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Sample records for pseudomonas screening assay

  1. Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

    Directory of Open Access Journals (Sweden)

    Wim Reybroeck

    Full Text Available Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

  2. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

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

  3. Screening of Gibberellic Acid Production by Pseudomonas SPP

    International Nuclear Information System (INIS)

    Khine Zar Wynn Myint; Khin Mya Lwin; Myo Myint

    2010-12-01

    The microbial gibberellic acid (GA3) production of Pseudomonas spp., was studied and qualitatively indentified by UV spectrophotometer. 20 strains of Pseudomonas spp., were isolated and screened the gibberellic acid productivily in King's B medium. Among them, only four strains can produce microbial gibberellic acid. The Rf values and colour appearance under UV were the same as authentic gibberellic acid. Moreover, the gibberellic acid producer strains were identified as Pseudomonas spp., by cultural, biochemical and drug sensitivity pattern.

  4. Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.

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    Anthony Arnoldo

    2008-02-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS, a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

  5. Metabolomics-Based Screening of Biofilm-Inhibitory Compounds against Pseudomonas aeruginosa from Burdock Leaf

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    Zaixiang Lou

    2015-09-01

    Full Text Available Screening of anti-biofilm compounds from the burdock leaf based on metabolomics is reported here. The crystal violet assay indicated 34% ethanol elution fraction of burdock leaf could completely inhibit biofilm formation of Pseudomonas aeruginosa at 1 mg·mL−1. Then, the chemical composition of burdock leaf fraction was analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS and 11 active compounds (chlorogenic acid, caffeic acid, p-coumaric acid, quercetin, ursolic acid, rutin, cynarin, luteolin, crocin, benzoic acid, and Tenacissoside I were identified. Lastly, UPLC-MS analysis was employed to obtain the metabolic fingerprints of burdock leaf fractions before and after inhibiting the biofilm of Pseudomonas aeruginosa. The metabolic fingerprints were transformed to data, analyzed with PLS-DA (partial least squares discriminant analysis and the peaks whose area was significantly changed were found out. Thus, 81 compounds were screened as potential anti-biofilm ingredients. Among them, rutin, ursolic acid, caffeic acid, p-coumaric acid and quercetin were identified and confirmed as the main anti-biofilm compounds in burdock leaf. The study provided basic anti-biofilm profile data for the compounds in burdock leaf, as well as provided a convenient method for fast screening of anti-biofilm compounds from natural plants.

  6. Spectrophotometric Enzyme Assays for High-Throughput Screening

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    Jean-Louis Reymond

    2004-01-01

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

  7. Screening of thermophilic neutral lipase-producing Pseudomonas ...

    African Journals Online (AJOL)

    From oil-contaminated soil, three lipase-producing microorganisms were selected as good lipase producers using rhodamine B-olive oil plate agar and they were identified as from Pseudomonas, Burkholderia and Klebsiella genera by morphology, biochemical characterization and 16S rRNA gene sequencing. Among the ...

  8. Rapid screening assay for calcium bioavailability studies

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  9. A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

    Science.gov (United States)

    2010-01-01

    Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum. PMID:20637114

  10. A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Murphy Anna

    2010-07-01

    Full Text Available Abstract Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE, 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.

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

    Science.gov (United States)

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

  12. Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

    Science.gov (United States)

    Saha, Ratul; Donofrio, Robert S; Bagley, Susan T

    2010-08-01

    A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

  13. Genome-wide screen of Pseudomonas aeruginosa In Saccharomyces cerevisiae identifies new virulence factors

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    Rafat eZrieq

    2015-11-01

    Full Text Available Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. 51 candidates were selected in a three-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec. By testing the cytotoxicity of wild type P. aeruginosa vs pec mutants towards macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

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

    Science.gov (United States)

    Winborn, Jessica; Kerrigan, Sarah

    2017-06-01

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

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  16. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

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

    2011-09-01

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

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

    KAUST Repository

    Soufan, Othman

    2015-12-14

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

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

    KAUST Repository

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

    2015-01-01

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

  19. Simple screening tests for the detection of metallo-β-lactamase (MBL production in clinical isolates of Pseudomonas and Acinetobacter

    Directory of Open Access Journals (Sweden)

    Shaheda Anwar

    2010-01-01

    Full Text Available There are no standard methods for the detection of metallo-b-lactamase (MBL production in gram negative organism in routine microbiology practice. The present study was undertaken to evaluate the screening tests like double disk synergy test (DDST and disk potentiation test (DPT using ceftazidime (CAZ and imipenem (IPM disks with chelating agents like EDTA, 2-mercaptopropionic acid (2-MPA. A total of 132 Pseudomonas and 76 Acinetobacter isolates were obtained from Bangabandhu Sheikh Mujib Medical University (BSMMU and Bangladesh Institute of Research and Rehabilitation for Diabetes, Endocrine and Metabolic Disorders (BIRDEM hospitals of Dhaka city. A total of 53 and 29 IPM resistant Pseudomonas and Acinetobacter isolates were selected. EDTA-IPM microdilution minimum inhibitory concentration (EDTA-IPM MIC method detected MBL in 44 (83% IPM resistant Pseudomonas and 19 (65.5% Acinetobacter isolates. DDST with CAZ-0.1M EDTA and CAZ-2-MPA detected MBL in 73.6% and 67.9% of IPM resistant Pseudomonas and 55.2% and 48.3% of Acinetobacter isolates respectively. The detection rate was 67.9% and 66.1% in Pseudomonas and 51.7% and 44.8% in Acinetobacter isolates by EDTA-IPM and IPM-2-MPA methods respectively. In comparison to DDST, DPT with CAZ-0.1M EDTA showed higher sensitivity (89.7% and specificity (100% for detection of MBL in Pseudomonas and Acinetobacter. The results showed that simple screening tests like DPT with 0.1M EDTA was able to detect MBL producing Pseudomonas and Acinetobacter from clinical samples with high sensitivity and specificity. Ibrahim Med. Coll. J. 2010; 4(1: 26-30

  20. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

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    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

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

    Science.gov (United States)

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

    2017-05-01

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

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

    Science.gov (United States)

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

  3. Indole-based assay to assess the effect of ethanol on Pseudomonas putida F1 dioxygenase activity.

    Science.gov (United States)

    da Silva, Márcio Luis Busi; Alvarez, Pedro J J

    2010-06-01

    Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 +/- 0.0006 OD(610) min(-1). The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  6. A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

    Science.gov (United States)

    Li, Z; Chang, S; Lin, L; Li, Y; An, Q

    2011-08-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

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

    KAUST Repository

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

    2016-01-01

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

  8. Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay.

    Science.gov (United States)

    Heussler, Gary E; Miller, Jon L; Price, Courtney E; Collins, Alan J; O'Toole, George A

    2016-11-15

    CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer. Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in

  9. Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation.

    Science.gov (United States)

    Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug

  10. Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation.

    Directory of Open Access Journals (Sweden)

    Vasudevan Aparna

    Full Text Available Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    NARCIS (Netherlands)

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

    2009-01-01

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

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

    Science.gov (United States)

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

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

    OpenAIRE

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

    2016-01-01

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

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

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

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

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

    Science.gov (United States)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-15

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Stéphanie Simon

    2015-11-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Seri Jeong

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

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

    Science.gov (United States)

    Jeong, Seri; Yang, Heeyoung; Hwang, Hyunyong

    2017-01-01

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

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

    Science.gov (United States)

    2013-01-01

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

  6. A Rapid Phenotypic Whole Cell Screening Approach for the Identification of Small Molecule Inhibitors that Counter Beta-lactamase Resistance in Pseudomonas aeruginosa

    Science.gov (United States)

    Collia, Deanna; Bannister, Thomas D.; Tan, Hao; Jin, Shouguang; Langaee, Taimour; Shumate, Justin; Scampavia, Louis; Spicer, Timothy P.

    2017-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen which is prevalent in hospitals and continues to develop resistance to multiple classes of antibiotics. Historically, β-lactam antibiotics have been the first line of therapeutic defense. However, the emergence of multidrug-resistant (MDR) strains of P. aeruginosa, such as AmpC β-lactamase overproducing mutants, limits the effectiveness of current antibiotics. Among AmpC hyper producing clinical isolates, inactivation of AmpG, which is essential for the expression of AmpC, increases bacterial sensitivity to β-lactam antibiotics. We hypothesize that inhibition of AmpG activity will enhance the efficacy of β-lactams against P. aeruginosa. Here, using a highly drug resistant AmpC inducible laboratory strain PAO1, we describe an ultra-high throughput whole cell turbidity assay designed to identify small molecule inhibitors of the AmpG. We screened 645K compounds to identify compounds with the ability to inhibit bacterial growth in the presence of Cefoxitin; an AmpC inducer, and identified 2,663 inhibitors which were also tested in the absence of Cefoxitin to determine AmpG specificity. The Z′ and S:B were robust at 0.87 ± 0.05 and 2.2 ± 0.2, respectively. Through a series of secondary and tertiary studies, including a novel luciferase based counterscreen, we ultimately identified 8 potential AmpG specific inhibitors. PMID:28850797

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

    OpenAIRE

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

    2015-01-01

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

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

    OpenAIRE

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

    2014-01-01

    Background The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. ...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2018-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jack Cuzick

    2015-12-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Guangchao Yu

    2014-01-01

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

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

    Science.gov (United States)

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

    2009-12-01

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

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2012-03-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Hampus Sunner

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Mary T. Bausch-Jurken

    2017-06-01

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

  9. The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

    Directory of Open Access Journals (Sweden)

    Bharani Manoharan

    Full Text Available The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    KAUST Repository

    Soufan, Othman

    2016-11-10

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

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

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alexandra Ibáñez Escribano

    2012-08-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-08-12

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

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

    Science.gov (United States)

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jie Lei

    2018-03-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

    Ishida, Akihiko; Yamada, Yasuko; Kamidate, Tamio

    2008-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Pieter W Smit

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

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

    Science.gov (United States)

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

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

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

    Directory of Open Access Journals (Sweden)

    Deepika Kanojia

    2017-11-01

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

  10. Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

    Science.gov (United States)

    Headey, Stephen J.; Vazirani, Mansha; Shouldice, Stephen R.; Coinçon, Mathieu; Tay, Stephanie; Morton, Craig J.; Simpson, Jamie S.; Martin, Jennifer L.

    2017-01-01

    At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors. PMID:28346540

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

    Science.gov (United States)

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

    2007-12-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

    Cancian, Mariana; Loreto, Elgion L S

    2018-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Ting Xiao

    2016-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Mirjana Bistan

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Prerna Grover

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

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

    Science.gov (United States)

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

    2011-09-19

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

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

    NARCIS (Netherlands)

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

    2005-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Asther Marcel

    2010-07-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Junaid Iqbal

    2013-01-01

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

  7. A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000.

    Directory of Open Access Journals (Sweden)

    Weiqing Zeng

    2011-10-01

    Full Text Available Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata, multiplication in the intercellular space (apoplast of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA, and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to

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

    Science.gov (United States)

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

    2018-03-20

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

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

    Science.gov (United States)

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

    1988-02-01

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

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

    Science.gov (United States)

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

    1997-01-15

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

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  12. Diagnostic multiplex polymerase chain reaction assay for the identification of Pseudomonas aeruginosa from the skin biopsy specimens in burn wound infections and detection of antibiotic susceptibility

    International Nuclear Information System (INIS)

    Mashouf, Rasoul Y.; Farahani, Hadi S.; Zamani, A.

    2008-01-01

    Objective was to identify Pseudomonas aeruginosa (P. aeruginosa) from the skin biopsy specimens in burn wound infections by multiplex polymerase chain reaction (M-PCR) and detection of antimicrobial susceptibility of isolates from culture. We conducted the cross-sectional study in 140 patients with wound infections who admitted to referral burn center of Motahari, Tehran, Iran, during a 12-month period from 2005-2006. Skin biopsy specimens were aseptically taken from each patient, one for PCR and one for bacterial culture. A M-PCR test based on simultaneous amplification of 2 lipoprotein genes: oprI and oprL, was used to directly detect fluorescent pseudomonades and P. aeruginosa in skin biopsy specimens. The susceptibility of P. aeruginosa isolates to 16 antibiotics was determined using the disc diffusion method. Out of 140 biopsy specimens, M-PCR detected 66 (47.2%) isolates, while culture detected 57 (40.7%) isolates as P. aeruginosa. Positive results for both genes which observed only for P. aeruginosa, while only one gene, oprI, was amplified from other fluorescent pseudomonades (n=12) and all other bacterial tested (n=62) were negative by the amplification test. The most effective antibiotics against isolate of P. aeruginosa were cefepime (79%), azetreonam (76%), ticarcillin-clavulanic acid (68%), tobramycin (62%) and amikacin (61%). Multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa from the burned skin biopsy specimens. Simultaneous amplification of 2 lipoprotein genes: oprI and oprL could detect P. aeruginosa and oprI gene only for other fluorescent pseudomonades. (author)

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Katsuyuki Hotta

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

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

    Directory of Open Access Journals (Sweden)

    P.M. O’Shea

    2017-04-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Cristiana Lalli

    2015-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

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

    NARCIS (Netherlands)

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

    2006-01-01

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

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

    Science.gov (United States)

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

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

    NARCIS (Netherlands)

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

    2009-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  4. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    Science.gov (United States)

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

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

    Directory of Open Access Journals (Sweden)

    Tom A. Ewing

    2018-01-01

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

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

    Science.gov (United States)

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

    2018-01-14

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

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

    Science.gov (United States)

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

    2018-02-01

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

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

    International Nuclear Information System (INIS)

    Sander, J.; Niehaus, C.

    1982-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    1992-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Helton J Wiggers

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

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

    Science.gov (United States)

    Wiggers, Helton J; Rocha, Josmar R; Fernandes, William B; Sesti-Costa, Renata; Carneiro, Zumira A; Cheleski, Juliana; da Silva, Albérico B F; Juliano, Luiz; Cezari, Maria H S; Silva, João S; McKerrow, James H; Montanari, Carlos A

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jing Lu

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    KAUST Repository

    Soufan, Othman

    2016-01-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

    Chiaravalli, Jeanne; Glickman, J Fraser

    2017-08-01

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

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

    Science.gov (United States)

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

    2004-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-06-01

    Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

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

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

    Science.gov (United States)

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

    2017-07-11

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

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

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  9. Pseudomonas - Fact Sheet

    OpenAIRE

    Public Health Agency

    2012-01-01

    Fact sheet on Pseudomonas, including:What is Pseudomonas?What infections does it cause?Who is susceptible to pseudomonas infection?How will I know if I have pseudomonas infection?How can Pseudomonas be prevented from spreading?How can I protect myself from Pseudomonas?How is Pseudomonas infection treated?

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    A simple, inexpensive and sensitive method for detecting organophosphate and carbamate insecticides is reported. Acetylcholinesterase was immobilized to PorexR Lateral-FloTM membrane material and remained active for several months at room temperature. The assay was sensitive ...

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-01-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage Lambda in Escherichia coli WP2s(Lambda), was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that were mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

    Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2017-03-01

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

  17. Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

    OpenAIRE

    Lewis, Russell E.; Liao, Guangling; Young, Katherine; Douglas, Cameron; Kontoyiannis, Dimitrios P.

    2014-01-01

    Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus f...

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

    Directory of Open Access Journals (Sweden)

    Windy A Boyd

    2007-12-01

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

  19. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  20. Characterisation of powerful antioxidants and synthetic iron ligands, as protective agents against oxidative damages, using new high throughput screening assays

    International Nuclear Information System (INIS)

    Meunier, St.

    2002-12-01

    This work was devoted to the development of pertinent high throughput screening assays in the aim of studying oxidative stress. Three screening assays have been developed for the evaluation of protective agents toward ROS generated by gamma irradiation, UV or by a Fenton-like system. 24 natural extracts and a library of 120 pure compounds, containing among the most powerful antioxidants known to date, have been readily studied using, these new techniques. We found that two pulvinic acid derivatives possess excellent protective properties, and especially a pigment of fungus named norbadione A. Beyond its in vitro activity, this molecule displays remarkable biological properties. In the aim of studying an alternative pathway of protection against oxidation induced by iron, ligands able to modify the redox properties of this metal, have been synthesised. We have developed a parallel synthesis allowing the variation of the architecture, denticity, chelating moieties and hydrophobicity of iron chelates. Using this strategy, 47 potential Fe(III) ligands were obtained. Their protective capacities have been studied using a fourth screening assay, demonstrating the effectiveness of some ligands. Finally, the immunoassay technique called SPI-RAD has been used in order to study a particular consequence of drastic oxidative stress, namely covalent crosslinks between proteins. Our results demonstrate that these linkages occur in the presence of metals (FeII or CuII) and hydrogen peroxide, as well as in the presence of NO . radical. Moreover, it has been demonstrated that tyrosines residues and disulfide bridges play an important role in these phenomena. (author)

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

    Directory of Open Access Journals (Sweden)

    Michael J Binks

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

  2. Screening of Riboflavin-Producing Lactobacilli by a Polymerase-Chain-Reaction-Based Approach and Microbiological Assay.

    Science.gov (United States)

    Thakur, Kiran; Tomar, Sudhir Kumar; Brahma, Biswajit; De, Sachinandan

    2016-03-09

    Riboflavin has an important role in various cellular metabolic activities through its participation in oxidation-reduction reactions. In this study, as many as 60 lactobacilli were screened for the presence or absence of riboflavin biosynthesis genes and riboflavin production. Of these, only 14 strains were able to grow in a commercial riboflavin-free medium. We observed that the presence of riboflavin biosynthesis genes is strain-specific across different species of lactobacilli. The microbiological assay was found to be appreciably reproducible, sensitive, rapid, and inexpensive and, hence, can be employed for screening the riboflavin-producing strains. The study thus represents a convenient and efficient method for selection of novel riboflavin producers. These riboflavin(+) strains thus identified and characterized could be explored as potent candidates for the development of a wide range of dairy- and cereal-based foods for the delivery of in situ riboflavin to consumers.

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

    International Nuclear Information System (INIS)

    Druwe, Ingrid; Freudenrich, Theresa M.; Wallace, Kathleen; Shafer, Timothy J.; Mundy, William R.

    2015-01-01

    High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery

  4. Cost-effectiveness of interferon-γ release assay versus chest X-ray for tuberculosis screening of employees.

    Science.gov (United States)

    Kowada, Akiko

    2011-12-01

    Currently, an annual chest X-ray examination (CXR) for detection of active tuberculosis (TB) in employees aged ≥40 years is recommended in the guidelines of the Japan Industrial Safety and Health Law. Interferon-γ release assays are new alternatives to the tuberculin skin test for detecting Mycobacterium tuberculosis infection, with higher specificity than the tuberculin skin test and without cross-reactivity with the Bacille Calmette-Guérin vaccine. This study aimed to assess the cost-effectiveness of employee TB screening using QuantiFERON-TB Gold In-Tube (QFT) versus CXR. Markov models were constructed. The target population was a hypothetical cohort of immunocompetent 40-year-old individuals, using a societal perspective and a lifetime horizon. All costs and clinical benefits were discounted at a fixed annual rate of 3%. In a base-case analysis, the QFT strategy was the most cost-effective ($US 262.84; 22.87049 quality-adjusted life-years [QALYs]) compared with no screening ($448.38; 22.85452 QALYs) and CXR ($543.50; 22.85453 QALYs) [year 2009 values]. The QFT strategy is currently robust for screening Bacille Calmette-Guérin- vaccinated employees in Japan. There appears to be little role for CXR. These findings may be applicable to other countries in terms of choosing optimal TB screening for employees. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  5. SCREENING FOR TOXIC INDUSTRIAL CHEMICALS USING SEMIPERMEABLE MEMBRANE DEVICES WITH RAPID TOXICITY ASSAYS

    Science.gov (United States)

    A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...

  6. VAPOR SAMPLING DEVICE FOR INTERFACE WITH MICROTOX ASSAY FOR SCREENING TOXIC INDUSTRIAL CHEMICALS

    Science.gov (United States)

    A time-integrated sampling system interfaced with a toxicity-based assay is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethyl sulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor...

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

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

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

  8. Screening assays to find out late latent syphilis cases – which is the ...

    African Journals Online (AJOL)

    non-treponemal) such as Rapid Plasma Reagin (RPR), and specific (treponemal) such as Treponema pallidum hemagglutination test (TPHA) and Enzyme-linked immunosorbent assay IgG, IgM (ELISA IgG, IgM) tests. The aim of this ...

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

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

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

  10. Comparison of a fluoroquinolone surface plasmon resonance biosensor screening assay with established methods

    NARCIS (Netherlands)

    Weigel, S.; Pikkemaat, M.G.; Elferink, J.W.A.; Mulder, P.P.J.; Huet, A.C.; Delahaut, P.; Schittko, S.; Flerus, R.; Nielen, M.W.F.

    2009-01-01

    The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  12. Development of a PCR assay suitable for Campylobacter spp. mass screening programs in broiler production

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Pedersen, Karl; Madsen, Mogens

    2001-01-01

    Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide. In order to comply with the demands of consumers for food free of bacterial pathogens, a mass screening program for Campylobacter in Danish broilers has been carried out based on conventional bacterial...

  13. Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

    Science.gov (United States)

    Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

  14. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  16. The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

    Science.gov (United States)

    Kimber, Ian; Dearman, Rebecca J; Betts, Catherine J; Gerberick, G Frank; Ryan, Cindy A; Kern, Petra S; Patlewicz, Grace Y; Basketter, David A

    2006-04-01

    The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.

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

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

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

  18. Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

    Science.gov (United States)

    Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

    2013-06-15

    The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-06-26

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

  20. DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

    Czech Academy of Sciences Publication Activity Database

    Navrátil, Václav; Schimer, Jiří; Tykvart, Jan; Knedlík, Tomáš; Vik, V.; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2017-01-01

    Roč. 45, č. 2 (2017), č. článku e10. ISSN 0305-1048 R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : quantitative PCR * enzyme detection * inhibitor screening Subject RIV: CE - Biochemistry OBOR OECD: Biochemical research methods Impact factor: 10.162, year: 2016 https:// academic .oup.com/nar/article-lookup/doi/10.1093/nar/gkw853

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    René van der Ploeg

    2015-07-01

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

  5. The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

    Science.gov (United States)

    Song, Lele; Jia, Jia; Peng, Xiumei; Xiao, Wenhua; Li, Yuemin

    2017-06-08

    The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

  6. High-Throughput Genetic Screen Reveals that Early Attachment and Biofilm Formation Are Necessary for Full Pyoverdine Production by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Donghoon Kang

    2017-09-01

    Full Text Available Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti

  7. Biological screening of Brazilian medicinal plants

    Directory of Open Access Journals (Sweden)

    Tânia Maria de Almeida Alves

    2000-06-01

    Full Text Available In this study, we screened sixty medicinal plant species from the Brazilian savanna ("cerrado" that could contain useful compounds for the control of tropical diseases. The plant selection was based on existing ethnobotanic information and interviews with local healers. Plant extracts were screened for: (a molluscicidal activity against Biomphalaria glabrata, (b toxicity to brine shrimp (Artemia salina L., (c antifungal activity in the bioautographic assay with Cladosporium sphaerospermum and (d antibacterial activity in the agar diffusion assay against Staphylococcus aureus, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. Forty-two species afforded extracts that showed some degree of activity in one or more of these bioassays.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

    Science.gov (United States)

    Heintz-Buschart, Anna; Eickhoff, Holger; Hohn, Erwin; Bilitewski, Ursula

    2013-03-10

    Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

    DEFF Research Database (Denmark)

    Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune

    2015-01-01

    suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed...... plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. RESULTS: The assay detected specific and saturable immunoglobulin G (IgG) binding to each...... cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients...

  11. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    Science.gov (United States)

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

    2016-08-05

    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  12. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  13. Novel heparan sulfate assay by using automated high-throughput mass spectrometry: Application to monitoring and screening for mucopolysaccharidoses.

    Science.gov (United States)

    Shimada, Tsutomu; Kelly, Joan; LaMarr, William A; van Vlies, Naomi; Yasuda, Eriko; Mason, Robert W; Mackenzie, William; Kubaski, Francyne; Giugliani, Roberto; Chinen, Yasutsugu; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji E; Fukao, Toshiyuki; Orii, Tadao; Tomatsu, Shunji

    2014-01-01

    Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity

  14. Pseudomonas aeruginosa PA14 pathogenesis in Caenorhabditis elegans.

    Science.gov (United States)

    Kirienko, Natalia V; Cezairliyan, Brent O; Ausubel, Frederick M; Powell, Jennifer R

    2014-01-01

    The nematode Caenorhabditis elegans is a simple model host for studying the interaction between bacterial pathogens such as Pseudomonas aeruginosa and the metazoan innate immune system. Powerful genetic and molecular tools in both C. elegans and P. aeruginosa facilitate the identification and analysis of bacterial virulence factors as well as host defense factors. Here we describe three different assays that use the C. elegans-P. aeruginosa strain PA14 host-pathogen system. Fast Killing is a toxin-mediated death that depends on a diffusible toxin produced by PA14 but not on live bacteria. Slow Killing is due to an active infection in which bacteria colonize the C. elegans intestinal lumen. Liquid Killing is designed for high-throughput screening of chemical libraries for anti-infective compounds. Each assay has unique features and, interestingly, the PA14 virulence factors involved in killing are different in each assay.

  15. An interferon-gamma release assay test performs well in routine screening for tuberculosis

    DEFF Research Database (Denmark)

    Vestergaard Danielsen, Allan; Fløe, Andreas; Lillebæk, Troels

    2014-01-01

    Introduction: A positive interferon-gamma release assay (IGRA) is regarded as proof of latent Mycobacterium tuberculosis infection. We conducted an evaluation of the IGRA test “T-SPOT.TB” to test its performance during clinical routine use by analysing the positivity rate and odds, effect of season...... and sensitivity. Material and methods: Data from T-SPOT.TB testing together with age and test indications (anti-tumour necrosis factor alpha (TNFα) candidate, contact investigation or suspicion of tuberculosis (TB)) were combined with mycobac­teria culture results. Results: A total of 1,809 patients were tested....... Conclusive results were achieved for 1,780 patients (98.4%). Among these, 4.6% of anti-TNFα candidates, 19.3% of contacts and 24.4% of TB suspects tested positive. Compared with anti-TNFα candidates, the odds for a positive result were significantly higher for contact investigations (odds ratio (OR), mean...

