other drug use, misuse, and dependence is essential in today's health care arena. Misuse of alcohol and other drugs is a significant problem for both individual health and societal economic welfare. The purpose of this article is to describe nursing faculty buy-in for the implementation of an evidence-based addiction training program at a university-based school of nursing. Derived from an academic-community partnership, the training program results suggest implications for continuing education and curriculum innovation in schools of nursing and clinical practice. The training content presented can be used in continuing education for nursing faculty across all types of nursing school programs and professional nursing staff employed in multiple settings. The training program was funded by the Health Resources and Services Administration. PMID:25153430

Puskar, Kathy; Mitchell, Ann M; Kane, Irene; Hagle, Holly; Talcott, Kimberly S



Non-Target Screening of Veterinary Drugs Using Tandem Mass Spectrometry on SmartMass  

NASA Astrophysics Data System (ADS)

Non-target screening of veterinary drugs using tandem mass spectrometric data was performed on the SmartMass platform. This newly developed software uses the characteristic fragmentation patterns (CFP) to identify chemicals, especially those containing particular substructures. A mixture of 17 sulfonamides was separated by ultra performance liquid chromatography (UPLC), and SmartMass was used to process the tandem mass spectrometry (MS/MS) data acquired on an Orbitrap mass spectrometer. The data were automatically extracted, and each sulfonamide was recognized and analyzed with a prebuilt analysis rule. By using this software, over 98 % of the false candidate structures were eliminated, and all the correct structures were found within the top 10 of the ranking lists. Furthermore, SmartMass could also be used to identify slightly modified contraband drugs and metabolites with simple prebuilt rules. [Figure not available: see fulltext.

Xia, Bing; Liu, Xin; Gu, Yu-Cheng; Zhang, Zhao-Hui; Wang, Hai-Yan; Ding, Li-Sheng; Zhou, Yan