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

  18. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    Science.gov (United States)

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  20. Tackling heterogeneity: a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco.

    Directory of Open Access Journals (Sweden)

    Natalia Piotrzkowski

    Full Text Available Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

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

    Science.gov (United States)

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

    2015-03-01

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

  2. Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

    Science.gov (United States)

    Matavulj, M; Molitoris, H P

    1992-12-01

    The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    2010-08-01

    Full Text Available High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  4. [Comparison of the clinical performance of the ECLusys HIV combi assay with the Lumipulse f and HISCL 2000-i HIV-1/2 ab screening assays].

    Science.gov (United States)

    Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

    2012-04-01

    We compared the ECLusys HIV combi assay (ECL HIV Ag/Ab) to the Lumipulse Forte (LPf HIV 1/2 Ab) and HISCL (HIS HIV 1/2 Ab) assays. In a dilution sensitivity test using dilution panels of WHO HIV antibody international reference panel (HIV-1 Subtype A, B, C, E, HIV-1 Group O, HIV-2) and HIV-1/2 Ab CE marked material(HIV-1, HIV-2) parent specimens, the ECL assay enabled detection at a higher level of sensitivity than either the LPf assay or the HIS assay for all dilution panels. In an early detection test in the early phase of infection in which a BBI HIV seroconversion panel was used, the ECL assay enabled detection 7 days after initial blood sample collection, whereas the LPf and HIS assays enabled detection after 27 days. In a specificity test using high RF positive specimens (n=33), pregnancy specimens (n=35), cytomegalovirus antibody positive specimens (n=36), and high M protein positive specimens (n=21) that were confirmed negative for HIV-1/2 antibodies by the LPf assay, negative results were obtained for all specimens on both the ECL assay and the HIS assay. In a correlation test using routinely collected clinical specimens (n=121), including positive stock specimens, the ECL and HIS assays demonstrated the highest agreement rate 98.3%. The above results confirmed that the fourth-generation reagent ECL assay, which simultaneously detects both HIV-1/2 antibodies and p24 antigens, is both highly sensitive and specific, and is a suitable assay for use in routine testing.

  5. Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Strange, Mette; Bräuner-Osborne, Hans; Jensen, Anders A.

    2006-01-01

    We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay...... assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors....

  6. Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

    Science.gov (United States)

    Schneider, Adele; Nakagawa, Sachiko; Keep, Rosanne; Dorsainville, Darnelle; Charrow, Joel; Aleck, Kirk; Hoffman, Jodi; Minkoff, Sherman; Finegold, David; Sun, Wei; Spencer, Andrew; Lebow, Johannah; Zhan, Jie; Apfelroth, Stephen; Schreiber-Agus, Nicole; Gross, Susan

    2009-11-01

    Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice. Copyright 2009 Wiley-Liss, Inc.

  7. Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

    Science.gov (United States)

    Meng, Juncai; Lai, Ming-Tain; Munshi, Vandna; Grobler, Jay; McCauley, John; Zuck, Paul; Johnson, Eric N; Uebele, Victor N; Hermes, Jeffrey D; Adam, Gregory C

    2015-06-01

    HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. © 2015 Society for Laboratory Automation and Screening.

  8. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

  9. [Comparison of the clinical performance of the ECLusys HBsAg II assay with the Lumipulse f and HISCL 2000-i HBsAg screening assays].

    Science.gov (United States)

    Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

    2012-02-01

    We compared the ECLusys HBsAgII (ECL HBsAg) assay to the Lumipulse Forte (LPf HBsAg) and HISCL (HIS HBsAg) assays. Measurement of dilution panels for which the WHO HBsAg international reference panel was the parent specimen revealed that the ECL and HIS assays enabled detection to a theoretical level of 0.04 IU/mL, whereas the LPf assay enabled detection to a level of 0.08 IU/mL. In a specificity test using high RF positive specimens (n = 33), pregnancy specimens (n = 35), cytomegalovirus antibody positive specimens (n = 36), and high M protein positive specimens (n = 21) that were confirmed negative for HBsAg by the LPf assay, negative results were obtained for all specimens on the HIS assay, but the ECL assay yielded a positive result for one of the high RF positive specimens. This individual was suggested on further testing to be an HBV carrier who was strongly positive for HBc antibody. In HBsAg mutants detection test, the detection rate was 92.3% with the ECL assay and 69.2% with the HIS assay. In a correlation test using routinely collected clinical specimens (n = 155), including positive stock specimens, aside from the one case where the LPf assay gave a negative result but both the ECL and HIS assays gave positive results, all of the results were consistent for all specimens. The above results confirmed that the ECL assay is both highly sensitive and specific, and also enables a high rate of HBsAg mutant detection.

  10. Feasibility and accuracy evaluation of three human papillomavirus assays for FTA card-based sampling: a pilot study in cervical cancer screening

    OpenAIRE

    Wang, Shao-Ming; Hu, Shang-Ying; Chen, Wen; Chen, Feng; Zhao, Fang-Hui; He, Wei; Ma, Xin-Ming; Zhang, Yu-Qing; Wang, Jian; Sivasubramaniam, Priya; Qiao, You-Lin

    2015-01-01

    Background Liquid-state specimen carriers are inadequate for sample transportation in large-scale screening projects in low-resource settings, which necessitates the exploration of novel non-hazardous solid-state alternatives. Studies investigating the feasibility and accuracy of a solid-state human papillomavirus (HPV) sampling medium in combination with different down-stream HPV DNA assays for cervical cancer screening are needed. Methods We collected two cervical specimens from 396 women, ...

  11. Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Hansen, Mette Reimert; Jakobsen, Tim H.; Bang, Claus Gunnar

    2015-01-01

    the pathogenesis and antibiotic tolerance of a bacterial biofilm. To identify the structural elements important for antagonistic or agonistic activity against the Pseudomonas aeruginosa LasR protein, we report the synthesis and screening of new triazole-containing mimics of natural N-acyl homoserine lactones....... A series of azide- and alkyne-containing homoserine lactone building blocks was used to prepare an expanded set of 123 homoserine lactone analogues through a combination of solution- and solid-phase synthesis methods. The resulting compounds were subjected to cell-based quorum sensing screening assays...

  12. Screens

    OpenAIRE

    2016-01-01

    This Sixth volume in the series The Key Debates. Mutations and Appropriations in European Film Studies investigates the question of screens in the context both of the dematerialization due to digitalization and the multiplication of media screens. Scholars offer various infomations and theories of topics such as the archeology of screen, film and media theories, contemporary art, pragmatics of new ways of screening (from home video to street screening).

  13. Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology.

    Science.gov (United States)

    Wang, Jing; Fang, Pengfei; Chase, Peter; Tshori, Sagi; Razin, Ehud; Spicer, Timothy P; Scampavia, Louis; Hodder, Peter; Guo, Min

    2017-01-01

    Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z' factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

  14. A Panel of High Resolution Melting (HRM Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

    Directory of Open Access Journals (Sweden)

    D. A. M. Heideman

    2009-01-01

    Full Text Available Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

  15. The BD Onclarity HPV assay on SurePath collected samples meets the International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

    DEFF Research Database (Denmark)

    Ejegod, Ditte; Bottari, Fabio; Pedersen, Helle

    2016-01-01

    This study describes a validation of the BD Onclarity HPV (Onclarity) assay using the international guidelines for HPV test requirements for cervical cancer screening of women 30 years and above using Danish SurePath screening samples. The clinical specificity (0.90, 95% CI: 0.88-0.91) and sensit......This study describes a validation of the BD Onclarity HPV (Onclarity) assay using the international guidelines for HPV test requirements for cervical cancer screening of women 30 years and above using Danish SurePath screening samples. The clinical specificity (0.90, 95% CI: 0.......88-0.91) and sensitivity (0.97, 95% CI: 0.87-1.0) of the Onclarity assay were shown to be non-inferior to the reference assay (specificity 0.90, 95% CI: 0.88-0.92, sensitivity 0.98, 95% CI: 0.91-1.0). The intra-laboratory reproducibility of Onclarity was 97% with a lower confidence bound of 96% (kappa value: 0...

  16. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    Science.gov (United States)

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

  17. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    International Nuclear Information System (INIS)

    Shin, Hyeong-Moo; Ernstoff, Alexi; Csiszar, Susan A.

    2015-01-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models

  18. ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SCREENING DIOXIN SOIL CONTAMINATION BY UNCONTROLLED COMBUSTION DURING INFORMAL RECYCLING IN SLUMS

    Science.gov (United States)

    Trindade, Mirta; Nording, Malin; Nichkova, Mikaela; Spinnel, Erik; Haglund, Peter; Last, Michael S.; Gee, Shirley; Hammock, Bruce; Last, Jerold A.; González-Sapienza, Gualberto; Brena, Beatriz M.

    2010-01-01

    Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly. PMID:18522475

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

    Directory of Open Access Journals (Sweden)

    Fariba Sharififar

    2017-03-01

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

  20. Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity

    International Nuclear Information System (INIS)

    Axon, Andrew; May, Felicity E.B.; Gaughan, Luke E.; Williams, Faith M.; Blain, Peter G.; Wright, Matthew C.

    2012-01-01

    Primary biliary cirrhosis (PBC) is a cholestatic liver disease of unknown cause that occurs most frequently in post-menopausal women. Since the female sex hormone oestrogen can be cholestatic, we hypothesised that PBC may be triggered in part by chronic exposure to xenoestrogens (which may be more active on a background of low endogenous oestrogen levels seen in post-menopausal women). A reporter gene construct employing a synthetic oestrogen response element predicted to specifically interact with oestrogen receptors (ER) was constructed. Co-transfection of this reporter into an ER null cell line with a variety of nuclear receptor expression constructs indicated that the reporter gene was trans-activated by ERα and ERβ, but not by the androgen, thyroid, progesterone, glucocorticoid or vitamin D receptors. Chemicals linked to PBC were then screened for xenoestrogen activity in the human ERα-positive MCF-7 breast cancer cell line. Using this assay, the coal-derived food and cosmetic colourings – sunset yellow and tartrazine – were identified as novel human ERα activators, activating the human ER with an EC 50% concentration of 220 and 160 nM, respectively.

  1. Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity.

    Science.gov (United States)

    Axon, Andrew; May, Felicity E B; Gaughan, Luke E; Williams, Faith M; Blain, Peter G; Wright, Matthew C

    2012-08-16

    Primary biliary cirrhosis (PBC) is a cholestatic liver disease of unknown cause that occurs most frequently in post-menopausal women. Since the female sex hormone oestrogen can be cholestatic, we hypothesised that PBC may be triggered in part by chronic exposure to xenoestrogens (which may be more active on a background of low endogenous oestrogen levels seen in post-menopausal women). A reporter gene construct employing a synthetic oestrogen response element predicted to specifically interact with oestrogen receptors (ER) was constructed. Co-transfection of this reporter into an ER null cell line with a variety of nuclear receptor expression constructs indicated that the reporter gene was trans-activated by ERα and ERβ, but not by the androgen, thyroid, progesterone, glucocorticoid or vitamin D receptors. Chemicals linked to PBC were then screened for xenoestrogen activity in the human ERα-positive MCF-7 breast cancer cell line. Using this assay, the coal-derived food and cosmetic colourings--sunset yellow and tartrazine--were identified as novel human ERα activators, activating the human ER with an EC(50%) concentration of 220 and 160 nM, respectively. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Landry Losi

    2010-08-01

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

  3. Fatty acid cosubstrates provide β-oxidation precursors for rhamnolipid biosynthesis in Pseudomonas aeruginosa, as evidenced by isotope tracing and gene expression assays.

    Science.gov (United States)

    Zhang, Lin; Veres-Schalnat, Tracey A; Somogyi, Arpad; Pemberton, Jeanne E; Maier, Raina M

    2012-12-01

    Rhamnolipids have multiple potential applications as "green" surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d(35) as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C(10) lipid chain were observed for octadecanoic acid-d(35) treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and β-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200- to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3- to 4-fold of the fadA β-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and β-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.

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

    Science.gov (United States)

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

    2014-03-01

    which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. A Two-Tiered-Testing Decision Tree for Assays in the USEPA-EDSP Screening Battery: Using 15 years of experience to improve screening and testing for endocrine active chemicals

    Science.gov (United States)

    Outline of the presentationEDCs – from 1991 to 1996 – Wingspread and Our Stolen Future 1996 – FQPA and SDWA mandates endocrine screening 1996-1998 – EDSTAC (the assays, debates over modes of action included) The final battery – EAT in vivo and in vit...

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

    Science.gov (United States)

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

    2013-05-13

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

  7. Screening non-classical 21-hydroxylase gene deficiency from patients diagnosed as polycystic ovary syndrome by gene assay

    Directory of Open Access Journals (Sweden)

    Jie HU

    2016-04-01

    Full Text Available Objective  To screen non-classical 21-hydroxylase deficiency (NC-21OHD from patients diagnosed as polycystic ovary syndrome (PCOS by gene assay. Methods  Ninety-eight patients with PCOS were enrolled according to 2003 Rotterdam criteria from Department of Endocrinology, Tangdu Hospital of Fourth Military Medical University, and they were divided into three groups according to the modified Ferriman-Gallway (mF-G score as follows: group A with score 0-2; group B with score 3-5, and group C with score ≥6. Meanwhile, 30 healthy subjects from the Medical Center of the Hospital were recruited as control group. Peripheral blood of all subjects were collected for extracting DNA, the CYP21A2 gene were amplified by 5 pairs of specific primers, and then the PCR products were sequenced by Shanghai Sangon Co. The subjects would accept test for serum cortisol and adrenocorticotropic hormone (ACTH at 8:00am if their CYP21A2 was proved to be abnormal. Results  Thirty subjects of control group had no any defects in CYP21A2, but 5 of 98 patients with PCOS were proved to be deficient in CYP21A2, and the genotypes were V281L/920-921insT (P1, V281L/I230M (P2, V281L/Normal (P3, P4, P5, respectively, and all of them were heterozygous mutations. The incidences of NC-21OHD in group C and B were 28.6% and 3.3%, respectively. Genotype P1 had been identified to belong to NC-21OHD, which was consistent with its clinical phenotype. All genotypes P3, P4 and P5 belonged to carriers. But for P2, since I230M hadn't been reported in literature, the patient with V281L/I230M couldn't be classified now. Serum biochemical results showed that only in P1 the cortisol was close to the normal lower level, and ACTH was close to the normal upper limit of the reported level in the literature, and the remainders were all normal. Conclusions  Although PCOS and NC-21OHD are very similar in clinical manifestations, they are different completely in the pathogenesis and treatment. So it

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

    Science.gov (United States)

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

    2009-03-01

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

  9. Screening health care workers with interferon-γ release assay versus tuberculin skin test: impact on costs and adherence to testing (the SWITCH study).

    Science.gov (United States)

    Wrighton-Smith, Peter; Sneed, Laurie; Humphrey, Frances; Tao, Xuguang; Bernacki, Edward

    2012-07-01

    To determine the price point at which an interferon-γ release assay (IGRA) is less costly than a tuberculin skin test (TST) for health care employee tuberculosis screening. A multidecision tree-based cost model incorporating inputs gathered from time-motion studies and parallel testing by IGRA and TST was conducted in a subset of our employees. Administering a TST testing program costs $73.20 per person screened, $90.80 per new hire, and $63.42 per annual screen. Use of an IGRA for employee health testing is cost saving at an IGRA test cost of $54.83 or less per test and resulted in higher completion rates because of the elimination of the need for a second visit to interpret the TST. Using an IGRA for employee health screening can be an institutional cost saving and results in higher compliance rates.

  10. Tuberculosis screening of new hospital employees: compliance, clearance to work time, and cost using tuberculin skin test and interferon-gamma release assays.

    Science.gov (United States)

    Foster-Chang, Sarah A; Manning, Mary L; Chandler, Laura

    2014-11-01

    Selection of the most suitable test(s) for detection of Mycobacterium tuberculosis (TB) infection should be based on purpose, setting, effectiveness, and cost. Two tests are available to screen for latent TB: the tuberculin skin test (TST) and the more recent interferon-gamma release assays (IGRAs). Based on the administrative, logistic, and technical ease of use, an IGRA trial was initiated by the occupational health department at an urban Veteran's Administration health care facility for TB screening of new employees. As a result, new employees completing the pre-placement process within the organization's designated 14 days increased from 77% to 97%, new employee clearance to work time decreased from 13.18 to 5.91 days, and new employee TB screening costs were reduced by 40%. The IGRA is an acceptable alternative to the TST and has significant potential to improve the process of pre-placement TB screening. Copyright 2014, SLACK Incorporated.

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

    Directory of Open Access Journals (Sweden)

    Michael J Smout

    Full Text Available BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

  12. Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm.

    Science.gov (United States)

    Kaga, Chiaki; Okochi, Mina; Tomita, Yasuyuki; Kato, Ryuji; Honda, Hiroyuki

    2008-03-01

    We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Science.gov (United States)

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

    2015-07-01

    Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work. © 2015 Society for Laboratory Automation and Screening.

  15. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    Directory of Open Access Journals (Sweden)

    Michael S Rogers

    Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

  16. Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

    Science.gov (United States)

    Tran, Nini; Zielke, Ryszard A; Vining, Oliver B; Azevedo, Mark D; Armstrong, Donald J; Banowetz, Gary M; McPhail, Kerry L; Sikora, Aleksandra E

    2013-09-01

    Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

  17. In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

    Directory of Open Access Journals (Sweden)

    Hong-Wei Ma

    2017-06-01

    Full Text Available Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE, but Hantaan virus (HTNV, the prototype hantavirus maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

  18. High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

    Science.gov (United States)

    Li, D Q; Zhao, J; Li, S P

    2014-06-06

    Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Molecular recognition and self-assembly special feature: A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin.

    Science.gov (United States)

    Shabbir, Shagufta H; Regan, Clinton J; Anslyn, Eric V

    2009-06-30

    A general approach to high-throughput screening of enantiomeric excess (ee) and concentration was developed by using indicator displacement assays (IDAs), and the protocol was then applied to the vicinal diol hydrobenzoin. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic acid-based receptors were screened by using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an artificial neural network to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified.

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

    KAUST Repository

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

    2016-01-01

    of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used

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

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

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

  2. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

    Science.gov (United States)

    Li, Baoguang; Liu, Huanli; Wang, Weimin

    2017-11-09

    Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

  3. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

    Science.gov (United States)

    Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

    2014-02-20

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

  4. Caenorhabditis elegans reveals novel Pseudomonas aeruginosa virulence mechanism

    NARCIS (Netherlands)

    Utari, Putri Dwi; Quax, Wim J.

    The susceptibility of Caenorhabditis elegans to different virulent phenotypes of Pseudomonas aeruginosa makes the worms an excellent model for studying host-pathogen interactions. Including the recently described liquid killing, five different killing assays are now available offering superb

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

    Science.gov (United States)

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

    2004-11-01

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

  6. Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay

    Directory of Open Access Journals (Sweden)

    Kwang Jin Lee

    2015-01-01

    Full Text Available This study investigated the antioxidant activity of one hundred kinds of pure chemical compounds found within a number of natural substances and oriental medicinal herbs (OMH. Three different methods were used to evaluate the antioxidant activity of DPPH radical-scavenging activity, ABTS radical-scavenging activity, and online screening HPLC-ABTS assays. The results indicated that 17 compounds exhibited better inhibitory activity against ABTS radical than DPPH radical. The IC50 rate of a more practical substance is determined, and the ABTS assay IC50 values of gallic acid hydrate, (+-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively. The ABTS assay is more sensitive to identifying the antioxidant activity since it has faster reaction kinetics and a heightened response to antioxidants. In addition, there was a very small margin of error between the results of the offline-ABTS assay and those of the online screening HPLC-ABTS assay. We also evaluated the effects of 17 compounds on the NO secretion in LPS-stimulated RAW 264.7 cells and also investigated the cytotoxicity of 17 compounds using a cell counting kit (CCK in order to determine the optimal concentration that would provide an effective anti-inflammatory action with minimum toxicity. These results will be compiled into a database, and this method can be a powerful preselection tool for compounds intended to be studied for their potential bioactivity and antioxidant activity related to their radical-scavenging capacity.

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

    OpenAIRE

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-03

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  10. A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae.

    Science.gov (United States)

    Teethaisong, Y; Eumkeb, G; Nakouti, I; Evans, K; Hobbs, G

    2016-08-01

    To validate a combined disc method along with resazurin chromogenic agar for early screening and differentiation of Klebsiella pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae. The combined disc test comprising of meropenem alone and with EDTA, phenylboronic acid or both EDTA and phenylboronic acid, and temocillin alone were evaluated with the resazurin chromogenic agar plate assay against a total of 86 molecularly confirmed Enterobacteriaceae clinical isolates (11 metallo-β-lactamases, eight Kl. pneumoniae carbapenemases, 11 OXA-48, 32 AmpC and 15 extended-spectrum-β-lactamase producers and nine co-producers of extended-spectrum-β-lactamase and AmpC). The inhibition zone diameters were measured and interpreted at 7 h for the presence of carbapenemase. All carbapenemase producers were phenotypically distinguished by this assay with 100% sensitivity and specificity. This early phenotypic method is very simple, inexpensive, and reliable in the detection and differentiation of carbapenemase-producing Enterobacteriaceae. It could be exploited in any microbiological laboratory for diagnosis of these recalcitrant bacteria. This assay poses excellent performance in discrimination of Kl. pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemases within 7 h, which is much faster than conventional disc diffusion methods. The rapid detection could help clinicians screen patients, control infection and provide epidemiological surveillance. © 2016 The Society for Applied Microbiology.

  11. Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed.

    Science.gov (United States)

    Bahrdt, C; Krech, A B; Wurz, A; Wulff, D

    2010-03-01

    For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD(abs)) GMOs in processed and unprocessed food, feed and seed samples with high efficiency.

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

    Science.gov (United States)

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

    2017-02-01

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

  13. Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

    Science.gov (United States)

    Wu, Szu-Huei; Yao, Chun-Hsu; Hsieh, Chieh-Jui; Liu, Yu-Wei; Chao, Yu-Sheng; Song, Jen-Shin; Lee, Jinq-Chyi

    2015-07-10

    Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 μM by high-throughput screening of 41,000 compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening.

    Science.gov (United States)

    Chung, Thomas D Y; Sergienko, Eduard; Millán, José Luis

    2010-04-27

    The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

  15. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia.

    Science.gov (United States)

    Noden, Bruce H; Martin, Jaclyn; Carrillo, Yisel; Talley, Justin L; Ochoa-Corona, Francisco M

    2018-01-01

    The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field

  16. AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

    Science.gov (United States)

    Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

    2017-09-29

    Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

  17. High-throughput screening assay used in pharmacognosy: Selection, optimization and validation of methods of enzymatic inhibition by UV-visible spectrophotometry

    Directory of Open Access Journals (Sweden)

    Graciela Granados-Guzmán

    2014-02-01

    Full Text Available In research laboratories of both organic synthesis and extraction of natural products, every day a lot of products that can potentially introduce some biological activity are obtained. Therefore it is necessary to have in vitro assays, which provide reliable information for further evaluation in in vivo systems. From this point of view, in recent years has intensified the use of high-throughput screening assays. Such trials should be optimized and validated for accurate and precise results, i.e. reliable. The present review addresses the steps needed to develop and validate bioanalytical methods, emphasizing UV-Visible spectrophotometry as detection system. Particularly focuses on the selection of the method, the optimization to determine the best experimental conditions, validation, implementation of optimized and validated method to real samples, and finally maintenance and possible transfer it to a new laboratory.

  18. Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.

    Science.gov (United States)

    Raudonis, Raimondas; Raudone, Lina; Jakstas, Valdas; Janulis, Valdimaras

    2012-04-13

    ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP - is based on the reduction of Fe(III)-tripyridyltriazine complex to Fe(II)-tripyridyltriazine at low pH by electron-donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeatable and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fragaria moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

    Science.gov (United States)

    Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

    2008-07-01

    The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

  20. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Supachai Sakkhachornphop

    2015-01-01

    Full Text Available The 3′-end processing (3′P of each viral long terminal repeat (LTR during human immunodeficiency virus type-1 (HIV-1 integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN, and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.

  1. Immunoassay of blood spot TSH; development of a rapid two-site immunoradiometric assay and comparison with radioimmunoassay as a screening method for neonatal hypothyroidism

    International Nuclear Information System (INIS)

    Sutherland, R.M.; Ratcliffe, J.G.; Chapman, R.S.

    1982-01-01

    The development of a two-site immunoradiometric assay (IRMA) for thyrotropin (TSH) eluted from dried blood filter paper discs is described and compared with a conventional TSH radioimmunoassay (RIA) as a screening procedure for neonatal hypothyroidism. The two-site IRMA involves a primary incubation of excess labelled TSH antibody and the blood disc for 16-18 h at pH 8 and a secondary 3 h incubation under agitation, with solid phase TSH antibody. Bound and free fractions are separated by a semi-automated washing procedure. It is concluded that the two-site TSH IRMA has advantages over conventional RIA in speed, sensitivity, precision and ruggedness and can be recommended as an efficient screening procedure for neonatal hypothyroidism. (Auth.)

  2. A filter paper dry blood spot procedure for acute intermittent porphyria population screening by use of whole blood uroporphyrinogen-I-synthase assay.

    Science.gov (United States)

    Johansson, L; Thunell, S; Wetterberg, L

    1984-03-13

    A filter paper dry blood spot procedure for the determination of whole blood uroporphyrinogen-I-synthase (UIS) activity is presented. The method is based on the concept of enzyme specific activity, the enzyme activity being related to the haemoglobin concentration of the assay sample. The diagnostic capacity with regard to the acute intermittent porphyria (AIP) gene carrier state is shown to be equivalent to that of a washed red cell reference method. On grounds of easy capillary blood sampling, uncomplicated and safe mail specimen transport and simple laboratory reception routines, the method is stated to be well adapted for use in AIP preadolescent population screening.

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

    Science.gov (United States)

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

    2017-10-07

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

  4. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Science.gov (United States)

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  5. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Gisder

    Full Text Available Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin or presumed (surfactin or no (paromomycin activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  6. Feasibility and accuracy evaluation of three human papillomavirus assays for FTA card-based sampling: a pilot study in cervical cancer screening

    International Nuclear Information System (INIS)

    Wang, Shao-Ming; Hu, Shang-Ying; Chen, Wen; Chen, Feng; Zhao, Fang-Hui; He, Wei; Ma, Xin-Ming; Zhang, Yu-Qing; Wang, Jian; Sivasubramaniam, Priya; Qiao, You-Lin

    2015-01-01

    Liquid-state specimen carriers are inadequate for sample transportation in large-scale screening projects in low-resource settings, which necessitates the exploration of novel non-hazardous solid-state alternatives. Studies investigating the feasibility and accuracy of a solid-state human papillomavirus (HPV) sampling medium in combination with different down-stream HPV DNA assays for cervical cancer screening are needed. We collected two cervical specimens from 396 women, aged 25–65 years, who were enrolled in a cervical cancer screening trial. One sample was stored using DCM preservative solution and the other was applied to a Whatman Indicating FTA Elute® card (FTA card). All specimens were processed using three HPV testing methods, including Hybrid capture 2 (HC2), careHPV™, and Cobas®4800 tests. All the women underwent a rigorous colposcopic evaluation that included using a microbiopsy protocol. Compared to the liquid-based carrier, the FTA card demonstrated comparable sensitivity for detecting high grade Cervical Intraepithelial Neoplasia (CIN) using HC2 (91.7 %), careHPV™ (83.3 %), and Cobas®4800 (91.7 %) tests. Moreover, the FTA card showed a higher specificity compared to a liquid-based carrier for HC2 (79.5 % vs. 71.6 %, P = 0.015), comparable specificity for careHPV™ (78.1 % vs. 73.0 %, P > 0.05), but lower specificity for the Cobas®4800 test (62.4 % vs. 69.9 %, P = 0.032). Generally, the FTA card-based sampling medium’s accuracy was comparable with that of liquid-based medium for the three HPV testing assays. FTA cards are a promising sample carrier for cervical cancer screening. With further optimization, it can be utilized for HPV testing in areas of varying economic development

  7. Feasibility and accuracy evaluation of three human papillomavirus assays for FTA card-based sampling: a pilot study in cervical cancer screening.

    Science.gov (United States)

    Wang, Shao-Ming; Hu, Shang-Ying; Chen, Wen; Chen, Feng; Zhao, Fang-Hui; He, Wei; Ma, Xin-Ming; Zhang, Yu-Qing; Wang, Jian; Sivasubramaniam, Priya; Qiao, You-Lin

    2015-11-04

    Liquid-state specimen carriers are inadequate for sample transportation in large-scale screening projects in low-resource settings, which necessitates the exploration of novel non-hazardous solid-state alternatives. Studies investigating the feasibility and accuracy of a solid-state human papillomavirus (HPV) sampling medium in combination with different down-stream HPV DNA assays for cervical cancer screening are needed. We collected two cervical specimens from 396 women, aged 25-65 years, who were enrolled in a cervical cancer screening trial. One sample was stored using DCM preservative solution and the other was applied to a Whatman Indicating FTA Elute® card (FTA card). All specimens were processed using three HPV testing methods, including Hybrid capture 2 (HC2), careHPV™, and Cobas®4800 tests. All the women underwent a rigorous colposcopic evaluation that included using a microbiopsy protocol. Compared to the liquid-based carrier, the FTA card demonstrated comparable sensitivity for detecting high grade Cervical Intraepithelial Neoplasia (CIN) using HC2 (91.7 %), careHPV™ (83.3 %), and Cobas®4800 (91.7 %) tests. Moreover, the FTA card showed a higher specificity compared to a liquid-based carrier for HC2 (79.5 % vs. 71.6 %, P = 0.015), comparable specificity for careHPV™ (78.1 % vs. 73.0 %, P > 0.05), but lower specificity for the Cobas®4800 test (62.4 % vs. 69.9 %, P = 0.032). Generally, the FTA card-based sampling medium's accuracy was comparable with that of liquid-based medium for the three HPV testing assays. FTA cards are a promising sample carrier for cervical cancer screening. With further optimization, it can be utilized for HPV testing in areas of varying economic development.

  8. Population-based V3 genotypic tropism assay: a retrospective analysis using screening samples from the A4001029 and MOTIVATE studies.

    Science.gov (United States)

    McGovern, Rachel A; Thielen, Alexander; Mo, Theresa; Dong, Winnie; Woods, Conan K; Chapman, Douglass; Lewis, Marilyn; James, Ian; Heera, Jayvant; Valdez, Hernan; Harrigan, P Richard

    2010-10-23

    The MOTIVATE-1 and 2 studies compared maraviroc (MVC) along with optimized background therapy (OBT) vs. placebo along with OBT in treatment-experienced patients screened as having R5-HIV (original Monogram Trofile). A subset screened with non-R5 HIV were treated with MVC or placebo along with OBT in a sister safety trial, A4001029. This analysis retrospectively examined the performance of population-based sequence analysis of HIV-1 env V3-loop to predict coreceptor tropism. Triplicate V3-loop sequences were generated using stored screening plasma samples and data was processed using custom software ('ReCall'), blinded to clinical response. Tropism was inferred using geno2pheno ('g2p'; 5% false positive rate). Primary outcomes were viral load changes after starting maraviroc; and concordance with prior screening Trofile results. Genotype and Trofile results were available for 1164 individuals with virological outcome data (N = 169 non-R5 by Trofile). Compared with Trofile, V3 genotyping had a specificity of 92.6% and a sensitivity of 67.4% for detecting non-R5 virus. However, when compared with clinical outcome, virological responses were consistently similar between Trofile and V3 genotype at weeks 8 and 24 following the initiation of therapy for patients categorized as R5. Despite differences in sensitivity for predicting non-R5 HIV, week 8 and 24 week virological responses were similar in this treatment-experienced population. These findings suggest the potential utility of V3 genotyping as an accessible assay to select patients who may benefit from maraviroc treatment. Optimization of the predictive tropism algorithm may lead to further improvement in the clinical utility of HIV genotypic tropism assays.

  9. A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action

    Directory of Open Access Journals (Sweden)

    Paul Priyesh Vijayakumar

    2015-06-01

    Full Text Available Lactic acid bacteria (LAB have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

  10. A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

    Science.gov (United States)

    Vijayakumar, Paul Priyesh; Muriana, Peter M

    2015-06-11

    Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

  11. Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

    Science.gov (United States)

    Marchand, Sophie; Vandriesche, Gonzalez; Coorevits, An; Coudijzer, Katleen; De Jonghe, Valerie; Dewettinck, Koen; De Vos, Paul; Devreese, Bart; Heyndrickx, Marc; De Block, Jan

    2009-07-31

    Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

  12. Contribution of two molecular assays as compared to selective culture for MRSA screening in a low MRSA prevalence population

    NARCIS (Netherlands)

    Nulens, E; Descheemaeker, P; Deurenberg, R H; Stobberingh, E E; Gordts, B

    BACKGROUND: As the prompt detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers upon admission is fundamental in the MRSA prevention strategy of our hospital, the infection control team is eagerly seeking the most sensitive and rapid screening method. The aim of this study was to

  13. Rapid Screening Assay for the Detection of Nivalenol and Deoxynivalenol using Monoclonal Antibody and Surface Plasmon Resonance

    Science.gov (United States)

    Nivalenol (NIV) and Deoxynivalenol (DON) are trichothecene mycotoxins produced by Fusarium spp that contaminate mainly cereal crops, such as wheat, barley, and maize. These mycotoxins are serious health hazards to human and domestic animals. The study reports a rapid screening method of NIV and DO...

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

    Science.gov (United States)

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

    2016-01-01

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

  15. High-sensitivity cardiac troponin I assay to screen for acute rejection in patients with heart transplant.

    Science.gov (United States)

    Patel, Parag C; Hill, Douglas A; Ayers, Colby R; Lavingia, Bhavna; Kaiser, Patricia; Dyer, Adrian K; Barnes, Aliessa P; Thibodeau, Jennifer T; Mishkin, Joseph D; Mammen, Pradeep P A; Markham, David W; Stastny, Peter; Ring, W Steves; de Lemos, James A; Drazner, Mark H

    2014-05-01

    A noninvasive biomarker that could accurately diagnose acute rejection (AR) in heart transplant recipients could obviate the need for surveillance endomyocardial biopsies. We assessed the performance metrics of a novel high-sensitivity cardiac troponin I (cTnI) assay for this purpose. Stored serum samples were retrospectively matched to endomyocardial biopsies in 98 cardiac transplant recipients, who survived ≥3 months after transplant. AR was defined as International Society for Heart and Lung Transplantation grade 2R or higher cellular rejection, acellular rejection, or allograft dysfunction of uncertain pathogenesis, leading to treatment for presumed rejection. cTnI was measured with a high-sensitivity assay (Abbott Diagnostics, Abbott Park, IL). Cross-sectional analyses determined the association of cTnI concentrations with rejection and International Society for Heart and Lung Transplantation grade and the performance metrics of cTnI for the detection of AR. Among 98 subjects, 37% had ≥1 rejection episode. cTnI was measured in 418 serum samples, including 35 paired to a rejection episode. cTnI concentrations were significantly higher in rejection versus nonrejection samples (median, 57.1 versus 10.2 ng/L; P<0.0001) and increased in a graded manner with higher biopsy scores (P(trend)<0.0001). The c-statistic to discriminate AR was 0.82 (95% confidence interval, 0.76-0.88). Using a cut point of 15 ng/L, sensitivity was 94%, specificity 60%, positive predictive value 18%, and negative predictive value 99%. A high-sensitivity cTnI assay seems useful to rule out AR in cardiac transplant recipients. If validated in prospective studies, a strategy of serial monitoring with a high-sensitivity cTnI assay may offer a low-cost noninvasive strategy for rejection surveillance. © 2014 American Heart Association, Inc.

  16. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides’ activity on various yeast species

    Czech Academy of Sciences Publication Activity Database

    Kodedová, Marie; Sychrová, Hana

    2016-01-01

    Roč. 233, Sep 10 (2016), s. 26-33 ISSN 0168-1656 R&D Projects: GA TA ČR(CZ) TA04010638; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : diS-C3(3) assay * antimicrobial peptide * Candida * membrane potential * microplate reader Subject RIV: EE - Microbiology, Virology Impact factor: 2.599, year: 2016

  17. Comparison of interferon {gamma} release assays and conventional screening tests before tumour necrosis factor {alpha} blockade in patients with inflammatory arthritis.

    LENUS (Irish Health Repository)

    Martin, J

    2012-02-01

    OBJECTIVE: To compare the performance of two interferon gamma release assays (IGRAs) and conventional screening tests in patients with inflammatory arthritis undergoing screening for latent tuberculosis infection (LTBI) before treatment with anti-tumour necrosis factor alpha (anti-TNFalpha) compounds. METHODS: Successive patients were subjected to conventional LTBI screening, including a tuberculin skin test (TST). The T-SPOT.TB test was performed on all patients and the QuantiFERON-TB Gold test was performed on a large subset. The results of the IGRAs were compared with the results of conventional screening tests. RESULTS: A total 150 patients were evaluated. The majority (57.9%) had rheumatoid arthritis. Previous vaccination with Bacille Calmette-Guerin was confirmed in 82% of patients. No patient had received prior anti-TB treatment. A total of 57 patients (38.0%) had at least one positive conventional risk factor. In contrast, an unequivocally positive T-SPOT.TB test was seen in only 14\\/143 (9.8%). There was 98.2% agreement between the two IGRAs. Statistically significant associations were found between each of the IGRAs and both TST and risk history, but not chest x-ray (CXR). A positive IGRA result was significantly associated with increased age. TB was not reactivated in any patient during the follow-up period. Interpretation: This study suggests that IGRAs may be useful when screening for LTBI before anti-TNFalpha therapy in patients with immune-mediated inflammatory diseases. The observations reported here also highlight the inadequate performance of CXR as a marker of LTBI.

  18. Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay

    Energy Technology Data Exchange (ETDEWEB)

    Schiliro, Tiziana, E-mail: tiziana.schiliro@unito.it [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Porfido, Arianna [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Spina, Federica; Varese, Giovanna Cristina [Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino (Italy); Gilli, Giorgio [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy)

    2012-08-15

    This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17{beta}-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.35 {+-} 1.68 ng/L pre-ozonation and 0.72 {+-} 0.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.18 {+-} 3.54 ng/L pre-ozonation and 2.53 {+-} 2.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p < 0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 67 {+-} 26% and 52 {+-} 27% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S = 0.650, p = 0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms. -- Highlights: Black-Right-Pointing-Pointer The two in vitro tests are suited for oestrogenic activity assessment in textile WWTP. Black-Right-Pointing-Pointer There is a significant correlation between the results of the two in vitro tests. Black-Right-Pointing-Pointer The oestrogenic activity of the effluent is reduced by ozonation. Black-Right-Pointing-Pointer The input of estrogenic substances into the river via textile WWTP is low.

  19. Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

    Science.gov (United States)

    Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

    2012-01-01

    The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and

  20. Design, synthesis, and validation of an in vitro platform peptide-whole cell screening assay using MTT reagent

    Directory of Open Access Journals (Sweden)

    Sahar Ahmed

    2017-05-01

    Full Text Available An in vitro platform to perform peptide screening against different cancer cell lines was designed. The strategy for this screening relied on the design and detection of high-affinity cancer-targeting peptides based on the sequences of NGR and P160. Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager. For proof of concept, a peptide library (132 spots, and 66 different peptides was designed, synthesized, and screened against different cancer cell lines. The current strategy assists in the identification of positive and negative peptides as well as the relative binding between positive ones. Better binding peptide sequences of the NGR motif were demonstrated to show up to a 2.6-fold increase in CD13+ cell lines with insignificant binding to CD13− ones. Comparable results were observed for P160 peptide sequences, to which different peptides had increased binding, with an up to 3-fold increase relative to the native P160 peptide. Based on our results, new peptide sequences for cancer targeting were identified, and the developed strategy was applied to two different peptide libraries.

  1. Evaluation of the Procleix Ultrio Elite Assay and the Panther-System for Individual NAT Screening of Blood, Hematopoietic Stem Cell, Tissue and Organ Donors

    Science.gov (United States)

    Heim, Albert

    2016-01-01

    Summary Background The performance of the multiplex Procleix Ultrio Elite assay as individual donor nucleic acid test (ID-NAT) for the detection of HIV-1, HIV-2, HCV, and HBV was evaluated in a retrospective, single center study. Methods ID-NAT results of 21,181 blood donors, 984 tissue donors, 293 hematopoietic stem cell donors and 4 organ donors were reviewed in synopsis with results of serological screening and additional discriminatory and repetitive NAT in case of positive donors. Results Specificity of the initial Procleix Ultrio Elite assay was 99.98% and after discriminatory testing 100.00%. Initially invalid results were observed in 75 of 21,181 blood donors (0.35%) but 16 of 984 tissue donors (1.62%, p donors. All these had valid negative ID-NAT results after repeated testing or testing of 1:5 diluted specimens in case of tissue donors. Occult hepatitis B (defined here as HBV DNAemia without HBsAg detection) was demonstrated by ID-NAT in two anti-HBc-positive tissue donors and suspected in two other tissue donors, where a definite diagnosis was not achieved due to the insufficient sample volumes available. Conclusion The Procleix Ultrio Elite assay proved to be specific, robust and rapid. Therefore, routine ID-NAT may also be feasible for organ and granulocyte donors. PMID:27403089

  2. Evaluation of the Procleix Ultrio Elite Assay and the Panther-System for Individual NAT Screening of Blood, Hematopoietic Stem Cell, Tissue and Organ Donors.

    Science.gov (United States)

    Heim, Albert

    2016-05-01

    The performance of the multiplex Procleix Ultrio Elite assay as individual donor nucleic acid test (ID-NAT) for the detection of HIV-1, HIV-2, HCV, and HBV was evaluated in a retrospective, single center study. ID-NAT results of 21,181 blood donors, 984 tissue donors, 293 hematopoietic stem cell donors and 4 organ donors were reviewed in synopsis with results of serological screening and additional discriminatory and repetitive NAT in case of positive donors. Specificity of the initial Procleix Ultrio Elite assay was 99.98% and after discriminatory testing 100.00%. Initially invalid results were observed in 75 of 21,181 blood donors (0.35%) but 16 of 984 tissue donors (1.62%, p donors. All these had valid negative ID-NAT results after repeated testing or testing of 1:5 diluted specimens in case of tissue donors. Occult hepatitis B (defined here as HBV DNAemia without HBsAg detection) was demonstrated by ID-NAT in two anti-HBc-positive tissue donors and suspected in two other tissue donors, where a definite diagnosis was not achieved due to the insufficient sample volumes available. The Procleix Ultrio Elite assay proved to be specific, robust and rapid. Therefore, routine ID-NAT may also be feasible for organ and granulocyte donors.

  3. Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

    Science.gov (United States)

    Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

    2016-01-01

    The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

  4. Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

    International Nuclear Information System (INIS)

    Maria Lina R; Andi Yasmon

    2011-01-01

    There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

  5. A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

    Science.gov (United States)

    Visser, M; Stephan, D; Jaynes, J M; Burger, J T

    2012-06-01

    Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. © 2012 The Authors. Letters in Applied Microbiology © 2012

  6. Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

    DEFF Research Database (Denmark)

    Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.

    2004-01-01

    In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested.......9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between...

  7. Effectiveness of halo-tolerant, auxin producing Pseudomonas and Rhizobium strains to improve osmotic stress tolerance in mung bean (Vigna radiata L.

    Directory of Open Access Journals (Sweden)

    Maqshoof Ahmad

    2013-12-01

    Full Text Available Halo-tolerant, auxin producing bacteria could be used to induce salt tolerance in plants. A number of Rhizobium and auxin producing rhizobacterial strains were assessed for their ability to tolerate salt stress by conducting osmoadaptation assay. The selected strains were further screened for their ability to induce osmotic stress tolerance in mung bean seedlings under salt-stressed axenic conditions in growth pouch/jar trials. Three most effective strains of Rhizobium and Pseudomonas containing ACC-deaminase were evaluated in combination, for their ability to induce osmotic stress tolerance in mung bean at original, 4, and 6 dS m-1 under axenic conditions. Results showed that sole inoculation of Rhizobium and Pseudomonas strains improved the total dry matter up to 1.4, and 1.9 fold, respectively, while the increase in salt tolerance index was improved up to 1.3 and 2.0 fold by the Rhizobium and Pseudomonas strains, respectively. However, up to 2.2 fold increase in total dry matter and salt tolerance index was observed due to combined inoculation of Rhizobium and Pseudomonas strains. So, combined application of Rhizobium and Pseudomonas strains could be explored as an effective strategy to induce osmotic stress tolerance in mung bean.

  8. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    DEFF Research Database (Denmark)

    Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon

    2015-01-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify....../oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models....

  9. An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

    International Nuclear Information System (INIS)

    Tang, J.C.T.; Li, S.H.

    1984-01-01

    Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

  10. Evaluation of a real-time PCR assay for rectal screening of OXA-48-producing Enterobacteriaceae in a general intensive care unit of an endemic hospital.

    Science.gov (United States)

    Fernández, J; Cunningham, S A; Fernández-Verdugo, A; Viña-Soria, L; Martín, L; Rodicio, M R; Escudero, D; Vazquez, F; Mandrekar, J N; Patel, R

    2017-07-01

    Carbapenemase-producing Enterobacteriaceae are increasing worldwide. Rectal screening for these bacteria can inform the management of infected and colonized patients, especially those admitted to intensive care units (ICUs). A laboratory developed, qualitative duplex real-time polymerase chain reaction assay for rapid detection of OXA-48-like and VIM producing Enterobacteriaceae, performed on rectal swabs, was designed and evaluated in an intensive care unit with endemic presence of OXA-48. During analytical assay validation, no cross-reactivity was observed and 100% sensitivity and specificity were obtained for both bla OXA-48-like and bla VIM in all spiked clinical samples. During the clinical part of the study, the global sensitivity and specificity of the real-time PCR assay for OXA-48 detection were 95.7% and 100% (P=0.1250), respectively, in comparison with culture; no VIM-producing Enterobacteriaceae were detected. Clinical features of patients in the ICU who were colonized or infected with OXA-48 producing Enterobacteriaceae, including outcome, were analyzed. Most had severe underlying conditions, and had risk factors for colonization with carbapenemase-producing Enterobacteriaceae before or during ICU admission, such as receiving previous antimicrobial therapy, prior healthcare exposure (including long-term care), chronic disease, immunosuppression and/or the presence of an intravascular catheter and/or mechanical ventilation device. The described real-time PCR assay is fast (~2-3hours, if DNA extraction is included), simple to perform and results are easy to interpret, features which make it applicable in the routine of clinical microbiology laboratories. Implementation in endemic hospitals could contribute to early detection of patients colonized by OXA-48 producing Enterobacteriaceae and prevention of their spread. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Schwarzenberger, T; Wolf, P; Brischwein, M; Kleinhans, R; Demmel, F; Becker, B; Wolf, B; Lechner, A

    2011-01-01

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

  12. Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (part I: virtual screening and activity assays.

    Directory of Open Access Journals (Sweden)

    Laura Guasch

    Full Text Available BACKGROUND: There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. METHODOLOGY/PRINCIPAL FINDINGS: We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity. Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual-screening protocol was successful in identifying novel

  13. DNA Comet Assay and Changes in Microflora Load as Screening Methods to Detect Irradiated Food in Egypt

    International Nuclear Information System (INIS)

    Hammad, A.A.; Abo El Nour, S.A.; Ibrahim, H.M.; Osman, M.E.; Abo El- Nasr, A.

    2014-01-01

    In the present study the microgel electrophoresis of single cells (DNA Comet Assay), and changes in microflora load were applied to detect irradiation treatment of strawberries and fresh-deboned chicken produced in Egypt. Strawberry samples were irradiated at 1.0, 2.0, 3.0 and 4.0 kGy, stored at 4 degree C±1 and analyzed at 0 and 7 days post.-irradiation. Fresh- deboned chicken meat samples were exposed to 2.0, 3.0, 4.0 and 5.0 kGy, stored at 4 degree C±1 and analyzed at 0, 7, 14 and 21 days post-irradiation. After electrophoresis performance, the accridine orange stain slides were seen under fluorescent microscope and the DNA comets were evaluated by photographic and image analysis. Changes in microflora load of irradiated samples were also evaluated. In all irradiated samples, the DNA fragments stretched or migrated out of the cells towards the anode of the agrose gel and appeared as a “comets” with tail. Whereas, DNA comets of all non-irradiated samples were almost intact, round without tail or had very short tail. Values of DNA % in tails and the tail length increased with increasing irradiation dose and storage times. The DNA comet assay could successfully be used to detect radiation treatment of strawberry and deboned-chicken meat samples up to 7 and 21 days post-irradiation, respectively. The absence of gram-negative bacteria and enterobacteriaceae group as well as the very low count of fungi (mostly yeasts) might be considered another evidence of radiation treatment of strawberries and fresh-deboned chicken.

  14. Colony to colorimetry in 6 h: ELISA detection of a surface-expressed Pseudomonas aeruginosa virulence factor using immobilized bacteria.

    Science.gov (United States)

    Adawi, Azmi; Neville, Lewis F

    2012-09-01

    A rapid ELISA employing intact Pseudomonas aeruginosa (PA) is described that allows discrimination between strains harboring flagellin type a or b. All 52 PA strains known to harbor flagellin type b were positive in this ELISA when screened with a fully human monoclonal antibody (LST-007) targeting flagellin type b. Completion of this assay in only 6 h, from picking a single bacterial colony to a colorimetric product, could easily be adapted to a clinical laboratory setting and permit the appropriate choice of therapeutic monoclonal antibody versus its homologous flagellin target in PA-infected patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR SCREENING OF MILK SAMPLES FOR SALMONELLA-TYPHIMURIUM IN DAIRY HERDS

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Wedderkopp, A.

    1995-01-01

    We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium epsiodes within the previous 7...... months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA...... positive (herd specificity, 0.9 and herd sensitivity, 1.0). A sig nificant correlation (P history of salmonellosis. It was concluded that ELISA testing of individual milk sam ples can be used for surveillance...

  16. Detection of bla-IMP-1 and bla-IMP-2 Genes Among Metallo-β-lactamase-Producing Pseudomonas Aeruginosa Isolated from Burn Patients in Isfahan

    Directory of Open Access Journals (Sweden)

    M. Pourbabaee

    2016-02-01

    Full Text Available Background: Pseudomonas aeruginosa is a nosocomial pathogen which especially causes infections among burn patients. Carbapenems are extensively used for the treatment of infections caused by multidrug-resistant P. aeruginosa isolates. The emergence of carbapenemases producing isolates is an outcome of increased utilization of carbapenems. The aim of this study was to determine the bla-IMP-1 and bla-IMP-2 genes in metallo-β-lactamase (MBL -producing Pseudomonas aeruginosa isolated from burn patients in Isfahan. Material and Methods: A total of 150 P. aeruginosa were isolated from burn patients hospitalized in Imam-Mousakazem hospital in Isfahan. Antimicrobial susceptibility was determined using disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI guidelines. Double Disk Synergy Test (DDST was carried out for screening of MBL production in imipenem-resistant strains. PCR assays were used for detection of bla-IMP-1 and bla-IMP-2 genes among metallo-β-lactamase-producing Pseudomonas aeruginosa isolates. The purified PCR products were sequenced. Results: Of 150 Pseudomonas aeruginosa isolates, %100 identified as multi-drug resistant strains. The most resistance rates were seen against ciprofloxacin, tobromycin, meropenem and imipenem. All of 144 imipenem-resistant Pseudomonas aeruginosa isolates were MBL producing by DDST test. Twenty-nine (19.3% and 8(5.3% MBL producing Pseudomonas aeruginosa isolates harbored bla-IMP-1 and bla-IMP-2 genes respectively. Conclusions: According to results of this study high level resistance to imipenem and MBl genes carriage was seen among Pseudomonas aeruginosa isolated from burn patient infections in our region.

  17. Diagnosis and screening of small hepatocellular carcinomas. Comparison of radionuclide imaging, ultrasound, computed tomography, hepatic angiography, and alpha 1-fetoprotein assay

    International Nuclear Information System (INIS)

    Takashima, T.; Matsui, O.; Suzuki, M.; Ida, M.

    1982-01-01

    Twenty-nine small (less than 5 cm) hepatocellular carcinomas in 18 patients were examined by radionuclide imaging (RN), ultrasound (US), computed tomography (CT), hepatic angiography, and serum alpha 1-fetoprotein (AFP) assay. Sensitivity was 39% with RN, 50% with US, 56% with CT, and 94% with angiography, including infusion hepatic angiography (IHA). Lesions larger than 3 cm could be detected by all of these methods; those between 2 and 3 cm were generally shown by US and CT but not RN. IHA was essential for diagnosis of lesions less than 2 cm, which were otherwise difficult or impossible to detect except with angiography. As a screening method, AFP was best, followed by US and CT. The authors recommend using AFP and US to minimize expense and radiation exposure. In questionable cases, IHA should be performed

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

    Science.gov (United States)

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

    2013-11-15

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

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

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

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

  20. The E-screen test and the MELN gene-reporter assay used for determination of estrogenic activity in fruits and vegetables in relation to pesticide residues.

    Science.gov (United States)

    Schilirò, Tiziana; Porfido, Arianna; Longo, Annalisa; Coluccia, Sara; Gilli, Giorgio

    2013-12-01

    Endocrine-disrupting chemicals (EDCs) may lead to adverse systemic effects by interfering with normal hormone homeostasis, and diet is considered to be among the main routes of EDC exposure. The present study investigated the total estrogenic activity of fruits and vegetables by calculating the 17-β-estradiol equivalent quantity (EEQ) using two in vitro tests: the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen test) and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay. Of the 24 analyzed fruits and vegetables, 14 contained from 1 to 4 pesticide residues in concentrations ranging from 0.02 to 1.19 ppm, whereas the other 10 did not contain any pesticide residues. The EEQ values for all positive samples ranged from 0.010 to 0.616 μg/100g for the above in vitro tests. Our study demonstrates that estrogenic activity was present in fruits and vegetables and that the concentration of allowable pesticide residues and EEQ values were positively correlated; however, no correlation was found by comparing the estrogenic activity and the intrinsic content of phytoestrogens obtained from the available literature. A theoretical adult dietary intake of 0.7-0.9 ng EEQ/L/day from fruits and vegetables was calculated. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Setting up a Bioluminescence Resonance Energy Transfer high throughput screening assay to search for protein/protein interaction inhibitors in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Cyril eCouturier

    2012-09-01

    Full Text Available Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed interactome. Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET technique was primarily developed to allow the dynamic monitoring of protein-protein interactions in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of protein-protein interactions and here is described why and how to set up and optimize a High Throughput Screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence substrate concentration, number of cells and medium composition used on the Z’ factor, and expected interferences for colored or fluorescent compounds.

  2. Development of a simple, low cost chronoamperometric assay for fructose based on a commercial graphite-nanoparticle modified screen-printed carbon electrode.

    Science.gov (United States)

    Nicholas, Phil; Pittson, Robin; Hart, John P

    2018-02-15

    This paper describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry with a 40µl mixture containing fructose, mediator and FDH, deposited onto the SPCE-G-COOH. The response was linear from 0.1mM to 1.0mM. A commercial fruit juice sample was analysed using the developed assay and the fructose concentration was calculated to be 477mM with a precision of 3.03% (n=5). Following fortification (477mM fructose) the mean recovery was found to be 97.12% with a coefficient of variation of 6.42% (n=5); consequently, the method holds promise for the analysis of commercial fruit juices. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Crystallization of the Membrane-Associated Annexin B1: Roles of Additive Screen, Dynamic Light Scattering, and Bioactivity Assay

    Energy Technology Data Exchange (ETDEWEB)

    Ding, F.; Xu, Y; Azzi, A; Zhu, D; Rehse, D; Chen, C; Sun, S; Lin, S

    2010-01-01

    Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A{sub 2}, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl{sub 2}) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 {angstrom}) has been collected successfully for crystals of L-selenomethionine labeled protein in the presence of Gn-HCl, to solve the structure with the single anomalous dispersion method (SAD). The unit cell parameters are a = 102.35 {angstrom}, b = 103.59 {angstrom}, c = 114.60 {angstrom}, {alpha} = {beta} = {gamma} = 90.00{sup o}.

  4. Screening assays of termite gut microbes that potentially as probiotic for human to digest cellulose as new food source

    Science.gov (United States)

    Abdullah, R.; Ananda, K. R. T.; Wijanarka

    2018-05-01

    According to UN, earth population will increase approximately 7.3 billion people up to 11.2 billion from 2015 until 2100. On the other side, food needs are not balance with the availability of food on earth. People of the world need solution for a new food source. By cellulose digesting ability, people analyzed can consume cellulose as the new food source to get glucose. The aims of research is obtaining termite gut cellulase bacteria selected which is potential as probiotic to split cellulose. Method used was as follows; isolation of termite gut microbes, microbial cellulase purification by screening method and probiotic test includes microbial pathogenicity test and human stomach acid and salt osmotic concentration resistance test. The result shows, 3 pure isolates of termite gut microbes can break down cellulose in the medium 1% CMC and 0.1% congo red (indicator of cellulose degradation activity) and life at pH 2- 2.5 and osmotic salt condition. Two isolates show the activity of gamma hemolysis (non-pathogenic in terms of pathogenicity on human blood). In conclusion, there are isolated termite gut microbes can be used as probiotic candidate for human to digest cellulose of the new food source for global food scarcity era.

  5. High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

    Science.gov (United States)

    Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

    2017-10-01

    The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust

    Directory of Open Access Journals (Sweden)

    Melanie Sanders

    2016-04-01

    Full Text Available A sample preparation method was developed for the screening of deoxynivalenol (DON in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA was compared to the sensor-based techniques of surface plasmon resonance (SPR and biolayer interferometry (BLI in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889 was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg.

  8. Evaluation of the tuberculin skin test and the interferon-γ release assay for TB screening in French healthcare workers

    Directory of Open Access Journals (Sweden)

    Raffi Francois

    2009-11-01

    Full Text Available Abstract Introduction Using French cut-offs for the Tuberculin Skin Test (TST, results of the TST were compared with the results of an Interferon-γ Release Assay (IGRA in Healthcare Workers (HCW after contact to AFB-positive TB patients. Methods Between May 2006 and May 2007, a total of 148 HCWs of the University Hospital in Nantes, France were tested simultaneously with IGRA und TST. A TST was considered to indicate recent latent TB infection (LTBI if an increase of >10 mm or if TST ≥ 15 mm for those with no previous TST result was observed. For those with a positive TST, chest X-ray was performed and preventive chemotherapy was offered. Results All HCWs were BCG-vaccinated. The IGRA was positive in 18.9% and TST ≥ 10 mm was observed in 65.5%. A recent LTBI was believed to be highly probable in 30.4% following TST. Agreement between IGRA and TST was low (kappa 0.041. In 10 (16.7% out of 60 HCWs who needed chest X-ray following TST the IGRA was positive. In 9 (20% out of 45 HCWs to whom preventive chemotherapy was offered following TST the IGRA was positive. Of those considered TST-negative following the French guidelines, 20.5% were IGRA-positive. In a two-step strategy - positive TST verified by IGRA - 18 out of 28 (64.3% IGRA-positive HCWs would not have been detected using French guidelines for TST interpretation. Conclusion The introduction of IGRA in contact tracings of BCG-vaccinated HCWs reduces X-rays and preventive chemotherapies. Increasing the cut-off for a positive TST does not seem to be helpful to overcome the effect of BCG vaccination on TST.

  9. Usefulness of FC-TRIPLEX Chagas/Leish IgG1 as confirmatory assay for non-negative results in blood bank screening of Chagas disease.

    Science.gov (United States)

    Campos, Fernanda Magalhães Freire; Repoles, Laura Cotta; de Araújo, Fernanda Fortes; Peruhype-Magalhães, Vanessa; Xavier, Marcelo Antônio Pascoal; Sabino, Ester Cerdeira; de Freitas Carneiro Proietti, Anna Bárbara; Andrade, Mariléia Chaves; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Gontijo, Célia Maria Ferreira

    2018-04-01

    A relevant issue in Chagas disease serological diagnosis regards the requirement of using several confirmatory methods to elucidate the status of non-negative results from blood bank screening. The development of a single reliable method may potentially contribute to distinguish true and false positive results. Our aim was to evaluate the performance of the multiplexed flow-cytometry anti-T. cruzi/Leishmania IgG1 serology/(FC-TRIPLEX Chagas/Leish IgG1) with three conventional confirmatory criteria (ELISA-EIA, Immunofluorescence assay-IIF and EIA/IIF consensus criterion) to define the final status of samples with actual/previous non-negative results during anti-T. cruzi ELISA-screening in blood banks. Apart from inconclusive results, the FC-TRIPLEX presented a weak agreement index with EIA, while a strong agreement was observed when either IIF or EIA/IIF consensus criteria were applied. Discriminant analysis and Spearman's correlation further corroborates the agreement scores. ROC curve analysis showed that FC-TRIPLEX performance indexes were higher when IIF and EIA/IIF consensus were used as a confirmatory criterion. Logistic regression analysis further demonstrated that the probability of FC-TRIPLEX to yield positive results was higher for inconclusive results from IIF and EIA/IIF consensus. Machine learning tools illustrated the high level of categorical agreement between FC-TRIPLEX versus IIF or EIA/IIF consensus. Together, these findings demonstrated the usefulness of FC-TRIPLEX as a tool to elucidate the status of non-negative results in blood bank screening of Chagas disease. Copyright © 2018. Published by Elsevier B.V.

  10. Use of interferon-gamma release assays in a health care worker screening program: experience from a tertiary care centre in the United States.

    Science.gov (United States)

    Joshi, Manish; Monson, Thomas P; Woods, Gail L

    2012-01-01

    Interferon-gamma release assays including the QuantiFERON-TB Gold In-Tube test (QFT-GIT [Cellestis Ltd, Australia]) may be used in place of the tuberculin skin test (TST) in surveillance programs for Mycobacterium tuberculosis infection control. However, data on performance and practicality of the QFT-GIT in such programs for health care workers (HCWs) are limited. To assess the performance, practicality and reversion rate of the QFT-GIT among HCWs at a tertiary health care institution in the United States. Retrospective chart review of HCWs at Central Arkansas Veterans Healthcare System (Arkansas, USA) who underwent QFT-GIT testing as a part of their employee screening between November 1, 2008 and October 31, 2009. QFT-GIT was used to screen 3290 HCWs. The initial QFT-GIT was interpreted as positive for 129 (3.9%) HCWs, negative for 3155 (95.9%) and indeterminate for six (0.2%). Testing with QFT-GIT was repeated in 45 HCWs who had positive results on the initial test. The QFT-GIT reverted to negative in 18 (40.0%) HCWs, all of whom had negative TST status and initial interferon-gamma values of 0.35 IU⁄mL to 2.0 IU⁄mL. The QFT-GIT test is feasible in large health care setting as an alternative to TST for M tuberculosis infection screening in HCWs but is not free from challenges. The major concerns are the high number of positive test results and high reversion rates on repeat testing, illustrating poor short-term reproducibility of positive QFT-GIT test results. These results suggest adopting a borderline zone between interferon-gamma values of 0.35 IU⁄mL to 2.0 IU⁄mL, and cautious clinical interpretation of values in this range.

  11. Identification of KX2-391 as an inhibitor of HBV transcription by a recombinant HBV-based screening assay.

    Science.gov (United States)

    Harada, Keisuke; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada

    2017-08-01

    Antiviral therapies for chronic hepatitis B virus (HBV) infection that are currently applicable for clinical use are limited to nucleos(t)ide analogs targeting HBV polymerase activity and pegylated interferon alpha (PEG-IFN). Towards establishing an effective therapy for HBV related diseases, it is important to develop a new anti-HBV agent that suppresses and eradicates HBV. This study used recombinant HBV encoding NanoLuc to screen anti-HBV compounds from 1827 US Food and Drug Administration approved compounds and identified several compounds that suppressed HBV infection. Among them, KX2-391, a non-ATP-competitive inhibitor of SRC kinase and tubulin polymerization, was identified as a lead candidate for an anti-HBV drug. Treatment of sodium taurocholate cotransporting polypeptide (NTCP) transduced-HepG2 (HepG2-NTCP) or primary human hepatocytes with KX2-391 suppressed HBV replication in a dose-dependent manner. The anti-HBV activity of KX2-391 appeared not to depend on SRC kinase activity because siRNA for SRC mRNA did not impair the HBV infection/replication. The anti-HBV activity of KX2-391 depended on the inhibitory effect of tubulin polymerization similar to other tubulin polymerization inhibitors, some of which were shown to inhibit HBV replication. KX2-391 inhibited HBV transcription driven by a HBV precore promoter in an HBV X protein-independent manner but did not inhibit the activity of HBV-S1, -S2, -X or cytomegalovirus promoters. Treatment with KX2-391 reduced the expression of several various factors including hepatocyte nuclear factor-4a. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2011-01-01

    and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative......OBJECTIVE: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. METHODS: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10...... assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable. CONCLUSION: The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay...

  13. Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

    Science.gov (United States)

    Bogestam, Katja; Vondracek, Martin; Karlsson, Mattias; Fang, Hong; Giske, Christian G

    2018-01-01

    Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

  14. Pseudomonas Lipopeptide Biosurfactants

    DEFF Research Database (Denmark)

    Bonnichsen, Lise

    Pseudomonas lipopetide biosurfactants are amphiphilic molecules with a broad range of natural functions. Due to their surface active properties, it has been suggested that Pseudomonas lipopetides potentially play a role in biodegradation of hydrophobic compounds and have essential functions...... lipopetide biosurfactants in pollutant biodegradation and natural roles in biofilm formation. The work presented is a combination of environmental microbiology and exploiting genetic manipulation of pure cultures to achieve insightinto the effects and mechanisms of lipopeptides on microbial processes...

  15. Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

    Science.gov (United States)

    Powers, Matthew J.; Sanabria-Valentín, Edgardo; Bowers, Albert A.

    2015-01-01

    ABSTRACT Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. IMPORTANCE Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that

  16. Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

    Science.gov (United States)

    Kim, Han-Shin; Park, Hee-Deung

    2013-01-01

    Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

  17. A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

    Directory of Open Access Journals (Sweden)

    Matthew Brecher

    2017-05-01

    Full Text Available The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2 in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV, West Nile virus (WNV, and Yellow fever virus (YFV on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and

  18. Antibacterial properties of Chinese herbal medicines against nosocomial antibiotic resistant strains of Pseudomonas aeruginosa in Taiwan.

    Science.gov (United States)

    Liu, Ching-Shen; Cham, Thau-Ming; Yang, Cheng-Hong; Chang, Hsueh-Wei; Chen, Chia-Hong; Chuang, Li-Yeh

    2007-01-01

    Pseudomonas aeruginosa is well-recognized as a nosocomial pathogen, which exhibits inherent drug resistance. In this study, the antibacterial activity of ethanol extracts of 58 Chinese herbal medicines used in Taiwan were tested against 89 nosocomial antibiotic resistant strains of Pseudomonas aeruginosa. The results gathered by the disc diffusion method showed that 26 out of the 58 herbal extracts exhibited antibacterial activity. Among the 26 herbal extracts, 10 extracts showed broad-spectrum antibacterial activities and were selected for further antibacterial property assay. The minimum inhibitory concentrations (MIC) of the active partition fractions ranged from 0.25 to 11.0 mg/L. The presence of flavonoid compounds in the active fractions of test herbal extracts was observed by the TLC-bioautography. The results from the time-kill assay revealed that most of the herbal extracts completely killed the test organisms within 4 hours. Exposure of the test strains to a sub-MIC level of the herbal extracts for 10 consecutive subcultures did not induce resistance to the active components. A combination of the active herbal fractions with antibiotics showed that one of the herbal medicines, the hexane fraction of Ramulus Cinnamomi, possessed a synergistic effect with tetracycline, gentamycin, and streptomycin. In conclusion, the tested Chinese medical herbs have the potential to be developed into natural antibiotics. This is the first evaluation for screening large amounts of medical plants against nosocomial antibiotic resistant bacteria in Taiwan.

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

    Directory of Open Access Journals (Sweden)

    Atsuya Yamashita

    2015-11-01

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

  20. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae...

  1. Complement activation by Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Jensen, E T; Kharazmi, A; Garred, P

    1993-01-01

    In chronic infections, such as the bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients, bacteria persist despite an intact host immune defense and frequent antibiotic treatment. An important reason for the persistence of the bacteria is their capacity for the biofilm...... mode of growth. In this study we investigated the role of biofilms in activation of complement, a major contributor to the inflammatory process. Complement activation by P. aeruginosa was examined in a complement consumption assay, production of C3 and factor B conversion products assessed by crossed...... immuno-electrophoresis, C5a generation tested by a PMN chemotactic assay, and terminal complement complex formation measured by ELISA. Two of the four assays showed that P. aeruginosa grown in biofilm activated complement less than planktonic bacteria, and all assays showed that activation by intact...

  2. Antimicrobial screening of Cichorium intybus seed extracts

    Directory of Open Access Journals (Sweden)

    Tauseef shaikh

    2016-11-01

    Full Text Available Medicinal plants play an important role in the field of natural products and human health care system. Chemical constituents present in the various parts of the plants can resist to parasitic attack by using several defense mechanisms. One such mechanism is the synthesis of antimicrobial compound. Cichorium intybus is one of the important medicinal plants which belong to Asteraceae family. In the present work, antimicrobial screening of C. intybus seed extract was studied by agar well diffusion assay by using aqueous and organic extracts. The pathogenic microorganisms tested include Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Escherichia coli. All the seed extracts showed antimicrobial activity against tested microorganisms whereas S. aureus was found to be most sensitive against aqueous extract and had the widest zone of inhibition. Ethyl acetate and ethanol extract were found to be significant against P. aeruginosa and S. aureus. The results obtained from antimicrobial screening scientifically support the effectiveness of the medicinal plant.

  3. Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutiérrez, Antonio; Marcús, Toni F; Soverini, Simona; Amat, Juan Carlos; Navarro-Palou, María; Ros, Teresa; Bex, Teresa; Ballester, Carmen; Bauça, Josep Miquel; SanFelix, Sara; Novo, Andrés; Vidal, Carmen; Santos, Carmen; Besalduch, Joan

    2012-03-01

    Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-10-03

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

  5. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  6. High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model.

    Science.gov (United States)

    Conery, Annie L; Larkins-Ford, Jonah; Ausubel, Frederick M; Kirienko, Natalia V

    2014-03-14

    In recent history, the nematode Caenorhabditis elegans has provided a compelling platform for the discovery of novel antimicrobial drugs. In this protocol, we present an automated, high-throughput C. elegans pathogenesis assay, which can be used to screen for anti-infective compounds that prevent nematodes from dying due to Pseudomonas aeruginosa. New antibiotics identified from such screens would be promising candidates for treatment of human infections, and also can be used as probe compounds to identify novel targets in microbial pathogenesis or host immunity. Copyright © 2014 John Wiley & Sons, Inc.

  7. Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

    Science.gov (United States)

    Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham

    2016-03-01

    To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Diversity and Abundance of Ice Nucleating Strains of Pseudomonas syringae in a Freshwater Lake in Virginia, USA.

    Science.gov (United States)

    Pietsch, Renée B; Vinatzer, Boris A; Schmale, David G

    2017-01-01

    The bacterium Pseudomonas syringae is found in a variety of terrestrial and aquatic environments. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as Ice+) allowing them to catalyze the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in France, little is known about the genetic diversity of P. syringae in natural aquatic habitats in North America. We collected samples of freshwater from three different depths in Claytor Lake, Virginia, USA between November 2015 and June 2016. Samples were plated on non-selective medium (TSA) and on medium selective for Pseudomonas (KBC) and closely related species to estimate the total number of culturable bacteria and of Pseudomonas , respectively. A droplet freezing assay was used to screen colonies for the Ice+ phenotype. Ice+ colonies were then molecularly identified based on the cts (citrate synthase) gene and the 16S rDNA gene. Phylogenetic analysis of cts sequences showed a surprising diversity of phylogenetic subgroups of P. syringae . Frequencies of Ice+ isolates on P. syringae selective medium ranged from 0 to 15% per sample with the highest frequency being found in spring. Our work shows that freshwater lakes can be a significant reservoir of Ice+ P. syringae . Future work is needed to determine the contribution of P. syringae from freshwater lakes to the P. syringae populations present in the atmosphere and on plants and, in particular, if freshwater lakes could be an inoculum source of P. syringae -caused plant disease outbreaks.

  9. Screening of latent tuberculosis infection among health care workers working in Hajj pilgrimage area in Saudi Arabia, using interferon gamma release assay and tuberculin skin test.

    Science.gov (United States)

    Bukhary, Zakeya A; Amer, Soliman M; Emara, Magdy M; Abdalla, Mohammad E; Ali, Sahar A

    2018-01-01

    Interferon gamma release assays (IGRA) is highly specific for Mycobacterium tuberculosis and is the preferred test in BCG-vaccinated individuals. The few studies that have screened health care workers (HCWs) in Saudi Arabia for latent tuberculosis infection (LTBI) using IGRA have varied in agreement with the traditional tuberculin skin test (TST). Assess the prevalence of LTBI among HCWs working in the Hajj pilgrimage using IGRA and TST and measuring their agreement. Cross-sectional prospective. Multiple non-tertiary care hospitals. HCWs who worked during the Hajj pilgrimage in Saudi Arabia in December 2015. Data was collected by standarized questionnaire. Samples were drawn and analyzed by standard methods. The prevalence of LTBI among HCW and the agreement by kappa statistic between QFT-GIT and TST. 520 subjects. Nurses accounted for 30.7% of the sample and physicians, 19.2%. The majority were BCG vaccinated (98.5%). There were a total of 56 positive by QFT-GIT and the LTBI rate was 10.8%. In 50 QFT positive/476 TST negative the LTBI rate was 10.5% in discordant tests, and in 6 QFT positive/44 TST positive it was 13.6% in concordant tests. The overall agreement between both tests was poor-83% and kappa was 0.02. LTBI prevalence was associated with longer employment (13.1 [9.2] years). The QFT-GIT positive test was significantly higher in physicians (P=.02) and in HCWs working in chest hospitals 16/76 (21.05%) (P=.001). Agreement between the tests was poor. QFT-GIT detected LTBI when TST was negative in HCWs who had a history of close contact with TB patients. A second step TST was not feasible within 2-3 weeks. None.

  10. Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples

    Energy Technology Data Exchange (ETDEWEB)

    Creusot, Nicolas; Kinani, Said; Maillot-Marechal, Emmanuelle; Porcher, Jean-Marc; Ait-Aissa, Selim [Unite Ecotoxicologie, INERIS, Verneuil-en-Halatte (France); Balaguer, Patrick [IRCM-UM1-CRLC Val d' Aurelle, INSERM U896, Montpellier (France); Tapie, Nathalie; LeMenach, Karyn; Budzinski, Helene [ISM/LPTC-UMR 5255 CNRS Universite Bordeaux 1, Talence (France)

    2010-01-15

    Many environmental endocrine-disrupting compounds act as ligands for nuclear receptors. Among these receptors, the human pregnane X receptor (hPXR) is well described as a xenobiotic sensor to various classes of chemicals, including pharmaceuticals, pesticides, and steroids. To assess the potential use of PXR as a sensor for aquatic emerging pollutants, we employed an in vitro reporter gene assay (HG5LN-hPXR cells) to screen a panel of environmental chemicals and to assess PXR-active chemicals in (waste) water samples. Of the 57 compounds tested, 37 were active in the bioassay and 10 were identified as new PXR agonists: triazin pesticides (promethryn, terbuthryn, terbutylazine), pharmaceuticals (fenofibrate, bezafibrate, clonazepam, medazepam) and non co-planar polychlorobiphenyls (PCBs; PCB101, 138, 180). Furthermore, we detected potent PXR activity in two types of water samples: passive polar organic compounds integrative sampler (POCIS) extracts from a river moderately impacted by agricultural and urban inputs and three effluents from sewage treatment works (STW). Fractionation of POCIS samples showed the highest PXR activity in the less polar fraction, while in the effluents, PXR activity was mainly associated with the dissolved water phase. Chemical analyses quantified several PXR-active substances (i.e., alkylphenols, hormones, pharmaceuticals, pesticides, PCBs, bisphenol A) in POCIS fractions and effluent extracts. However, mass-balance calculations showed that the analyzed compounds explained only 0.03% and 1.4% of biological activity measured in POCIS and STW samples, respectively. In effluents, bisphenol A and 4-tert-octylphenol were identified as main contributors of instrumentally derived PXR activities. Finally, the PXR bioassay provided complementary information as compared to estrogenic, androgenic, and dioxin-like activity measured in these samples. This study shows the usefulness of HG5LN-hPXR cells to detect PXR-active compounds in water samples

  11. Gentamicin in Pseudomonas aeruginosa

    African Journals Online (AJOL)

    infections by Ps. aeruginosa is contra-indicated. In our study only 2,3 % of the Ps. aeruginosa strains were resistant to gentamicin (MIC 25 Ilg/ml). In view of the synergy reported for combined gentamicin and carbeni- cillin therapy," a combination of these two drugs may be recommended in the treatment of all Pseudomonas.

  12. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

    Science.gov (United States)

    Rahme, Laurence G.; Tan, Man-Wah; Le, Long; Wong, Sandy M.; Tompkins, Ronald G.; Calderwood, Stephen B.; Ausubel, Frederick M.

    1997-01-01

    We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis. PMID:9371831

  13. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident

    Science.gov (United States)

    Semenova, Vera A.; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from −4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = −0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. PMID:27814939

  14. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

    Science.gov (United States)

    Semenova, Vera A; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. Published by Elsevier Ltd.

  15. Isolation and identification of Pseudomonas azotoformans for induced calcite precipitation.

    Science.gov (United States)

    Heidari Nonakaran, Siamak; Pazhouhandeh, Maghsoud; Keyvani, Abdullah; Abdollahipour, Fatemeh Zahra; Shirzad, Akbar

    2015-12-01

    Biomineralization is a process by which living organisms produce minerals. The extracellular production of these biominerals by microbes has potential for various bioengineering applications. For example, crack remediation and improvement of durability of concrete is an important goal for engineers and biomineral-producing microbes could be a useful tool in achieving this goal. Here we report the isolation, biochemical characterization and molecular identification of Pseudomonas azotoformans, a microbe that produces calcite and which potentially be used to repair cracks in concrete structures. Initially, 38 bacterial isolates were isolated from soil and cements. As a first test, the isolates were screened using a urease assay followed by biochemical tests for the rate of urea hydrolysis, calcite production and the insolubility of calcite. Molecular amplification and sequencing of a 16S rRNA fragment of selected isolates permitted us to identify P. azotoformans as a good candidate for preparation of biotechnological concrete. This species was isolated from soil and the results show that among the tested isolates it had the highest rate of urea hydrolysis, produced the highest amount of calcite, which, furthermore was the most adhesive and insoluble. This species is thus of interest as an agent with the potential ability to repair cracks in concrete.

  16. Extracellular toxins of pseudomonas aeruginosa. Pt. 4

    International Nuclear Information System (INIS)

    Obernesser, H.J.; Doering, G.

    1982-01-01

    A sensitive and specific solid phase radioimmunoassay (RIA) for detection of the elastase (Ela) of Pseudomonas aeruginosa (PA) was developed and the RIA was used to assay 10 PA strains of various origin and serotype. A great strain variability of Ela production was found which different from 94.1 to 0.1 μg per ml of culture supernatant fluid (CSF). The Ela and alkaline protease (AP) concentrations were converted to proteolytic activity and combined. The sum of the calculated enzymatic values of Ela and AP correlated well with the experimentally determined values of total proteolytic activity of the CSF. (orig.) [de

  17. Occurrence and Distribution of Pesticides in the St. Lucie River Watershed, South-Central Florida, 2000-01, Based on Enzyme-Linked Immunosorbent Assay (ELISA) Screening

    Science.gov (United States)

    Lietz, A.C.

    2003-01-01

    The St. Lucie River watershed is a valuable estuarine ecosystem and resource in south-central Florida. The watershed has undergone extensive changes over the last century because of anthropogenic activities. These activities have resulted in a complex urban and agricultural drainage network that facilitates the transport of contaminants, including pesticides, to the primary canals and then to the estuary. Historical data indicate that aquatic life criteria for selected pesticides have been exceeded. To address this concern, a reconnaissance was conducted to assess the occurrence and distribution of selected pesticides within the St. Lucie River watershed. Numerous water samples were collected from 37 sites among various land-use categories (urban/built-up, citrus, cropland/pastureland, and inte-grated). Samples were collected at inflow points to primary canals (C-23, C-24, and C-44) and at control structures along these canals from October 2000 to September 2001. Samples were screened for four pesticide classes (triazines, chloroacetanilides, chlorophenoxy compounds, and organophosphates) by using Enzyme-Linked Immunosorbent Assay (ELISA) screening. A temporal distribution of pesticides within the watershed was made based on samples collected at the integrated sites during different rainfall events between October 2000 and September 2001. Triazines were detected in 32 percent of the samples collected at the integrated sites. Chloroacetanilides were detected in 60 percent of the samples collected at the integrated sites, with most detections occurring at one site. Chlorophenoxy compounds were detected in 17 percent of the samples collected at the integrated sites. Organophosphates were detected in only one sample. A spatial distribution and range of concentration of pesticides at the 37 sampling sites in the watershed were determined among land-use categories. Triazine concentrations ranged from highest to lowest in the citrus, urban/built-up, and integrated areas

  18. Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

    Science.gov (United States)

    Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping

    2011-01-01

    Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121

  19. Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry

    NARCIS (Netherlands)

    Jonker, W.; Lamoree, M.H.; Houtman, C.J.; Hamers, T.; Somsen, G.W.; Kool, J.

    2015-01-01

    In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was

  20. Reexamining intra and extracellular metabolites produced by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Maria Shuja

    2016-02-01

    Full Text Available Objective: To isolate, screen and analyze bacteria from different areas of Pakistan for the production of antimicrobial compounds, zinc solubilization and bioplastic production. Methods: Isolation and purification was proceeding with streak plate method. Antagonistic assay was completed with well diffusion and thin-layer chromatography. In vivo analysis of bioplastic was analyzed with Nile blue fluorescence under UV and Sudan staining. Results: A total of 18 bacterial strains purified from soil samples while 148 strains form stock cultures were used. Out of 166 only 94 showed antimicrobial activity against each of Grampositive and Gram-negative; cocci and rods. In case of heavy metal (ZnO and Zn3(PO42.4H2O solubilization, 54 strains solubilized ZnO and 23 strains solubilized Zn3(PO42.4H2O, while 127 strains grown on polyhydroxyalkanoate detection meedia supplemented with Nile blue medium showed bioplastic production by producing fluorescence under UV light. Four bacterial strains (coded as 100, 101, 104 and 111 were selected for further characterization. Induction time assay showed that strains 101, 104, and 111 showed inhibitory activity after 4 h of incubation while strain 100 showed after 8 h. All four strains were tolerable to the maximum concentration of ZnO. Amplified products of both 16S rRNA and PhaC gene fragments of strain 111 were sequenced and submitted to GenBank as accession numbers EU781525 and EU781526. Conclusions: Bacterial strain Pseudomonas aeruginosa-111 has potential to utilize as biofertilize and bioplastic producer.

  1. Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp.

    Science.gov (United States)

    Federico, Baruzzi; Pinto, Loris; Quintieri, Laura; Carito, Antonia; Calabrese, Nicola; Caputo, Leonardo

    2015-12-23

    The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfected fresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptides in controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolates were collected from ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonas media, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas putida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was found when Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarole chicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papain and rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability. As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using the peptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced (P ≤ 0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTE lettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable cell density in the inoculated wounds. This is the first paper providing direct evidence regarding the application of an antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage.

  2. Combined inoculation of Pseudomonas fluorescens and Trichoderma harzianum for enhancing plant growth of vanilla (Vanilla planifolia).

    Science.gov (United States)

    Sandheep, A R; Asok, A K; Jisha, M S

    2013-06-15

    This study was conducted to evaluate the plant growth promoting efficiency of combined inoculation of rhizobacteria on Vanilla plants. Based on the in vitro performance of indigenous Trichoderma spp. and Pseudomonas spp., four effective antagonists were selected and screened under greenhouse experiment for their growth enhancement potential. The maximum percentage of growth enhancement were observed in the combination of Trichoderma harzianum with Pseudomonas fluorescens treatment followed by Pseudomonas fluorescens, Trichoderma harzianum, Pseudomonas putida and Trichoderma virens, respectively in decreasing order. Combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens registered the maximum length of vine (82.88 cm), highest number of leaves (26.67/plant), recorded the highest fresh weight of shoots (61.54 g plant(-1)), fresh weight of roots (4.46 g plant(-1)) and dry weight of shoot (4.56 g plant(-1)) where as the highest dry weight of roots (2.0806 g plant(-1)) were achieved with treatments of Pseudomonas fluorescens. Among the inoculated strains, combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens recorded the maximum nitrogen uptake (61.28 mg plant(-1)) followed by the combined inoculation of Trichoderma harzianum (std) and Pseudomonas fluorescens (std) (55.03 mg plant(-1)) and the highest phosphorus uptake (38.80 mg plant(-1)) was recorded in dual inoculation of Trichoderma harzianum and Pseudomonas fluorescens.

  3. Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

    Science.gov (United States)

    Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

    2015-05-01

    In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  4. Evaluation of mericon E. coli O157 Screen Plus and mericon E. coli STEC O-Type Pathogen Detection Assays in Select Foods: Collaborative Study, First Action 2017.05.

    Science.gov (United States)

    Bird, Patrick; Benzinger, M Joseph; Bastin, Benjamin; Crowley, Erin; Agin, James; Goins, David; Armstrong, Marcia

    2018-05-01

    QIAGEN mericon Escherichia coli O157 Screen Plus and mericon E. coli Shiga toxin-producing E. coli (STEC) O-Type Pathogen Detection Assays use Real-Time PCR technology for the rapid, accurate detection of E. coli O157 and the "big six" (O26, O45, O103, O111, O121, O145) (non-O157 STEC) in select food types. Using a paired study design, the assays were compared with the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook Chapter 5.09 reference method for the detection of E. coli O157:H7 in raw ground beef. Both mericon assays were evaluated using the manual and an automated DNA extraction method. Thirteen technicians from five laboratories located within the continental United States participated in the collaborative study. Three levels of contamination were evaluated. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference between laboratories POD (dLPOD) value with a 95% confidence interval of 0.00 (-0.12, 0.12) for the mericon E. coli O157 Screen Plus with manual and automated extraction and mericon E. coli STEC O-Type with manual extraction and -0.01 (-0.13, 0.10) for the mericon E. coli STEC O-Type with automated extraction. The dLPOD results indicate equivalence between the candidate methods and the reference method.

  5. A microfluidic device for rapid screening of E. coli O157:H7 based on IFAST and ATP bioluminescence assay for water analysis

    CSIR Research Space (South Africa)

    Ngamsom, B

    2017-08-01

    Full Text Available We present a simple microfluidic system for rapid screening of Escherichia coli (E. coli) O157:H7 employing the specificity of immunomagnetic separation (IMS) via immiscible filtration assisted by surface tension (IFAST), and the sensitivity...

  6. Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

    NARCIS (Netherlands)

    Perneel, M.; Heyrman, J.; Adiobo, A.; Maeyer, de K.; Raaijmakers, J.M.; Vos, de P.; Höfte, M.

    2007-01-01

    Aim: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. Material and Results: Forty pseudomonads were isolated from the rhizosphere

  7. Prevention of Post Transfusion Hepatitis Employing Sensitive Assay for Hepatitis B Surface Antigen Screening(Topics in Transfusion Medicine 1990 : Autologous Transfusion and Post-Transfusion Hepatitis)

    OpenAIRE

    小島, 秀男; 大竹, 幸子; 富樫, 和枝; 石口, 重子; 山田, 恵子; 品田, 章二; Kojima, Hideo; Ohtake, Sachiko; Togashi, Kazue; Ishiguchi, Shigeko; Yamada, Keiko; Shinada, Shoji

    1990-01-01

    Post transfusion Hepatitis (PTH) is one of serious side effects and some times lead to fulminant hepatic failure in case transfused blood contain very low level (under the sensitivity of usual screening method) of hepatitis B virus (HBV). Redcross blood center and blood transfusion devision of our hospital have been employed reverse passive hemmaglutination method (RPHA) for HBsAg screening. Authors employed EIA for sensitive HBsAg test system and compared with RPHA method. Of 2,255 sera from...

  8. Toxicity screening of soils from different mine areas—A contribution to track the sensitivity and variability of Arthrobacter globiformis assay

    Energy Technology Data Exchange (ETDEWEB)

    Marques, Catarina R., E-mail: crmarques@ua.pt [Departamento de Biologia and CESAM (Centro de Estudos do Ambiente e do Mar), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro (Portugal); Caetano, Ana L. [Departamento de Biologia and CESAM (Centro de Estudos do Ambiente e do Mar), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro (Portugal); Haller, Andreas [ECT Oekotoxikologie GmbH, Böttgerstraße 2–14, D-65439 Flörsheim a. M. (Germany); Gonçalves, Fernando [Departamento de Biologia and CESAM (Centro de Estudos do Ambiente e do Mar), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro (Portugal); Pereira, Ruth [Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto (Portugal); Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Römbke, Jörg [ECT Oekotoxikologie GmbH, Böttgerstraße 2–14, D-65439 Flörsheim a. M. (Germany)

    2014-06-01

    Highlights: • The assay gave rapid and feasible discrimination of toxic soils to A. globiformis. • Sensitive and low variability response to soils from different regions. • Soil properties may interfere with metal toxicity and fluorescence measurements. • Proposal of a toxicity threshold for the contact assay regarding soils. • A. globiformis assay should be included in the Tier I of risk assessment frameworks. - Abstract: This study used the Arthrobacter globiformis solid-contact test for assessing the quality of soils collected in areas subjected to past and present mine activities in Europe (uranium mine, Portugal) and North Africa (phosphogypsum pile, Tunisia; iron mine, Morocco). As to discriminate the influence of soils natural variability from the effect of contaminants, toxicity thresholds were derived for this test, based on the dataset of each study area. Furthermore, the test sensitivity and variability was also evaluated. As a result, soils that inhibited A. globiformis dehydrogenase activity above 45% or 50% relatively to the control, were considered to be toxic. Despite the soil metal content determined, the properties of soils seemed to influence dehydrogenase activity. Overall, the contact test provided a coherent outcome comparing to other more time-consuming and effort-demanding ecotoxicological assays. Our results strengthened the feasibility and ecological relevance of this assay, which variability was quite reduced hence suggesting its potential integration within the test battery of tier 1 of soil risk assessment schemes.

  9. Toxicity screening of soils from different mine areas—A contribution to track the sensitivity and variability of Arthrobacter globiformis assay

    International Nuclear Information System (INIS)

    Marques, Catarina R.; Caetano, Ana L.; Haller, Andreas; Gonçalves, Fernando; Pereira, Ruth; Römbke, Jörg

    2014-01-01

    Highlights: • The assay gave rapid and feasible discrimination of toxic soils to A. globiformis. • Sensitive and low variability response to soils from different regions. • Soil properties may interfere with metal toxicity and fluorescence measurements. • Proposal of a toxicity threshold for the contact assay regarding soils. • A. globiformis assay should be included in the Tier I of risk assessment frameworks. - Abstract: This study used the Arthrobacter globiformis solid-contact test for assessing the quality of soils collected in areas subjected to past and present mine activities in Europe (uranium mine, Portugal) and North Africa (phosphogypsum pile, Tunisia; iron mine, Morocco). As to discriminate the influence of soils natural variability from the effect of contaminants, toxicity thresholds were derived for this test, based on the dataset of each study area. Furthermore, the test sensitivity and variability was also evaluated. As a result, soils that inhibited A. globiformis dehydrogenase activity above 45% or 50% relatively to the control, were considered to be toxic. Despite the soil metal content determined, the properties of soils seemed to influence dehydrogenase activity. Overall, the contact test provided a coherent outcome comparing to other more time-consuming and effort-demanding ecotoxicological assays. Our results strengthened the feasibility and ecological relevance of this assay, which variability was quite reduced hence suggesting its potential integration within the test battery of tier 1 of soil risk assessment schemes

  10. Degradation of polynuclear aromatic hydrocarbons by two strains of Pseudomonas.

    Science.gov (United States)

    Nwinyi, Obinna C; Ajayi, Oluseyi O; Amund, Olukayode O

    2016-01-01

    The goal of this investigation was to isolate competent polynuclear aromatic hydrocarbons degraders that can utilize polynuclear aromatic hydrocarbons of former industrial sites at McDoel Switchyard in Bloomington, Indiana. Using conventional enrichment method based on soil slurry, we isolated, screened and purified two bacterial species strains PB1 and PB2. Applying the ribotyping technique using the 16S rRNA gene analysis, the strains were assigned to the genus Pseudomonas (Pseudomonas plecoglossicida strain PB1 and Pseudomonas sp. PB2). Both isolates showed promising metabolic capacity on pyrene sprayed MS agar plates during the preliminary investigations. Using time course studies in the liquid cultures at calculated concentrations 123, 64, 97 and 94ppm for naphthalene, chrysene, fluroanthene and pyrene, P. plecoglossicida strain PB1 and Pseudomonas sp. PB2 showed partial utilization of the polynuclear aromatic hydrocarbons. Naphthalene was degraded between 26% and 40%, chrysene 14% and 16%, fluroanthene 5% and 7%; pyrene 8% and 13% by P. plecoglossicida strain PB1 and Pseudomonas sp. PB2 respectively. Based on their growth profile, we developed a model R(2)=1 to predict the degradation rate of slow polynuclear aromatic hydrocarbon-degraders where all the necessary parameters are constant. From this investigation, we confirm that the former industrial site soil microbial communities may be explored for the biorestoration of the industrial site. Copyright © 2016. Published by Elsevier Editora Ltda.

  11. Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Mullasseril, Praseeda; Dawit, Sara

    2010-01-01

    N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe...... a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal...

  12. Indole-3-acetic acid (IAA) producing Pseudomonas isolates inhibit seed germination and α-amylase activity in durum wheat (Triticum turgidum L.)

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabaei, S.; Ehsanzadeh, P.; Etesami, H.; Alikhani, H.A.; Glick, B.R.

    2016-11-01

    The role of plant-associated bacteria in plant physiology and metabolism is well documented, but little has been known about the roles played by Pseudomonas in durum wheat (Triticum turgidum L. var durum) growth and development. An in vitroexperiment was conducted to observe the effect of the inoculation of four indole-3-acetic acid (IAA)-producing Pseudomonas isolates and exogenous IAA on seed germination traits and α-amylase activity of durum wheat. The results showed inoculation with all bacterial isolates led to a decrease in the germination percent, although the extent of the depression varied with the isolate. A significant relationship between concentrations of bacterial IAA and the germination inhibition percent in durum wheat seeds by different bacteria strains was observed. The results of this assay showed the effect of bacterial isolates on α-amylase activity after six and 8 days of inoculation was significant, while effect of these isolates on α-amylase activity after two and 4 days of inoculation was not meaningful. In addition, the exogenously applied IAA displayed a concentration-dependent effect on seed germination attributes and α-amylase activity, consistent with the possibility that the inhibitory effect of bacterial inoculation on seed germination was in consequence of bacteria-produced IAA. Therefore, it may suggested that the inhibitory role of IAA in seed germination and α-amylase activity should be taken into account during the screening of IAA-producing Pseudomonas isolates for durum wheat growth promoting agents. (Author)

  13. Assessment of a New Lower-Cost Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus: Useful for Cervical Screening in Limited-Resource Settings?

    Science.gov (United States)

    Fokom Domgue, Joel; Schiffman, Mark; Wentzensen, Nicolas H; Gage, Julia C; Castle, Philip E; Raine-Bennett, Tina R; Fetterman, Barbara; Lorey, Thomas; Poitras, Nancy E; Befano, Brian; Xie, Yi; Miachon, Lais S; Dean, Michael

    2017-08-01

    Inexpensive and easy-to-perform human papillomavirus (HPV) tests are needed for primary cervical cancer screening in lower-resource regions. In a convenience sample of 516 residual exfoliative cervical specimens from the Kaiser Permanente Northern California and U.S. National Cancer Institute Persistence and Progression Study, we assessed the agreement and clinical performance of a simple, inexpensive real-time PCR assay for the detection of 13 carcinogenic HPV types (the H13 assay; Hybribio, Hong Kong) that is marketed in limited-resource settings compared to previous testing by the Hybrid Capture 2 assay (HC2; Qiagen, Germantown, MD) and the Onclarity assay (BD Diagnostics, Sparks, MD). The test set was chosen to include many HPV-positive specimens. The reference standard was a combination of HC2 and Onclarity results for HPV detection and histologic diagnosis of controls (less than cervical intraepithelial neoplasia grade 2 [screening in limited-resource settings. Copyright © 2017 American Society for Microbiology.

  14. Feeding behaviour of Caenorhabditis elegans is an indicator of Pseudomonas aeruginosa PAO1 virulence

    Directory of Open Access Journals (Sweden)

    Shawn Lewenza

    2014-08-01

    Full Text Available Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secretion (T3S system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced virulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.

  15. Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil.

    Science.gov (United States)

    Frasson, David; Opoku, Michael; Picozzi, Tara; Torossi, Tanja; Balada, Stefanie; Smits, Theo H M; Hilber, Urs

    2017-08-01

    Within the frame of a biotechnological screening, we isolated two Pseudomonas strains from forest soil. 16S rRNA gene sequence analysis indicated that strain CCOS 864T shared 99.8 % similarity with Pseudomonas donghuensis HYST, while strain CCOS 865T shared 99.0 % similarity with Pseudomonas putida DSM 291T and lower similarity with other P. putida group type strains. Based on multilocus sequence analysis, the two strains were genotypically distinct from each other, each forming a separate clade. Strains CCOS 864T and CCOS 865T were Gram-stain-negative, motile and rod-shaped, growing at a temperature range of 4-37 °C. Strain CCOS 864T could be phenotypically distinguished from P. putida group species by the combination of gelatinase-positive reaction and positive growth on N-acetyl-d-glucosamine, p-hydroxyphenylacetic acid and inosine but lack of fluorescein production on King's B medium, while strain CCOS 865T could be distinguished from P. putida group species by the combination of positive growth with saccharic acid and negative growth with p-hydroxyphenylacetic acid and l-pyroglutamic acid. The major polar lipid for both strains was phosphatidylethanolamine; the major quinone was ubiquinone Q-9. DNA-DNA hybridization and average nucleotide identities confirmed the novel species status for the two strains. The DNA G+C contents of CCOS 864T and CCOS 865T were 62.1 and 63.8 mol%, respectively. The phenotypic, phylogenetic and DNA-DNA relatedness data support the suggestion that CCOS 864T and CCOS 865T represent two novel Pseudomonas species. The names Pseudomonas wadenswilerensis sp. nov. (type strain CCOS 864T=LMG 29327T) and Pseudomonas reidholzensis sp. nov. (type strain CCOS 865T=LMG 29328T) are proposed.

  16. Fiscal 2000 research and development of technologies for intelligent infrastructure creation and utilization. Development of high-precision screening assay system for endocrine disrupting chemicals and construction of database; 2000 nendo chiteki kiban sose riyo gijutsu kenkyu kaihatsu. Naibunpi kakuran busshitsu no koseido screening shiken hoho no kaihatsu oyobi data kiban seibi

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Research and development is carried out for the establishment of a system for assaying endocrine disruption now presenting itself as a hazard and for the application of the assay system to important chemicals and chemicals under development for the detection of presence of endocrine disrupting action in them. In this fiscal year, for the establishment of a reporter-gene assay system, cell characteristics were clarified through the determination of the expression sequence of the produced cell, the determination of the stable conservation period of the cell, and the study of the sustenance of activity. Studies were conducted, targeted at the stable supply of cells usable for high-throughput screening. Basic data were collected for the Hershberger assay being developed by OECD (Organization for Economic Cooperation and Development) and for the repeated administration test method for assaying thyroid hormone action by use of pubertal rats. For the assay of impact on environmental organisms, moreover, basic data were collected for the establishment of testing methods using fishes and amphibians. (NEDO)

  17. A Fast, Sensitive and Accurate High Resolution Melting (HRM Technology-Based Assay to Screen for Common K-ras Mutations

    Directory of Open Access Journals (Sweden)

    D. Kramer

    2009-01-01

    Full Text Available Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.

  18. Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa.

    OpenAIRE

    Trias, J; Dufresne, J; Levesque, R C; Nikaido, H

    1989-01-01

    The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa was shown to have decreased permeability to imipenem but not to cephaloridine. These experiments were performed with intact cells and liposomes containing imipenem-hydrolyzing beta-lactamase derived from Pseudomonas maltophilia, in both cases utilizing an imipenem concentration of 50 microM. In contrast, liposome swelling assays using imipenem at 8 mM detected no significant difference between the imipenem-resistant mu...

  19. Biosurfactant production by Pseudomonas strains isolated from floral nectar.

    Science.gov (United States)

    Ben Belgacem, Z; Bijttebier, S; Verreth, C; Voorspoels, S; Van de Voorde, I; Aerts, G; Willems, K A; Jacquemyn, H; Ruyters, S; Lievens, B

    2015-06-01

    To screen and identify biosurfactant-producing Pseudomonas strains isolated from floral nectar; to characterize the produced biosurfactants; and to investigate the effect of different carbon sources on biosurfactant production. Four of eight nectar Pseudomonas isolates were found to produce biosurfactants. Phylogenetic analysis based on three housekeeping genes (16S rRNA gene, rpoB and gyrB) classified the isolates into two groups, including one group closely related to Pseudomonas fluorescens and another group closely related to Pseudomonas fragi and Pseudomonas jessenii. Although our nectar pseudomonads were able to grow on a variety of water-soluble and water-immiscible carbon sources, surface active agents were only produced when using vegetable oil as sole carbon source, including olive oil, sunflower oil or waste frying sunflower oil. Structural characterization based on thin layer chromatography (TLC) and ultra high performance liquid chromatography-accurate mass mass spectrometry (UHPLC-amMS) revealed that biosurfactant activity was most probably due to the production of fatty acids (C16:0; C18:0; C18:1 and C18:2), and mono- and diglycerides thereof. Four biosurfactant-producing nectar pseudomonads were identified. The active compounds were identified as fatty acids (C16:0; C18:0; C18:1 and C18:2), and mono- and diglycerides thereof, produced by hydrolysis of triglycerides of the feedstock. Studies on biosurfactant-producing micro-organisms have mainly focused on microbes isolated from soils and aquatic environments. Here, for the first time, nectar environments were screened as a novel source for biosurfactant producers. As nectars represent harsh environments with high osmotic pressure and varying pH levels, further screening of nectar habitats for biosurfactant-producing microbes may lead to the discovery of novel biosurfactants with broad tolerance towards different environmental conditions. © 2015 The Society for Applied Microbiology.

  20. Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions

    Directory of Open Access Journals (Sweden)

    Uppalapati Srinivasa R

    2011-10-01

    Full Text Available Abstract Background The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens. Results In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1 the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS and phytotoxin coronatine (COR; 2 the effector-triggered immunity; and 3 Arabidopsis mutants affected in salicylic acid (SA- and pathogen-associated molecular pattern (PAMPs-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2 in NHR. Conclusions The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to

  1. Tools for the identification of bioactives impacting the metabolic syndrome: Screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell based assays

    Science.gov (United States)

    Buehrer, Benjamin M.; Duffin, David J.; Lea-Currie, Y. Renee; Ribnicky, David; Raskin, Ilya; Stephens, Jacqueline M.; Cefalu, William T.; Gimble, Jeffrey M.

    2011-01-01

    Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis. PMID:21543201

  2. Validation of the AlamarBlue® assay as a fast screening method to determine the antimicrobial activity of botanical extracts

    NARCIS (Netherlands)

    Tyc, O.; Tomás-Menor, L.; Garbeva, P.V.; Barrajón-Catalán, E.; Micol, V.

    2016-01-01

    Plant compounds are a potential source of new antimicrobial molecules against a variety of infections. Plant extracts suppose complex phytochemical libraries that may be used for the first stages of the screening process for antimicrobials. However, their large variability and complexity require

  3. Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

    2016-04-19

    A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

  4. Using the Larval Zebrafish Locomotor Assay in Functional Neurotoxicity Screening: Light Intensity and the Order of Stimulus Presentation Affect the Outcome

    Science.gov (United States)

    The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals using 6 day old zebrafish (Danio rerio) as an alternative test model for detecting neurotoxic chemicals. We use a behavioral testing paradigm that simultaneously tes...

  5. Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer

    DEFF Research Database (Denmark)

    Burford, B; Gentry-Maharaj, A; Graham, R

    2013-01-01

    Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoanti...

  6. Pseudomonas aeruginosa biofilm infections

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim

    2014-01-01

    Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self-produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display...... a remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device-associated infections, and chronic infections in individuals who are immune-compromised or otherwise impaired in the host defense. Because...... the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...

  7. Screening for simian foamy virus infection by using a combined antigen Western blot assay: evidence for a wide distribution among Old World primates and identification of four new divergent viruses

    International Nuclear Information System (INIS)

    Hussain, Althaf I.; Shanmugam, Vedapuri; Bhullar, Vinod B.; Beer, Brigitte E.; Vallet, Dominique; Gautier-Hion, Annie; Wolfe, Nathan D.; Karesh, William B.; Kilbourn, Annelisa M.; Tooze, Zeena; Heneine, Walid; Switzer, William M.

    2003-01-01

    Simian foamy viruses (SFVs) belong to a genetically and antigenically diverse class of retroviruses that naturally infect a wide range of nonhuman primates (NHPs) and can also be transmitted to humans occupationally exposed to NHPs. Current serologic detection of SFV infection requires separate Western blot (WB) testing by using two different SFV antigens [SFV AGM (African green monkey) and SFV CPZ (chimpanzee)]. However, this method is labor intensive and validation is limited to only small numbers of NHPs. To facilitate serologic SFV testing, we developed a WB assay that combines antigens from both SFV AGM and SFV CPZ . The combined-antigen WB (CA-WB) assay was validated with 145 serum samples from 129 NHPs (32 African and Asian species) and 16 humans, all with known SFV infection status determined by PCR. Concordant CA-WB results were obtained for all 145 PCR-positive or -negative primate and human specimens, giving the assay a 100% sensitivity and specificity. In addition, no reactivity was observed in sera from persons positive for human immunodeficiency virus or human T cell lymphotropic virus (HIV/HTLV) (n = 25) or HIV/HTLV-negative U.S. blood donors (n = 100). Using the CA-WB assay, we screened 360 sera from 43 Old World primate species and found an SFV prevalence of about 68% in both African and Asian primates. We also isolated SFV from the blood of four seropositive primates (Allenopithecus nigroviridis, Trachypithecus francoisi, Hylobates pileatus, and H. leucogenys) not previously known to be infected with SFV. Phylogenetic analysis of integrase sequences from these isolates confirmed that all four SFVs represent new, distinct, and highly divergent lineages. These results demonstrate the ability of the CA-WB assay to detect infection in a large number of NHP species, including previously uncharacterized infections with divergent SFVs

  8. Antimicrobial activity screening of marine bacteria isolated from Port Klang and Port Tanjung Pelepas

    Science.gov (United States)

    Ibrahim, Nik Nuraznida Nik; Usup, Gires; Ahmad, Asmat

    2018-04-01

    Over the past ten years, marine natural product researchers have expanded the scope of their studies from macroorganisms such as algae to marine microorganisms. The marine environment is believed to be able to provide novel lead against pathogenic microbes that are evolving and developing resistance to existing pharmaceutical agents. In this study, a total of 150 bacterial isolates isolated from Port Klang and Port Tanjung Pelepas were screened for antimicrobial activity against Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Entrococcus, faecalis, Pseudomonas aeruginosa and Methicillin-Resistance Staphylococcus aureus (MRSA). Only 10 isolates: PW01, PW02, PB03, and PS (04, 05, 06, 07, 08, 09, and 10) showed strong antibacterial activity. Based on the strongest activity, isolates PW01 and PW02 were selected for secondary screening using well diffusion assay. The dichloromethane extract of Pseudomonas sp. PW01 showed activity against S. aureus (15±0 mm), V. parahaemolyticus (25±1.63 mm) and MRSA (18±0.81 mm). Meanwhile, the diethyl ether extract of Pseudomonas sp. PW02 showed active activity against S. aureus (10±0 mm), V. parahaemolyticus (30±0.94 mm), MRSA (30±0.94 mm), E. coli (22±1.25 mm) and E. faecalis (26±0 mm). Through this study, it was suggested that marine microorganisms may represent an untapped reservoir of biodiversity capable of synthesizing antimicrobial molecules.

  9. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay.

    Science.gov (United States)

    Hördegen, P; Cabaret, J; Hertzberg, H; Langhans, W; Maurer, V

    2006-11-03

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyl-tetrazolium (MTT) reduction assay. Pyrantel tartrate was used as reference anthelmintic. Bromelain, the enzyme complex of the stem of Ananas comosus (Bromeliaceae), the ethanolic extracts of seeds of Azadirachta indica (Meliaceae), Caesalpinia crista (Caesalpiniaceae) and Vernonia anthelmintica (Asteraceae), and the ethanolic extracts of the whole plant of Fumaria parviflora (Papaveraceae) and of the fruit of Embelia ribes (Myrsinaceae) showed an anthelmintic efficacy of up to 93%, relative to pyrantel tartrate. Based on these results obtained with larval Haemonchus contortus, the modified MTT reduction assay could be a possible method for testing plant products with anthelmintic properties.

  10. Development of a genotype-by-sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure.

    Science.gov (United States)

    Donaldson, Michael E; Rico, Yessica; Hueffer, Karsten; Rando, Halie M; Kukekova, Anna V; Kyle, Christopher J

    2018-01-01

    Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing (HTS) technologies, such as genotype-by-sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization-based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to "capture" specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300-kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on-target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer-read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune-related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation

  11. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay

    OpenAIRE

    Hördegen, P.; Cabaret, J.; Hertzberg, H.; Langhans, W.; Maurer, V.

    2006-01-01

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyltetrazolium (MTT) reduction assay. Pyrantel tartrate was u...

  12. Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors

    Science.gov (United States)

    Seol, Yeonee; Zhang, Hongliang; Agama, Keli; Lorence, Nicholas; Pommier, Yves; Neuman, Keir C.

    2015-01-01

    Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anti-cancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors. PMID:26351326

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

    Czech Academy of Sciences Publication Activity Database

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

    2007-01-01

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

  14. ANTAGONISTIC POTENTIAL OF FLUORESCENT Pseudomonas ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    GROWTH OF TOMATO CHALLENGED WITH PHTOPATHOGENS ... This study focused on the antagonistic potential of fluorescent Pseudomonas in vitro, and its inoculation effect on growth .... the 5 days old culture in starch agar with Lugol's.

  15. Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

    Science.gov (United States)

    Rajkumar, M; Lee, Wang Hyu; Lee, Kui Jae

    2005-01-01

    The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici. ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

  16. Pesticide tolerant and phosphorus solubilizing Pseudomonas sp. strain SGRAJ09 isolated from pesticides treated Achillea clavennae rhizosphere soil.

    Science.gov (United States)

    Rajasankar, R; Manju Gayathry, G; Sathiavelu, A; Ramalingam, C; Saravanan, V S

    2013-05-01

    In this study, an attempt was made to identify an effective phosphate solubilizing bacteria from pesticide polluted field soil. Based on the formation of solubilization halo on Pikovskaya's agar, six isolates were selected and screened for pesticide tolerance and phosphate (P) solubilization ability through liquid assay. The results showed that only one strain (SGRAJ09) obtained from Achillea clavennae was found to tolerate maximum level of the pesticides tested and it was phylogenetically identified as Pseudomonas sp. It possessed a wide range of pesticide tolerance, ranging from 117 μg mL(-1) for alphamethrin to 2,600 μg mL(-1) for endosulfan. The available P concentrations increased with the maximum and double the maximum dose of monocrotophos and imidacloprid, respectively. On subjected to FT-IR and HPLC analysis, the presence of organic acids functional group in the culture broth and the production of gluconic acid as dominant acid aiding the P solubilization were identified. On comparison with control broth, monocrotophos and imidacloprid added culture broth showed quantitatively high organic acids production. In addition to gluconic acid production, citric and acetic acids were also observed in the pesticide amended broth. Furthermore, the Pseudomonas sp. strain SGRAJ09 possessed all the plant growth promoting traits tested. In presence of monocrotophos and imidacloprid, its plant growth promoting activities were lower than that of the pesticides unamended treatment.

  17. Plant-associated fluorescent Pseudomonas from red lateritic soil: Beneficial characteristics and their impact on lettuce growth.

    Science.gov (United States)

    Maroniche, Guillermo A; Rubio, Esteban J; Consiglio, Adrián; Perticari, Alejandro

    2016-11-25

    Fluorescent Pseudomonas are ubiquitous soil bacteria that usually establish mutualistic associations with plants, promoting their growth and health by several mechanisms. This makes them interesting candidates for the development of crop bio-inoculants. In this work, we isolated phosphate-solubilizing fluorescent Pseudomonas from the rhizosphere and inner tissues of different plant species growing in red soil from Misiones, Argentina. Seven isolates displaying strong phosphate solubilization were selected for further studies. Molecular identification by rpoD genotyping indicated that they belong to different species within the P. fluorescens and P. putida phylogenetic groups. Screening for in vitro traits such as phosphate solubilization, growth regulators synthesis or degradation, motility and antagonism against phytopathogens or other bacteria, revealed a unique profile of characteristics for each strain. Their plant growth-promoting potential was assayed using lettuce as a model for inoculation under controlled and greenhouse conditions. Five of the strains increased the growth of lettuce plants. Overall, the strongest lettuce growth promoter under both conditions was strain ZME4, isolated from inner tissues of maize. No clear association between lettuce growth promotion and in vitro beneficial traits was detected. In conclusion, several phosphate solubilizing pseudomonads from red soil were isolated that display a rich array of plant growth promotion traits, thus showing a potential for the development of new inoculants.

  18. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

    DEFF Research Database (Denmark)

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or ...... also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS....

  19. Oak (Quercus frainetto Ten. Honeydew Honey—Approach to Screening of Volatile Organic Composition and Antioxidant Capacity (DPPH and FRAP Assay

    Directory of Open Access Journals (Sweden)

    Igor Jerković

    2010-05-01

    Full Text Available Two samples of oak honeydew honey were investigated. Headspace solid-phase microextraction (HS-SPME combined with GC and GC/MS enabled identification of the most volatile organic headspace compounds being dominated by terpenes(mainly cis- and trans-linalool oxides. The volatile and less-volatile organic composition of the samples was obtained by ultrasonic assisted extraction (USE with two solvents (1:2 (v/v pentane -diethyl ether mixture and dichloromethane followed by GC and GC/MS analysis. Shikimic pathway derivatives are of particular interest with respect to the botanical origin of honey and the most abundant was phenylacetic acid (up to 16.4%. Antiradical activity (DPPH assay of the honeydew samples was 4.5 and 5.1 mmol TEAC/kg. Ultrasonic solvent extracts showed several dozen times higher antiradical capacity in comparison to the honeydew. Antioxidant capacity (FRAP assay of honeydew samples was 4.8 and 16.1 mmol Fe2+/kg, while the solvent mixture extracts showed antioxidant activity of 374.5 and 955.9 Fe2+/kg, respectively, and the dichloromethane extracts 127.3 and 101.5 mmol Fe2+/kg.

  20. Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

    Directory of Open Access Journals (Sweden)

    Sarika Kedar Marathe

    2018-06-01

    Full Text Available In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37 °C for 48 h with a constant agitation of 120 rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1 M ice cold CaCl2 and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60 s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production. Keywords: Alkaline protease, Skim milk agar, Bacillus, Alcaligenes, Pseudomonas, Isoelectric precipitation, Protease activity, Enzyme immobilization, Detergent compatibility

  1. High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening.

    Science.gov (United States)

    Shcherbakova, Elena G; Brega, Valentina; Lynch, Vincent M; James, Tony D; Anzenbacher, Pavel

    2017-07-26

    A simple and efficient method for determination of the yield, enantiomeric/diasteriomeric excess (ee/de), and absolute configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach utilizes a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan methyl ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophysical properties and allow for fluorescence-based ee determination of molecules containing a 1,2- or 1,3-diol moiety. This method has been utilized for the screening of ee in a number of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asymmetric hydrogenation of benzil was performed in a highly parallel fashion with errors products from the parallel asymmetric synthesis in real time and in a high-throughput screening (HTS) fashion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.

    Science.gov (United States)

    Woldemichael, Girma M; Vasselli, James R; Gardella, Roberta S; McKee, Tawnya C; Linehan, W Marston; McMahon, James B

    2006-09-01

    Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

  3. Evaluation of Endocrine Disrupting Effects of Nitrate after In Utero Exposure in Rats and of Nitrate and Nitrite in the H295R and T-Screen Assay

    DEFF Research Database (Denmark)

    Hansen, Pernille Reimer; Taxvig, Camilla; Christiansen, Sofie

    2009-01-01

    /l. At GD21, fetuses were examined for anogenital distance, plasma thyroxine levels, testicular and plasma levels of testosterone and progesterone, and testicular testosterone production and histopathology. In addition, endocrine disrupting activity of nitrate and nitrite were studied in two in vitro assays......Animal studies have shown that nitrate acts as an endocrine disrupter affecting the androgen production in adult males. This raises a concern for more severe endocrine disrupting effects after exposure during the sensitive period of prenatal male sexual development. As there are no existing studies...... of effects of nitrate on male sexual development, the aim of the study was to examine how in utero exposure to nitrate would affect male rat fetuses. Pregnant dams were dosed with nitrate in the drinking water from gestational day (GD) 7 to GD21 at the following dose levels 17.5, 50, 150, 450, and 900 mg...

  4. An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants.

    Science.gov (United States)

    Salazar, Carolina; Armenta, Jenny M; Shulaev, Vladimir

    2012-07-06

    In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The

  5. An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

    Directory of Open Access Journals (Sweden)

    Carolina Salazar

    2012-07-01

    Full Text Available In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the

  6. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    International Nuclear Information System (INIS)

    Wang Lei; Sasai, Ken; Akagi, Tsuyoshi; Tanaka, Shinya

    2008-01-01

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development

  7. Screening of Indian medicinal plants for cytotoxic activity by Brine Shrimp Lethality (BSL assay and evaluation of their total phenolic content

    Directory of Open Access Journals (Sweden)

    Mahesh Biradi

    2014-01-01

    Full Text Available Objective: Plant-derived cytotoxic constituents and polyphenolic compounds have played an important role in the development of clinically useful anticancer agents. In this context, we have selected six Indian medicinal plants based on the literature claims and an attempt was made to evaluate the cytotoxic potential and total phenolic content (TPC of their methanol extracts and fractions. Materials and Methods: Six plants have been selected for the study, namely, Artemisia absinthium Linn. (Asteraceae, Oroxylum indicum (Linn. Vent. (Bignoniaceae, Heliotropium indicum Linn. (Boraginaceae, Amorphophallus sylvaticus (Roxb. Kunth. (Araceae, Mimosa pudica Linn. (Mimosaceae, and Premna serratifolia Linn. (Verbenaceae. Authenticated plant materials were subjected to extraction with methanol by cold maceration and hot percolation methods. The extracts were fractionated into four fractions (F1, F2, F3, and F4. Preliminary phytochemical investigation was carried out for all extracts and fractions. All extracts and their fractions were subjected to cytotoxicity screening by brine shrimp lethality (BSL bioassay. The plants with significant cytotoxicity were evaluated for TPC by using Folin-Ciocalteu reagent. Results: F1, F2, and F3 fractions of A. absinthium and P. serratifolia and F1 fraction of M. pudica have shown significant cytotoxicity (lethal concentration (LC 50 < 100 ppm compared with other fractions. F1, F2, and F3 fractions of A. absinthium show the LC 50 values 32.52, 14.27, and 24.02, respectively; F1, F2, and F3 of P. serratifolia show LC 50 values 7.61, 4.01, and 10.91 and same for F1 fraction of M. pudica was 34.82 μg/ml, respectively. TPC was found to be significantly higher (39.11 mg gallic acid equivalent (GAE/g in P. serratifolia compared with other two plants. Conclusion: The cytotoxicity screening system confirmed the proposed anticancer plants used by traditional healers and literature claims.

  8. Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin.

    Science.gov (United States)

    Matthijs, Sandra; Vander Wauven, Corinne; Cornu, Bertrand; Ye, Lumeng; Cornelis, Pierre; Thomas, Christopher M; Ongena, Marc

    2014-10-01

    Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas a large collection of Pseudomonas strains of worldwide origin, consisting of 117 Pseudomonas type strains and 461 strains isolated from different biological origins, was screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD(+) strains from different geographic and biological origin were identified. They all produced mupirocin and were strongly antagonistic against Staphylococcus aureus. Phylogenetic analysis showed that mupirocin production is limited to a single species. Inactivation of mupirocin production leads to complete loss of in vitro antagonism against S. aureus, except on certain iron-reduced media where the siderophore pyoverdine is responsible for the in vitro antagonism of a mupirocin-negative mutant. In addition to mupirocin some of the strains produced lipopeptides of the massetolide group. These lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing strains against S. aureus. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  9. Assessment of Pseudomonas aeruginosa N5,N10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase as a potential antibacterial drug target.

    Directory of Open Access Journals (Sweden)

    Thomas C Eadsforth

    Full Text Available The bifunctional enzyme methylenetetrahydrofolate dehydrogenase - cyclohydrolase (FolD is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.

  10. Screening for antibacterial and antiprotozoal activities of crude ...

    African Journals Online (AJOL)

    The antibacterial properties of organic and aqueous extracts of these plants were determined by the microdilution method and the microplate alamar blue assay against Stenotrophomonas maltophilia, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae, ...

  11. "Hot Tub Rash" and "Swimmer's Ear" (Pseudomonas)

    Science.gov (United States)

    Facts About “Hot Tub Rash” and “Swimmer’s Ear” (Pseudomonas) What is Pseudomonas and how can it affect me? Pseudomonas (sue-doh- ... a major cause of infections commonly known as “hot tub rash” and “swimmer’s ear.” This germ is ...

  12. UV/H2O2 advanced oxidation for abatement of organophosphorous pesticides and the effects on various toxicity screening assays.

    Science.gov (United States)

    Parker, Austa M; Lester, Yaal; Spangler, Emily K; von Gunten, Urs; Linden, Karl G

    2017-09-01

    Advanced oxidation processes (AOPs) are utilized due to their ability to treat emerging contaminants with the fast reacting and non-selective hydroxyl radical (OH). Organophosphorous insecticides are common drinking water contaminants, with 12 different compounds of this class being found on the US EPA's most recent Candidate Contaminant List (CCL4). The use of the AOP UV/H 2 O 2 for the treatment of organophosphorous insecticides was explored in this study, by coupling biological and analytical tools to follow the abatement of the target compounds. Four insecticides were explored for advanced oxidation treatment: acephate, dicrotophos, fenamiphos, and methamidophos. All four compounds were fast reacting with OH, all reacting with second order rate constants ≥5.5 × 10 9  M -1 s -1 . Three major endpoints of toxicity were studied: estrogenicity, genotoxicity (mutagenicity) and neurotoxicity. None of the target compounds showed any estrogenic activity, while all compounds showed an active genotoxic (mutagenic) response (AMES II assay) and most compounds had some level of neurotoxic activity. AOP treatment did not induce any estrogenic activity, and reduced the compounds' neurotoxicity and genotoxicity in all but one case. Methamidophos degradation by UV/H 2 O 2 resulted in an increase in genotoxicity, likely due to the formation of toxic transformation products. The increase in toxicity gradually decreased with time, possibly due to hydrolysis of the transformation products formed. This study provides insights into parent compound abatement and the changes in toxicity due to transformation products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The Abbott RealTime High Risk HPV test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies.

    Science.gov (United States)

    Poljak, Mario; Oštrbenk, Anja

    2013-01-01

    Human papillomavirus (HPV) testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. We reviewed the most important validation studies of a next-generation real-time polymerase chain reaction-based assay, the RealTime High Risk HPV test (RealTime)(Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older published in peer-reviewed journals from 2009 to 2013. RealTime is designed to detect 14 high-risk HPV genotypes with concurrent distinction of HPV-16 and HPV-18 from 12 other HPV genotypes. The test was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of HPV. We concisely reviewed validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay: the Abbott RealTime High Risk HPV test. Eight validation studies of RealTime in referral settings showed its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3-100%) and CIN3+ (range 93.0-100%), as well as comparative clinical sensitivity relative to the currently most widely used HPV test: the Qiagen/Digene Hybrid Capture 2 HPV DNA Test (HC2). Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to HC2. Four validation studies of RealTime performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and specificity relative to HC2 and GP5+/6+ PCR. RealTime has been extensively evaluated in the last 4 years. RealTime can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older.

  14. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for first-line screening.

    Science.gov (United States)

    Fenollar, Florence; Laouira, Sonia; Lepidi, Hubert; Rolain, Jean-Marc; Raoult, Didier

    2008-09-01

    Whipple disease (WD) is a chronic infectious disease caused by Tropheryma whipplei. WD DNA has been found in stool and saliva specimens from patients and asymptomatic carriers. A total of 4418 samples that were sent to our center for determination of WD were tested by a T. whipplei-specific quantitative polymerase chain reaction (PCR) based on repetitive sequences. Definite WD was diagnosed in 71 patients, including 55 patients with classic WD (defined by positive results of periodic acid-Schiff staining and/or specific immunohistochemistry of small-bowel biopsy specimens) and 16 patients with localized WD (including patients with endocarditis, neurologic infection, and uveitis). Of the persons without WD, 2.3% had stool specimens positive for T. whipplei by PCR and 0.2% had saliva specimens positive for T. whipplei by PCR. Diagnosis of WD was likely in patients with positive results of both PCR of saliva specimens and PCR of stool specimens (positive predictive value, 95.2%). When the bacterial load was >10(4) colony-forming units per g of stool, the positive predictive value was 100%. A negative result of PCR of a saliva or stool specimen had a negative predictive value of 99.2% for classic WD. For localized WD, positive results of both PCR of saliva specimens and PCR of stool specimens had a sensitivity of 58% (compared with 94% for classic WD). The positive predictive value of testing of blood, cerebrospinal fluid, and urine specimens was 100% for each, and the positive predictive value for testing of duodenal biopsy specimens was 97.5%. T. whipplei-specific quantitative PCR of saliva and stool specimens should be performed as first-line noninvasive screening for WD. When the results for both types of specimens are positive, diagnosis of classic WD should be highly suspected, especially if a high bacterial load is detected. Because PCR of saliva and stool specimens lacks sensitivity for determination of localized WD, invasive samples should be tested on the

  15. Biodegradation of propargite by Pseudomonas putida, isolated from tea rhizosphere.

    Science.gov (United States)

    Sarkar, Soumik; Seenivasan, Subbiah; Asir, Robert Premkumar Samuel

    2010-02-15

    Biodegradation of miticide propargite was carried out in vitro by selected Pseudomonas strains isolated from tea rhizosphere. A total number of 13 strains were isolated and further screened based on their tolerance level to different concentrations of propargite. Five best strains were selected and further tested for their nutritional requirements. Among the different carbon sources tested glucose exhibited the highest growth promoting capacity and among nitrogen sources ammonium nitrate supported the growth to the maximum. The five selected Pseudomonas strain exhibited a range of degradation capabilities. Mineral salts medium (MSM) amended with glucose provided better environment for degradation with the highest degradation potential in strain SPR 13 followed by SPR 8 (71.9% and 69.0% respectively).

  16. The Pseudomonas Quinolone Signal (PQS)

    DEFF Research Database (Denmark)

    Sams, Thomas; Baker, Ysobel; Hodgkinson, James

    2015-01-01

    Pseudomonas aeruginosa is an opportunistichuman pathogen that routinely appears near the top ofpublic health threat lists worldwide. P. aeruginosa causes in-fections by secreting a wealth of exceptionally active exo-products, leading to tissue damage. The synthesis of manyof these virulence factors...

  17. Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability.

    OpenAIRE

    Studemeister, A E; Quinn, J P

    1988-01-01

    The permeability of the outer membranes of imipenem-susceptible and imipenem-resistant clinical isolates of Pseudomonas aeruginosa was investigated by the liposome swelling assay. Sugars and cephaloridine penetrated rapidly, whereas imipenem penetrated poorly into liposomes constructed from porin-rich outer membrane fractions of the resistant isolates.

  18. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    Science.gov (United States)

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  19. Lethality and Developmental Delay of Drosophila melanogaster Following Ingestion of Selected Pseudomonas fluorescens Strains

    Science.gov (United States)

    Pseudomonas fluorescens secretes antimicrobial compounds that promote plant health and provide protection from pathogens. We used a non-invasive feeding assay to study the toxicity of P. fluorescens strains Pf0-1, SBW25, and Pf-5 to Drosophila melanogaster. The three strains of P. fluorescens varie...

  20. Three Strains of Pseudomonas fluorescens Exhibit Differential Toxicity Against Drosophila melanogaster

    Science.gov (United States)

    Three strains of Pseudomonas fluorescens were tested for toxicity to Drosophila melanogaster in an insect feeding assay. Insect eggs were placed on the surface of a non-nutritive agar plate supplemented with a food source that was non-inoculated or inoculated with P. fluorescens Pf0-1, SBW25, or Pf-...

  1. Phenotypic detection of metallo-β-lactamase among the clinical isolates of imipenem resistant Pseudomonas and Acinetobacter in tertiary care hospitals of Dhaka city

    Directory of Open Access Journals (Sweden)

    Shaheda Anwar

    2010-07-01

    Full Text Available The rapid spread of Metallo-b-lactamase (MBL producing Gram negative bacilli represents a matter of great concern worldwide. The study analyzed the occurrence of MBL production in carbapenem resistant Pseudomonas and Acinetobacter isolates over one year period. A total of 132 Pseudomonas and 76 Acinetobacter isolates were obtained from two tertiary care hospitals of Dhaka city. A total of 53 Pseudomonas and 29 Acinetobacter isolates were selected because of their resistance to carbapenem specially imipenem (IPM. Screening for MBL production was performed in these isolates by IPM-EDTA microdilution MIC method. 44 (83% IPM resistant Pseudomonas and 19 (65.5% Acinetobacter isolates were MBL producer by IPM-EDTA microdilution MIC method. These results suggest that MBL producing Pseudomonas and Acinetobacter isolates are emerging in our country and it is essential to screen carbapenem resistant isolates for MBL production. Ibrahim Med. Coll. J. 2010; 4(2: 63-65

  2. Role of mungbean root nodule associated fluorescent Pseudomonas and rhizobia in suppressing the root rotting fungi and root knot nematodes in chickpea (Cicer arietinum L.)

    International Nuclear Information System (INIS)

    Noreen, R.; Shafique, A.; Haque, S.E.; Ali, S.A.

    2016-01-01

    Three isolates each of fluorescent Pseudomonas (NAFP-19, NAFP-31 and NAFP-32) and rhizobia (NFB- 103, NFB-107 and NFB-109) which were originally isolated from root nodules of mungbean (Vigna radiata) showed significant biocontrol activity in the screen house and under field condition, against root rotting fungi viz., Macrophomina phaseolina, Fusarium solani, F. oxysporum and Rhizoctonia solani evaluated on chickpea. Biocontrol potential of these isolates was also evaluated against Meloidogyne incognita, the root knot nematode. Application of Pseudomonas and rhizobial isolates as a soil drench, separately or mixed significantly reduced root rot disease under screen house and field conditions. Nematode penetration in roots was also found significantly less in rhizobia or Pseudomonas treatments used separately or mixed as compared to control. Fluorescent Pseudomonas treated plants produced greater number of nodules per plant than control plants and about equal to rhizobia treated plants, indicating that root nodule associated fluorescent Pseudomonas enhance root nodulation. (author)

  3. The Role of Glutamate Dehydrogenase (GDH Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China.

    Directory of Open Access Journals (Sweden)

    Jing-Wei Cheng

    Full Text Available The incidence and severity of Clostridium difficile infection (CDI in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0% and specificity (92.8%, compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7% and positive predictive value (97.2%, but low sensitivity (45.0% and negative predictive value (88.3%, compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing.

  4. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  5. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  6. Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Fong, July; Yuan, Mingjun; Jakobsen, Tim Holm

    2017-01-01

    Since its discovery 22 years ago, the bacterial cell-to-cell communication system, termed quorum sensing (QS), has shown potential as antipathogenic target. Previous studies reported that ajoene from garlic inhibits QS in opportunistic human pathogen Pseudomonas aeruginosa. In this study, screening...

  7. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

    DEFF Research Database (Denmark)

    Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza

    2009-01-01

    Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendere...

  8. MEDICINAL PLANTS FROM BRAZILIAN CAATINGA: ANTIBIOFILM AND ANTIBACTERIAL ACTIVITIES AGAINST Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    DANIELLE SILVA TRENTIN

    2014-01-01

    Full Text Available The Caatinga biome covers a vast area in northeastern Brazil and presents a high level of biodiversity. It is known that about 400 plant species are used by semi-arid local communities for medical purposes. Based on ethnopharmacological reports, this study aims to screen 24 species from Caatinga regarding the ability to prevent biofilm formation and to inhibit the growth of Pseudomonas aeruginosa - a major opportunistic human pathogen and an important causative agent of morbidity and mortality. The effects of aqueous extracts, at 0.4 and 4.0 mg mL-1, on biofilm formation and on growth of P. aeruginosa ATCC 27853 were studied using the crystal violet assay and the OD600 absorbance, respectively. The most active extracts were analyzed by thinlayer chromatography and high performance liquid chromatography. Our investigation pointed extracts of four species with potential application for the control of P. aeruginosa: Anadenanthera colubrina (Vell. Brenan, Commiphora leptophloeos (Mart. J.B. Gillett, Myracrodruoun urundeuva Allemão, whose antibiofilm effects (89%, 56% and 79% inhibition of biofilm, respectively were associated with complete inhibition of bacterial growth, and Pityrocarpa moniliformis (Benth. Luckow & R.W. Jobson, which were able avoid 68% of biofilm formation and inhibited 30% bacterial growth. The qualitative phytochemical analyses reveal the complexity of the samples as well as the presence of compounds with high molecular weight.

  9. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

    Science.gov (United States)

    Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

    2009-01-01

    Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

  10. Elicitation of Induced Resistance against Pectobacterium carotovorum and Pseudomonas syringae by Specific Individual Compounds Derived from Native Korean Plant Species

    Directory of Open Access Journals (Sweden)

    Choong-Min Ryu

    2013-10-01

    Full Text Available Plants have developed general and specific defense mechanisms for protection against various enemies. Among the general defenses, induced resistance has distinct characteristics, such as broad-spectrum resistance and long-lasting effectiveness. This study evaluated over 500 specific chemical compounds derived from native Korean plant species to determine whether they triggered induced resistance against Pectobacterium carotovorum supsp. carotovorum (Pcc in tobacco (Nicotiana tabacum and Pseudomonas syringae pv. tomato (Pst in Arabidopsis thaliana. To select target compound(s with direct and indirect (volatile effects, a new Petri-dish-based in vitro disease assay system with four compartments was developed. The screening assay showed that capsaicin, fisetin hydrate, jaceosidin, and farnesiferol A reduced the disease severity significantly in tobacco. Of these four compounds, capsaicin and jaceosidin induced resistance against Pcc and Pst, which depended on both salicylic acid (SA and jasmonic acid (JA signaling, using Arabidopsis transgenic and mutant lines, including npr1 and NahG for SA signaling and jar1 for JA signaling. The upregulation of the PR2 and PDF1.2 genes after Pst challenge with capsaicin pre-treatment indicated that SA and JA signaling were primed. These results demonstrate that capsaicin and jaceosidin can be effective triggers of strong induced resistance against both necrotrophic and biotrophic plant pathogens.

  11. Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

    Science.gov (United States)

    Zhang, N; Stewart, B G; Moore, J C; Greasham, R L; Robinson, D K; Buckland, B C; Lee, C

    2000-10-01

    Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

  12. Discovery and biophysical characterization of 2-amino-oxadiazoles as novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence.

    Science.gov (United States)

    Zender, Michael; Klein, Tobias; Henn, Claudia; Kirsch, Benjamin; Maurer, Christine K; Kail, Dagmar; Ritter, Christiane; Dolezal, Olan; Steinbach, Anke; Hartmann, Rolf W

    2013-09-12

    The human pathogen Pseudomonas aeruginosa employs alkyl quinolones for cell-to-cell communication. The Pseudomonas quinolone signal (PQS) regulates various virulence factors via interaction with the transcriptional regulator PqsR. Therefore, we consider the development of PqsR antagonists a novel strategy to limit the pathogenicity of P. aeruginosa. A fragment identification approach using surface plasmon resonance screening led to the discovery of chemically diverse PqsR ligands. The optimization of the most promising hit (5) resulted in the oxadiazole-2-amine 37 showing pure antagonistic activity in Escherichia coli (EC50 = 7.5 μM) and P. aeruginosa (EC50 = 38.5 μM) reporter gene assays. 37 was able to diminish the production of the PQS precursor HHQ in a PqsH-deficient P. aeruginosa mutant. The level of the major virulence factor pyocyanin was significantly reduced in wild-type P. aeruginosa. In addition, site-directed mutagenesis in combination with isothermal titration calorimetry and NMR INPHARMA experiments revealed that the identified ligands bind to the same site of PqsR by adopting different binding modes. These findings will be utilized in a future fragment-growing approach aiming at novel therapeutic options for the treatment of P. aeruginosa infections.

  13. Application of endocrine disruptor screening program fish short-term reproduction assay: Reproduction and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus) exposed to Bermuda pond sediment.

    Science.gov (United States)

    Fort, Douglas J; Mathis, Michael; Fort, Chelsea E; Fort, Hayley M; Bacon, Jamie P

    2015-06-01

    A modified tier 1 Endocrine Disruptor Screening Program (EDSP) 21-d fish short-term reproduction assay (FSTRA) was used to evaluate the effects of sediment exposure from freshwater and brackish ponds in Bermuda on reproductive fecundity and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus). Reproductively active male and female fish were exposed to control sediment and sediment from 2 freshwater ponds (fathead minnow) and 2 marine ponds (killifish) contaminated with polyaromatic hydrocarbons and metals via flow-through exposure for 21 d. Reproductive fecundity was monitored daily. At termination, the status of the reproductive endocrine system was assessed by the gonadosomatic index, gonadal histology, plasma steroids (estrogen [E2], testosterone [T], and 11-ketotestosterone [11-KT]), steroidogenic enzymes (aromatase and combined 3β/17β -hydroxysteroid dehydrogenase [3β/17β-HSD]), and plasma vitellogenin (VTG). Decreased reproductive fecundity, lower male body weight, and altered endocrinological measures of reproductive status were observed in both species. Higher plasma T levels in female minnows and 11-KT levels in both male and female minnows and female killifish exposed to freshwater and brackish sediments, respectively. Decreased female E2 and VTG levels and gonadal cytochrome P19 (aromatase) activity were also found in sediment exposed females from both species. No effect on female 3β/17β-HSD activity was found in either species. The FSTRA provided a robust model capable of modification to evaluate reproductive effects of sediment exposure in fish. © 2015 SETAC.

  14. Comparison of Microscopy, Nested-PCR, and Real-Time-PCR Assays Using High-Throughput Screening of Pooled Samples for Diagnosis of Malaria in Asymptomatic Carriers from Areas of Endemicity in Myanmar

    Science.gov (United States)

    Wang, Bo; Han, Soe-Soe; Cho, Cho; Han, Jin-Hee; Cheng, Yang; Lee, Seong-Kyun; Galappaththy, Gawrie N. L; Thimasarn, Krongthong; Soe, Myat Thu; Oo, Htet Wai; Kyaw, Myat Phone

    2014-01-01

    Asymptomatic infection is an important obstacle for controlling disease in countries where malaria is endemic. Because asymptomatic carriers do not seek treatment for their infections, they can have high levels of gametocytes and constitute a reservoir available for new infection. We employed a sample pooling/PCR-based molecular detection strategy for screening malaria infection in residents from areas of Myanmar where malaria is endemic. Blood samples (n = 1,552) were collected from residents in three areas of malaria endemicity (Kayin State, Bago, and Tanintharyi regions) of Myanmar. Two nested PCR and real-time PCR assays showed that asymptomatic infection was detected in about 1.0% to 9.4% of residents from the surveyed areas. The sensitivities of the two nested PCR and real-time PCR techniques were higher than that of microscopy examination (sensitivity, 100% versus 26.4%; kappa values, 0.2 to 0.5). Among the three regions, parasite-positive samples were highly detected in subjects from the Bago and Tanintharyi regions. Active surveillance of residents from regions of intense malaria transmission would reduce the risk of morbidity and mitigate transmission to the population in these areas of endemicity. Our data demonstrate that PCR-based molecular techniques are more efficient than microscopy for nationwide surveillance of malaria in countries where malaria is endemic. PMID:24648557

  15. Methods of detecting and controlling mucoid Pseudomonas biofilm production

    Science.gov (United States)

    Yu, Hongwei D. (Inventor); Qiu, Dongru (Inventor)

    2013-01-01

    Compositions and methods for detecting and controlling the conversion to mucoidy in Pseudomonas aeruginosa are disclosed. The present invention provides for detecting the switch from nonmucoid to mucoid state of P. aeruginosa by measuring mucE expression or MucE protein levels. The interaction between MucE and AlgW controls the switch to mucoidy in wild type P. aeruginosa. Also disclosed is an alginate biosynthesis heterologous expression system for use in screening candidate substances that inhibit conversion to mucoidy.

  16. Use of polymeric dyes in lignin biodegradation assays

    International Nuclear Information System (INIS)

    Gold, M.H.; Alic, M.; Glenn, J.K.

    1988-01-01

    This paper reviews the historical use of various 14 C-radiolabeled and unlabeled substrates to screen for ligninolytic activity. The disadvantages of these assays are presented. The authors describe the development of assays utilizing polymeric dyes

  17. Pseudomonas-follikulitis efter badning i spabad

    DEFF Research Database (Denmark)

    Uldall Pallesen, Kristine Appel; Andersen, Klaus Ejner; Mørtz, Charlotte Gotthard

    2012-01-01

    . We describe a 23-year-old healthy woman who developed a pustular rash and general malaise after using a spa bath contaminated with Pseudomonas aeruginosa. Bacterial culture from a pustule confirmed Pseudomonas folliculitis and the patient was treated with ciprofloxacin with rapid good effect....

  18. Pseudomonas Septic Arthritis | Thanni | Nigerian Journal of ...

    African Journals Online (AJOL)

    BACKGROUND: Septic arthritis due to pseudomonas species is unusual and when it occurs, there is often an underlying cause like immune depression, intravenous drug abuse or a penetrating injury. PATIENT AND METHOD: We report a case of pseudomonas septic arthritis complicating cannulation of a leg vein following ...

  19. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  20. Depression Screening

    Science.gov (United States)

    ... Depression Screening Substance Abuse Screening Alcohol Use Screening Depression Screening (PHQ-9) - Instructions The following questions are ... this tool, there is also text-only version . Depression Screening - Manual Instructions The following questions are a ...

  1. Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light

    International Nuclear Information System (INIS)

    McBeth, D.L.

    1989-01-01

    The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded

  2. Preliminary phytochemical and antimicrobial screening of 50 ...

    African Journals Online (AJOL)

    Ethanolic extracts of 50 plant species were screened for their antimicrobial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The results indicated that of the 50 plant extracts, 28 plant extracts inhibited the growth of one or more test pathogens.

  3. Screening of thermophilic neutral lipase-producing Pseudomonas ...

    African Journals Online (AJOL)

    akpobome uruemuesiri

    2013-02-27

    Feb 27, 2013 ... Full Length Research Paper ... The main objective of this research was to isolate and ... liquid-state fermentation in shake flask. ..... lues generated signals, which were large in comparison .... production in a bioreactor. Proc.

  4. Draft genome sequence of Pseudomonas mosselii Gil3, isolated from catfish and antagonistic against hypervirulent Aeromonas hydrophila

    Science.gov (United States)

    Pseudomonas mosselii Gil3 was isolated from a catfish that survived from lethal challenge with hypervirulent Aeromonas hydrophila (vAh). When assayed in vitro, the bacterium showed antagonism against vAh. Sequence analysis revealed that the genome of P. mosselii Gil3 encodes numerous aromatic metabo...

  5. Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa.

    Science.gov (United States)

    Sabaeifard, Parastoo; Abdi-Ali, Ahya; Soudi, Mohammad Reza; Gamazo, Carlos; Irache, Juan Manuel

    2016-10-10

    Amikacin is a very effective aminoglycoside antibiotic but according to its high toxicity, the use of this antibiotic has been limited. The aim of this study was to formulate and characterize amikacin loaded PLGA nanoparticles. Nanoparticles were synthetized using a solid-in-oil-in-water emulsion technique with different ratio of PLGA 50:50 (Resomer 502H) to drug (100:3.5, 80:3.5 and 60:3.5), two different concentrations of stabilizer (pluronic F68) (0.5% or 1%) and varied g forces to recover the final products. The most efficient formulation based on drug loading (26.0±1.3μg/mg nanoparticle) and encapsulation efficiency (76.8±3.8%) was the one obtained with 100:3.5 PLGA:drug and 0.5% luronic F68, recovered by 20,000×g for 20min. Drug release kinetic study indicated that about 50% of the encapsulated drug was released during the first hour of incubation in phospahte buffer, pH7.4, 37°C, 120rpm. Using different cell viability/cytotoxicity assays, the optimized formulation showed no toxicity against RAW macrophages after 2 and 24h of exposure. Furthermore, released drug was active and maintained its bactericidal activity against Pseudomonas aeruginosa in vitro. These results support the effective utilization of the PLGA nanoparticle formulation for amikacin in further in vivo studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. The Versatile Mutational Resistome of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Carla López-Causapé

    2018-04-01

    Full Text Available One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  7. The Versatile Mutational Resistome of Pseudomonas aeruginosa.

    Science.gov (United States)

    López-Causapé, Carla; Cabot, Gabriel; Del Barrio-Tofiño, Ester; Oliver, Antonio

    2018-01-01

    One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  8. Silver against Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Kirketerp-Møller, K.; Kristiansen, S.

    2007-01-01

    bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa......, but that the silver concentration is important. A concentration of 5-10 ig/mL silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 ig/mL) had no effect. The bactericidal concentration of silver required to eradicate the bacterial biofilm was 10-100 times higher than that used to eradicate...... planktonic bacteria. These observations strongly indicate that the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. It is suggested that clinicians and manufacturers of the said wound dressings consider whether they are treating wounds...

  9. Pseudomona pseudomallei community acquired pneumonia

    International Nuclear Information System (INIS)

    Severiche, Diego

    1998-01-01

    This is the first published case report en Colombia about pseudomona pseudomallei community acquired pneumonia. This uncommon pathogen is from the epidemiological standpoint a very important one and medical community should be aware to look after it in those patients where no other etiological pathogen is recovered. A brief summary about epidemiology is showed, emphasizing those regions where it can be found. Likewise, comments about the differential diagnosis are important since it should be considered in those patients where tuberculosis is suspected. This is particularly representative for countries with high tuberculosis rates. Furthermore, a microbiological review is shown, emphasizing on isolation techniques, descriptions about therapeutics and other regarding treatment issues according international standards. Finally; a description about the clinical picture, laboratory findings, treatment and evolution of the case reported are shown for discussion

  10. Glyphosate catabolism by Pseudomonas sp

    International Nuclear Information System (INIS)

    Shinabarger, D.L.

    1986-01-01

    The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3- 14 C] glyphosate revealed that approximately 50-59% of the C3 carbon was oxidized to CO 2 . Fractionation of stationary phase cells labeled with [3- 14 C]glyphosate revealed that from 45-47% of the assimilated C3 carbon is distributed to proteins and that amino acids methionine and serine are highly labeled. The nucleic acid bases adenine and guanine received 90% of the C3 label that was incorporated into nucleic acids, and the only pyrimidine base labeled was thymine. Pulse labeling of PG2982 cells with [3- 14 C]glyphosate revealed that [3- 14 C]sarcosine is an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of an enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. Phosphonate utilization by Pseudomonas sp. PG2982 was investigated. Each of the ten phosphonates tested were utilized as a sole source of phosphorus by PG2982. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. PG2982 cultures degraded phenylphosphonate to benzene and produced methane from methylphosphonate. The data indicate that PG2982 is capable of cleaving the carbon-phosphorus bond of several structurally different phosphonates

  11. Pseudomonas fluorescens' view of the periodic table.

    Science.gov (United States)

    Workentine, Matthew L; Harrison, Joe J; Stenroos, Pernilla U; Ceri, Howard; Turner, Raymond J

    2008-01-01

    Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of magnitude. In this study, a high-throughput metal toxicity screen was initiated with the aim of correlating biological toxicity data in planktonic and biofilm cells to the physiochemical properties of metal ions. To this end, Pseudomonas fluorescens ATCC 13525 was grown in the Calgary Biofilm Device (CBD) and biofilms and planktonic cells of this microorganism were exposed to gradient arrays of different metal ions. These arrays included 44 different metals with representative compounds that spanned every group of the periodic table (except for the halogens and noble gases). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) values were obtained after exposing the biofilms to metal ions for 4 h. Using these values, metal ion toxicity was correlated to the following ion-specific physicochemical parameters: standard reduction-oxidation potential, electronegativity, the solubility product of the corresponding metal-sulfide complex, the Pearson softness index, electron density and the covalent index. When the ions were grouped according to outer shell electron structure, we found that heavy metal ions gave the strongest correlations to these parameters and were more toxic on average than the other classes of the ions. Correlations were different for biofilms than for planktonic cells, indicating that chemical mechanisms of metal ion toxicity differ between the two modes of growth. We suggest that biofilms can specifically counter the toxic effects of certain physicochemical parameters, which may contribute to the increased ability of biofilms to withstand metal toxicity.

  12. Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia induce distinct host responses.

    Science.gov (United States)

    McConnell, Kevin W; McDunn, Jonathan E; Clark, Andrew T; Dunne, W Michael; Dixon, David J; Turnbull, Isaiah R; Dipasco, Peter J; Osberghaus, William F; Sherman, Benjamin; Martin, James R; Walter, Michael J; Cobb, J Perren; Buchman, Timothy G; Hotchkiss, Richard S; Coopersmith, Craig M

    2010-01-01

    Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities. Prospective, randomized controlled study. Animal laboratory in a university medical center. Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points. The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs. Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a

  13. Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Samuel Takashi Saito

    2012-01-01

    Full Text Available Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS. Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI<3 only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

  14. Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

    Science.gov (United States)

    Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

    2012-01-01

    Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications. PMID:22548121

  15. The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery

    International Nuclear Information System (INIS)

    Moynie, Lucille; Schnell, Robert; McMahon, Stephen A.; Sandalova, Tatyana; Boulkerou, Wassila Abdelli; Schmidberger, Jason W.; Alphey, Magnus; Cukier, Cyprian; Duthie, Fraser; Kopec, Jolanta; Liu, Huanting; Jacewicz, Agata; Hunter, William N.; Naismith, James H.; Schneider, Gunter

    2012-01-01

    A focused strategy has been directed towards the structural characterization of selected proteins from the bacterial pathogen P. aeruginosa. The objective is to exploit the resulting structural data, in combination with ligand-binding studies, and to assess the potential of these proteins for early-stage antimicrobial drug discovery. Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns

  16. Simultaneous and quantitative monitoring of co-cultured Pseudomonas aeruginosa and Staphylococcus aureus with antibiotics on a diffusometric platform

    Science.gov (United States)

    Chung, Chih-Yao; Wang, Jhih-Cheng; Chuang, Han-Sheng

    2017-04-01

    Successful treatments against bacterial infections depend on antimicrobial susceptibility testing (AST). However, conventional AST requires more than 24 h to obtain an outcome, thereby contributing to high patient mortality. An antibiotic therapy based on experiences is therefore necessary for saving lives and escalating the emergence of multidrug-resistant pathogens. Accordingly, a fast and effective drug screen is necessary for the appropriate administration of antibiotics. The mixed pathogenic nature of infectious diseases emphasizes the need to develop an assay system for polymicrobial infections. On this basis, we present a novel technique for simultaneous and quantitative monitoring of co-cultured microorganisms by coupling optical diffusometry with bead-based immunoassays. This simple integration simultaneously achieves a rapid AST analysis for two pathogens. Triple color particles were simultaneously recorded and subsequently analyzed by functionalizing different fluorescent color particles with dissimilar pathogen-specific antibodies. Results suggested that the effect of the antibiotic, gentamicin, on co-cultured Pseudomonas aeruginosa and Staphylococcus aureus was effectively distinguished by the proposed technique. This study revealed a multiplexed and time-saving (within 2 h) platform with a small sample volume (~0.5 μL) and a low initial bacterial count (50 CFU per droplet, ~105 CFU/mL) for continuously monitoring the growth of co-cultured microorganisms. This technique provides insights into timely therapies against polymicrobial diseases in the near future.

  17. Production and characterization of biosurfactant from Pseudomonas ...

    African Journals Online (AJOL)

    Further characterization of biosurfactant using Fourier transform infrared spectroscopy (FTIR) revealed it as a rhamnolipid. Keywords: Mangrove ecosystems, Pseudomonas aeruginosa, biosurfactant, critical micelle concentration (CMC), FT-IR fourier transform infrared spectroscopy (FTIR). African Journal of Biotechnology, ...

  18. Pseudomonas aeruginosa (Family Pseudomonadaceae) is an ...

    African Journals Online (AJOL)

    Pseudomonas aeruginosa (Family Pseudomonadaceae) is an obligate aerobic, motile, gram negative bacillus.which is able to grow and survive in almost any environment and resistant to temperature extremes. It is involved in the etiology of several diseases i.

  19. Growth of Pseudomonas fluorescens on Cassava Starch ...

    African Journals Online (AJOL)

    Michael Horsfall

    ABSTRACT: The potential of local strains of microorganism (Pseudomonas fluorescens) in polyhydroxbutyrate production ... The demand for the use of biopolymers ... This work therefore investigates the production of polyhydroxybutyrate from.

  20. Antibiotics Susceptibility Pattern of Pseudomonas aeruginosa ...

    African Journals Online (AJOL)

    ABSTRACT: This work investigated the prevalence and antibiotics sensitivity of Pseudomonas aeruginosa isolated from ... skin triggers coagulation and an acute inflammatory response ... agents with anti-pseudomonal activity, life-threatening.

  1. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  2. Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection.

    Science.gov (United States)

    Périnet, Simone; Jeukens, Julie; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Charette, Steve J; Levesque, Roger C

    2016-01-12

    Mechanisms underlying the success of Pseudomonas aeruginosa in chronic lung infection among cystic fibrosis (CF) patients are poorly defined. The modA gene was previously linked to in vivo competitiveness of P. aeruginosa by a genetic screening in the rat lung. This gene encodes a subunit of transporter ModABC, which is responsible for extracellular uptake of molybdate. This compound is essential for molybdoenzymes, including nitrate reductases. Since anaerobic growth conditions are known to occur during CF chronic lung infection, inactivation of a molybdate transporter could inhibit proliferation through the inactivation of denitrification enzymes. Hence, we performed phenotypic characterization of a modA mutant strain obtained by signature-tagged mutagenesis (STM_modA) and assessed its virulence in vivo with two host models. The STM_modA mutant was in fact defective for anaerobic growth and unable to use nitrates in the growth medium for anaerobic respiration. Bacterial growth and nitrate usage were restored when the medium was supplemented with molybdate. Most significantly, the mutant strain showed reduced virulence compared to wild-type strain PAO1 according to a competitive index in the rat model of chronic lung infection and a predation assay with Dictyostelium discoideum amoebae. As the latter took place in aerobic conditions, the in vivo impact of the mutation in modA appears to extend beyond its effect on anaerobic growth. These results support the modABC-encoded transporter as important for the pathogenesis of P. aeruginosa, and suggest that enzymatic machinery implicated in anaerobic growth during chronic lung infection in CF merits further investigation as a potential target for therapeutic intervention.

  3. 5-Aminolevulinic acid induced photodynamic inactivation on Staphylococcus aureus and Pseudomonas aeruginosa.

    Science.gov (United States)

    Hsieh, Chien-Ming; Huang, Yen-Hao; Chen, Chueh-Pin; Hsieh, Bo-Chuan; Tsai, Tsuimin

    2014-09-01

    The aim of the present study was to develop a simple and fast screening technique to directly evaluate the bactericidal effects of 5-aminolevulinic acid (ALA)-mediated photodynamic inactivation (PDI) and to determine the optimal antibacterial conditions of ALA concentrations and the total dosage of light in vitro. The effects of PDI on Staphylococcus aureus and Pseudomonas aeruginosa in the presence of various concentrations of ALA (1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM) were examined. All bacterial strains were exponentially grown in the culture medium at room temperature in the dark for 60 minutes and subsequently irradiated with 630 ± 5 nm using a light-emitting diode (LED) red light device for accumulating the light doses up to 216 J/cm 2 . Both bacterial species were susceptible to the ALA-induced PDI. Photosensitization using 1.0 mM ALA with 162 J/cm 2 light dose was able to completely reduce the viable counts of S. aureus. A significant decrease in the bacterial viabilities was observed for P. aeruginosa, where 5.0 mM ALA was photosensitized by accumulating the light dose of 162 J/cm 2 . We demonstrated that the use of microplate-based assays-by measuring the apparent optical density of bacterial colonies at 595 nm-was able to provide a simple and reliable approach for quickly choosing the parameters of ALA-mediated PDI in the cell suspensions. Copyright © 2013. Published by Elsevier B.V.

  4. Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

    Science.gov (United States)

    El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

    2000-02-15

    We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

  5. Bioreporter pseudomonas fluorescens HK44 immobilized in a silica matrix

    Directory of Open Access Journals (Sweden)

    Trogl J.

    2003-01-01

    Full Text Available The bioluminescent bioreporter Pseudomonas fluorescens HK44, the whole cell bacterial biosensor that responds to naphthalene and its metabolites via the production of visible light, was immobilized into a silica matrix by the sol-gel technique. The bioluminescence intensities were measured in the maximum of the bioluminescence band at X = 500 nm. The immobilized cells (>105 cells per g silica matrix produced light after induction by salicylate (cone. > 10 g/l, naphthalene and aminobenzoic acid. The bioluminescence intensities induced by 2,3-dihydroxynaphthalene 3-hydroxybenzoic acid and 4-hydroxybenzoic acid were comparable to a negative control. The cells in the silica layers on glass slides produced light in response to the presence of an inductor at least 8 months after immobilization, and >50 induction cycles. The results showed that these test slides could be used as assays for the multiple determination of water pollution.

  6. OXIDATION OF POLYCHLORINATED BIPHENYLS BY PSEUDOMONAS SP. STRAIN LB400 AND PSEUDOMONAS PSEUDOALCALIGENES KF707

    Science.gov (United States)

    Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in th...

  7. Evaluación de las técnicas de detección del VPH en los programas de cribado para cáncer de cuello uterino Assessment of hpv detection assays for use in cervical cancer screening programs

    Directory of Open Access Journals (Sweden)

    M Paz Cañadas

    2006-10-01

    Full Text Available OBJETIVO: La identificación de la infección por tipos de alto riesgo del virus del papiloma humano (VPH es una herramienta útil para el cribado de cáncer del cuello uterino. Las distintas técnicas aplicadas para su detección deben contrastarse y validarse para su empleo en la tamización poblacional. MATERIAL Y MÉTODOS: Se evalúan tres técnicas para la detección del VPH en 166 muestras cervicales procedentes de mujeres atendidas en una clínica de dermatología en Oviedo (España: a PCR-EIA mediante consensos MY09/MY011; b PCR con line blot hybridization (PCR-LBH con consensos PGMY; y c hybrid capture 2. RESULTADOS: El ADN-VPH se reconoció en 29.5%, 25.3% y 24.7%, de acuerdo con el ensayo. La concordancia global entre PCR-EIA, PCR-LBH y HC2 fue de 73.5% con los valores de kappa superiores a 0.56 entre los ensayos (pOBJECTIVE: Detection of high-risk human papillomavirus types (HPV infection is an important tool in the screening of cervical cancer and triage of cytological abnormalities. The different techniques for detection of this cancer need to be contrasted and validated for use in population screening. MATERIAL AND METHODS: Cervical cell samples were collected from 166 women attending a dermatology clinic in Oviedo (Spain. We evaluated the performance of three different assays for VPH detection. The methods utilized were 1 In-house PCR-EIA using L1 consensus primers MY09/MY11, 2 A PCR-reverse line blot hybridization (PCR-LBH that uses L1 consensus PGMY primers. 3 Hybrid Capture 2. All assays were performed blinded. The kappa statistic was used to test for global agreement between assay pairs. RESULTS: HPV DNA was detected in 24,7%, 25,3% and 29,5% of the women, respective to the assay. The overall agreement between the in-house PCR, PCR-LBH and HC2 was (73.5% with all kappa values between assay pairs exceeding 0.56 (p<0.001. CONCLUSION: The three HPV assays were equally accurate in estimating high-risk HPV prevalence and HPV

  8. Capsule production by Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Lynn, A.R.

    1984-01-01

    Mucoid strains of Pseudomonas aeruginosa, associated almost exclusively with chronic respiratory infections in patients with cystic fibrosis, possess a capsule composed of alginic acid similar to one produced by Azotobacter vinelandii. Recent reports have provided evidence that the biosynthetic pathway for alginate in P. aeruginosa may differ from the pathway proposed for A. vinelandii in that synthesis in P. aeruginosa may occur by way of the Entner-Doudoroff pathway. Incorporation of isotope from (6-/sup 14/C)glucose into alginate by both P. aueroginosa and A. vinelandii was 10-fold greater than that from either (1-/sup 14/C)/sup -/ or (2-/sup 14/C)glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate biosynthesis. These data strongly suggest that the Entner-Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii. The enzymes of carbohydrate metabolism in mucoid strains of P. aeruginosa appear to be unchanged whether alignate is actively produced or not and activities do not differ significantly from nonmucoid strain PAO.

  9. Assessing Pseudomonas virulence with a nonmammalian host: Drosophila melanogaster.

    Science.gov (United States)

    Haller, Samantha; Limmer, Stefanie; Ferrandon, Dominique

    2014-01-01

    Drosophila melanogaster flies represent an interesting model to study host-pathogen interactions as: (1) they are cheap and easy to raise rapidly and do not bring up ethical issues, (2) available genetic tools are highly sophisticated, for instance allowing tissue-specific alteration of gene expression, e.g., of immune genes, (3) they have a relatively complex organization, with distinct digestive tract and body cavity in which local or systemic infections, respectively, take place, (4) a medium throughput can be achieved in genetic screens, for instance looking for Pseudomonas aeruginosa mutants with altered virulence. We present here the techniques used to investigate host-pathogen relationships, namely the two major models of infections as well as the relevant parameters used to monitor the infection (survival, bacterial titer, induction of host immune response).

  10. Antibacterial efficacies of some plant extracts against Aeromonas and Pseudomonas diseases of farmed catfish (Heterobranchus longifilis

    Directory of Open Access Journals (Sweden)

    Albert P. Ekanem

    2011-11-01

    Full Text Available Aeromonas and Pseudomonas diseases are responsible for mortalities of some farmed catfish in Nigeria. The objective of the study is to investigate the efficacies of extracts of some plants against Aeromonas and Pseudomonas diseases of Heterobranchus longifilis. Ethanol extracts of Phyllanthus amarus, Allium sativum, Artemisia annua, Citrus limon, Moringa oleifera, Allium cepa and Azadirachta indica were tested against Aeromonas hydrophila and Pseudomonas flourescens of H. longifilis by disc diffusion assay. Extracts of P. amarus, A. sativum, A. annua and C. limon were significantly (P<0.05 more sensitive to A. hydrophila and P. flourescens than M. oleifera, A. cepa and A. indica which were effective against P. flourescens. Minimum inhibitory concentrations (MIC of the extracts were 25mg/ml for P. amarus and A. annua; 25 and 100mg/ml for C. lemon and A. cepa respectively and 50mg/ml for A. indica.  Alkaloid was demonstrated in all plants except A. annua by qualitative methods. Moderate amount (++ of cardiac glycosides was demonstrated in A. sativum, M. oleifera and P. amarus. Saponin (+++ was present in M. oleifera and A. indica while, tannin (++ was present in M. oleifera, P. amarus and A. indica respectively. Phlobatanins and Anthraquinones (++ were present in P. amarus and M. oleifera respectively.  Extracts of aforementioned plants have potentials as therapy against Aeromonas hydrophila and Pseudomonas flourescens of farmed catfish.

  11. Antimicrobial Activity of Various Plant Extracts on Pseudomonas Species Associated with Spoilage of Chilled Fish

    Directory of Open Access Journals (Sweden)

    Osan Bahurmiz

    2016-11-01

    Full Text Available The antimicrobial activity of various plant extracts on Pseudomonas bacteria isolated from spoiled chilled tilapia (Oreochromis sp. was evaluated in this study. In the first stage of this study, red tilapia was subjected to chilled storage (4°C for 3 weeks, and spoilage bacteria were isolated and identified from the spoiled fish. Pseudomonas was the dominant bacteria isolated from the spoiled fish and further identification revealed that P. putida, P. fluorescens and Pseudomonas spp. were the main species of this group. In the second stage, methanolic extracts of 15 selected plant species were screened for their antimicrobial activity, by agar disc diffusion method, against the Pseudomonas isolates. Results indicated that most of the extracts had different degrees of activity against the bacterial isolates. The strongest activity was exhibited by bottlebrush flower (Callistemon viminalis extract. This was followed by extracts from guava bark (Psidium guajava and henna leaf (Lawsonia inermis. Moderate antimicrobial activities were observed in extracts of clove (Syzygium aromaticum, leaf and peel of tamarind (Tamarindus indica, cinnamon bark (Cinnamomum zeylanicum, wild betel leaf (Piper sarmentosum and fresh thyme (Thymus spp.. Weak or no antimicrobial activity was observed from the remaining extracts. The potential antimicrobial activity shown by some plant extracts in this study could significantly contribute to the fish preservation.

  12. Genotypische diversiteit en rhizosfeerkolonisatie van DAPG-producerende Pseudomonas spp.

    NARCIS (Netherlands)

    Bergsma-Vlami, M.

    2009-01-01

    Het antibioticum 2,4-diacetylphloroglucinol (DAPG) speelt een belangrijke rol in biologische bestrijding van verschillende plantenpathogenen door fluorescerende Pseudomonas-soorten. DAPG-producerende Pseudomonas-stammen zijn effectief in biologische bestrijding, maar hun saprofytisch vermogen is

  13. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis

    DEFF Research Database (Denmark)

    Johansen, Helle Krogh; Gøtzsche, Peter C

    2013-01-01

    Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed.......Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed....

  14. Dynamics of development and dispersal in sessile microbial communities: examples from Pseudomonas aeruginosa and Pseudomonas putida model biofilms

    DEFF Research Database (Denmark)

    Klausen, M.; Gjermansen, Morten; Kreft, J.-U.

    2006-01-01

    Surface-associated microbial communities in many cases display dynamic developmental patterns. Model biofilms formed by Pseudomonas aeruginosa and Pseudomonas putida in laboratory flow-chamber setups represent examples of such behaviour. Dependent on the experimental conditions the bacteria...

  15. 21 CFR 866.3415 - Pseudomonas spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pseudomonas spp. serological reagents. 866.3415... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3415 Pseudomonas spp. serological reagents. (a) Identification. Pseudomonas spp. serological reagents are devices that...

  16. Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans

    Institute of Scientific and Technical Information of China (English)

    Prabhakaran Priyaja; Puthumana Jayesh; Neil Scolastin Correya; Balachandran Sreelakshmi; Naduthalmuriparambil S Sudheer; Rosamma Philip; Isaac Sarogeni Bright Singh

    2014-01-01

    Objective: To abrogate pathogenic vibrios in aquaculture by testing the potential of Pseudomonas isolates from fresh water, brackish and marine environments as probiotics.Methods:Antagonistic activity of the compound against 7 Vibrio spp. was performed. Influence of salinity on the production of pyocyanin and the toxicity was done through the compound using brine shrimp lethality assay. Molecular characterization was performed to confirm that the isolates werePseudomonas aeruginosa. Results: Salinity was found to regulate the levels of pyocyanin production, with 5-10 g/L as the optimum. All Pseudomonas isolates grew at salinities ranging from 5 to 70 g/L. Isolates of marine origin produced detectable levels of pyocyanin up to 45 g/L salinity. Brackish and freshwater isolates ceased to produce pyocyanin at salinities above 30 g/L and 20 g/L, respectively. Culture supernatants of all 5 Pseudomonas isolates possessed the ability to restrict the growth of Vibrio spp. and maximum antagonistic effect on Vibrio harveyi was obtained when they were grown at salinities of 5 to 10 g/L. The marine isolate MCCB117, even when grown at a salinity of 45 g/L possessed the ability to inhibit Vibrio spp.Conclusions:Purification and structural elucidation of antagonistic compound were carried out. ideal for application in freshwater, MCCB102 and MCCB103 in brackish water and MCCB117 and The present investigation showed that Pseudomonas aeruginosa MCCB119 would be MCCB118 in marine aquaculture systems as putative probiotics in the management of vibrios.

  17. Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans

    Directory of Open Access Journals (Sweden)

    Prabhakaran Priyaja

    2014-01-01

    Full Text Available Objective: To abrogate pathogenic vibrios in aquaculture by testing the potential of Pseudomonas isolates from fresh water, brackish and marine environments as probiotics. Methods: Purification and structural elucidation of antagonistic compound were carried out. Antagonistic activity of the compound against 7 Vibrio spp. was performed. Influence of salinity on the production of pyocyanin and the toxicity was done through the compound using brine shrimp lethality assay. Molecular characterization was performed to confirm that the isolates were Pseudomonas aeruginosa. Results: Salinity was found to regulate the levels of pyocyanin production, with 5-10 g/L as the optimum. All Pseudomonas isolates grew at salinities ranging from 5 to 70 g/L. Isolates of marine origin produced detectable levels of pyocyanin up to 45 g/L salinity. Brackish and freshwater isolates ceased to produce pyocyanin at salinities above 30 g/L and 20 g/L, respectively. Culture supernatants of all 5 Pseudomonas isolates possessed the ability to restrict the growth of Vibrio spp. and maximum antagonistic effect on Vibrio harveyi was obtained when they were grown at salinities of 5 to 10 g/L. The marine isolate MCCB117, even when grown at a salinity of 45 g/L possessed the ability to inhibit Vibrio spp. Conclusions: The present investigation showed that Pseudomonas aeruginosa MCCB119 would be ideal for application in freshwater, MCCB102 and MCCB103 in brackish water and MCCB117 and MCCB118 in marine aquaculture systems as putative probiotics in the management of vibrios.

  18. Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Haagensen, Janus Anders Juul; Klausen, M; Ernst, RK

    2007-01-01

    During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicell......During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom......-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell...... that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties....

  19. Pseudomonas aeruginosa Population Structure Revisited

    Science.gov (United States)

    Pirnay, Jean-Paul; Bilocq, Florence; Pot, Bruno; Cornelis, Pierre; Zizi, Martin; Van Eldere, Johan; Deschaght, Pieter; Vaneechoutte, Mario; Jennes, Serge; Pitt, Tyrone; De Vos, Daniel

    2009-01-01

    At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P

  20. Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

    Directory of Open Access Journals (Sweden)

    Palloma Rodrigues Marinho

    2009-08-01

    Full Text Available Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3 isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

  1. Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2007-01-01

    The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 degrees C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.

  2. Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria.

    Science.gov (United States)

    Marinho, Palloma Rodrigues; Moreira, Ana Paula Barbosa; Pellegrino, Flávia Lúcia Piffano Costa; Muricy, Guilherme; Bastos, Maria do Carmo de Freire; Santos, Kátia Regina Netto dos; Giambiagi-deMarval, Marcia; Laport, Marinella Silva

    2009-08-01

    Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

  3. Pseudomonas helleri sp. nov. and Pseudomonas weihenstephanensis sp. nov., isolated from raw cow's milk.

    Science.gov (United States)

    von Neubeck, M; Huptas, C; Glück, C; Krewinkel, M; Stoeckel, M; Stressler, T; Fischer, L; Hinrichs, J; Scherer, S; Wenning, M

    2016-03-01

    Analysis of the microbiota of raw cow's milk and semi-finished milk products yielded seven isolates assigned to the genus Pseudomonas that formed two individual groups in a phylogenetic analysis based on partial rpoD and 16S rRNA gene sequences. The two groups could be differentiated from each other and also from their closest relatives as well as from the type species Pseudomonas aeruginosa by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. ANIb values within the groups were higher than 97.3 %, whereas similarity values to the closest relatives were 85 % or less. The major cellular lipids of strains WS4917T and WS4993T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q-9 in both strains, with small amounts of Q-8 in strain WS4917T. The DNA G+C contents of strains WS4917T and WS4993T were 58.08 and 57.30 mol%, respectively. Based on these data, strains WS4917T, WS4995 ( = DSM 29141 = LMG 28434), WS4999, WS5001 and WS5002 should be considered as representatives of a novel species of the genus Pseudomonas, for which the name Pseudomonas helleri sp. nov. is proposed. The type strain of Pseudomonas helleri is strain WS4917T ( = DSM 29165T = LMG 28433T). Strains WS4993T and WS4994 ( = DSM 29140 = LMG 28438) should be recognized as representing a second novel species of the genus Pseudomonas, for which the name Pseudomonas weihenstephanensis sp. nov. is proposed. The type strain of Pseudomonas weihenstephanensis is strain WS4993T ( = DSM 29166T = LMG 28437T).

  4. Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide.

    OpenAIRE

    Burns, F R; Paterson, C A; Gray, R D; Wells, J T

    1990-01-01

    Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL...

  5. Biodegradation of chlorpyrifos by bacterial genus Pseudomonas.

    Science.gov (United States)

    Gilani, Razia Alam; Rafique, Mazhar; Rehman, Abdul; Munis, Muhammad Farooq Hussain; Rehman, Shafiq Ur; Chaudhary, Hassan Javed

    2016-02-01

    Chlorpyrifos is an organophosphorus pesticide commonly used in agriculture. It is noxious to a variety of organisms that include living soil biota along with beneficial arthropods, fish, birds, humans, animals, and plants. Exposure to chlorpyrifos may cause detrimental effects as delayed seedling emergence, fruit deformities, and abnormal cell division. Contamination of chlorpyrifos has been found about 24 km from the site of its application. There are many physico-chemical and biological approaches to remove organophosphorus pesticides from the ecosystem, among them most promising is biodegradation. The 3,5,6-trichloro-2-pyridinol (TCP) and diethylthiophosphate (DETP) as primary products are made when chlorpyrifos is degraded by soil microorganisms which further break into nontoxic metabolites as CO(2), H(2)O, and NH(3). Pseudomonas is a diversified genus possessing a series of catabolic pathways and enzymes involved in pesticide degradation. Pseudomonas putida MAS-1 is reported to be more efficient in chlorpyrifos degradation by a rate of 90% in 24 h among Pseudomonas genus. The current review analyzed the comparative potential of bacterial species in Pseudomonas genus for degradation of chlorpyrifos thus, expressing an ecofriendly approach for the treatment of environmental contaminants like pesticides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Evaluation of gamma irradiation effect and Pseudomonas ...

    African Journals Online (AJOL)

    Antagonistic effect of Pseudomonas fluorescens and influence of gamma irradiation on the development of Penicillium expansum, the causal agent of postharvest disease on apple fruit was studied. P. fluorescens was originally isolated from rhizosphere of the apple trees. Suspension of P. fluorescens and P. expansum ...

  7. Extracytoplasmic function sigma factors in Pseudomonas syringae

    DEFF Research Database (Denmark)

    Kiil, Kristoffer; Oguiza, J.A.; Ussery, D.W.

    2005-01-01

    Genome analyses of the plant pathogens Pseudomonas syringae pv. tomato DC3000, pv. syringae B728a and pv. phaseolicola 1448A reveal fewer extracytoplasmic function (ECF) sigma factors than in related Pseudomonads with different lifestyles. We highlight the presence of a P. syringae-specific ECF...

  8. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protease production was 37°C at pH 9, with 2% inoculum in the medium for 24 h. .... Positive. Catalase test. Positive ... The enzyme activity gradually decreases from ... Effect of temperature on protease production by Pseudomonas fluorescens. 0 .... between RNA polymerase and upstream promotes DNA.

  9. Comparative evaluation of organic formulations of Pseudomonas ...

    African Journals Online (AJOL)

    An experiment was conducted in the laboratory and farm of the Department of Biotechnology, Gauhati University, to explore the potentiality of various organic formulations of Pseudomonas fluorescens (Pf) and to manage bacterial wilt disease of brinjal (Solanum melongena L.) under local conditions. Different organic ...

  10. High pressure inactivation of Pseudomonas in black truffle - comparison with Pseudomonas fluorescens in tryptone soya broth

    Science.gov (United States)

    Ballestra, Patricia; Verret, Catherine; Cruz, Christian; Largeteau, Alain; Demazeau, Gerard; El Moueffak, Abdelhamid

    2010-03-01

    Pseudomonas is one of the most common genera in black Perigord truffle. Its inactivation by high pressure (100-500 MPa/10 min) applied on truffles at sub-zero or low temperatures was studied and compared with those of Pseudomonas fluorescens in tryptone soya broth. Pressurization of truffles at 300 MPa/4 °C reduced the bacterial count of Pseudomonas by 5.3 log cycles. Higher pressures of 400 or 500 MPa, at 4 °C or 20 °C, allowed us to slightly increase the level of destruction to the value of ca. 6.5 log cycles but did not permit us to completely inactivate Pseudomonas. The results showed a residual charge of about 10 CFU/g. Pressure-shift freezing of truffles, which consists in applying a pressure of 200 MPa/-18 °C for 10 min and then quickly releasing this pressure to induce freezing, reduced the population of Pseudomonas by 3.3 log cycles. The level of inactivation was higher than those obtained with conventional freezing. Endogenous Pseudomonas in truffle was shown to be more resistant to high pressure treatments than P. fluorescens used for inoculation of broths.

  11. Genomic and Genetic Diversity within the Pseudomonas fluorescens Complex.

    Directory of Open Access Journals (Sweden)

    Daniel Garrido-Sanz

    Full Text Available The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been described as plant growth-promoting rhizobacteria (PGPR with potential applications in biocontrol and biofertilization. So far the phylogeny of this complex has been analyzed according to phenotypic traits, 16S rDNA, MLSA and inferred by whole-genome analysis. However, since most of the type strains have not been fully sequenced and new species are frequently described, correlation between taxonomy and phylogenomic analysis is missing. In recent years, the genomes of a large number of strains have been sequenced, showing important genomic heterogeneity and providing information suitable for genomic studies that are important to understand the genomic and genetic diversity shown by strains of this complex. Based on MLSA and several whole-genome sequence-based analyses of 93 sequenced strains, we have divided the P. fluorescens complex into eight phylogenomic groups that agree with previous works based on type strains. Digital DDH (dDDH identified 69 species and 75 subspecies within the 93 genomes. The eight groups corresponded to clustering with a threshold of 31.8% dDDH, in full agreement with our MLSA. The Average Nucleotide Identity (ANI approach showed inconsistencies regarding the assignment to species and to the eight groups. The small core genome of 1,334 CDSs and the large pan-genome of 30,848 CDSs, show the large diversity and genetic heterogeneity of the P. fluorescens complex. However, a low number of strains were enough to explain most of the CDSs diversity at core and strain-specific genomic fractions. Finally, the identification and analysis of group-specific genome and the screening for distinctive characters revealed a phylogenomic distribution of traits among the groups that provided insights into biocontrol and bioremediation applications as well as their role as

  12. Study on screening of anti-predator rhizosphere bacterium against Caenorhabditis elegans and its anti predation mechanism

    Directory of Open Access Journals (Sweden)

    HE Qingling

    2016-08-01

    Full Text Available Althoughmicrobial fertilizer is multi-effect,environmental friendly and long-term efficient,its practical application effect is but decreased for being prey by the other creators living in soil frequently.Many bacterium have developed their mechanisms that expel or kill worms to defend themselves from predators.Screening of anti-predator rhizosphere bacterium helps us to find out competitive plant growth promoting rhizobacteria(PGPR.Using Caenorhabditis elegans as sample,this study roughly observed two strains of biocontrol:Pseudomonas aurantiaca JD37 and Pseudomonas fluorescens P13.Using Escherichia coli OP50 as control group,we find the preference order of worms,from highest to lowest,is P13,OP50 and JD37.In slow killing assay,the death rate of worms for JD37 and P13 are 26.12% and 18.66% respectively.The activity and reproduction rate of C.elegans decrease when it is fed on JD37.The results of chemical and micro-biological study show that JD37 cannot produce any currently studied second metabolites which kill worms,while P13 can produce Hydrogen cyanide (HCN.All these results show that JD37 has the ability of anti-predator,and is more competitive under predation pressure,which suggests its broad application prospect as microbial fertilizer.

  13. Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity.

    Science.gov (United States)

    Shahbazi, Razieh; Kasra-Kermanshahi, Roha; Gharavi, Sara; Moosavi-Nejad, Zahra; Borzooee, Faezeh

    2013-06-01

    Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS-degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

  14. Phytochemical screening and antibacterial evaluation of stem bark ...

    African Journals Online (AJOL)

    SERVER

    2007-07-04

    Jul 4, 2007 ... Mallotus philippinensis var. Tomentosus is a medicinal plant, which was tested against Escherichia coli,. Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi and Bacillus subtilis. Phytochemi- cal screening of the stem bark of M. philippinensis indicates the presence of secondary ...

  15. An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

    Science.gov (United States)

    Koeva, Martina; Gutu, Alina D; Hebert, Wesley; Wager, Jeffrey D; Yonker, Lael M; O'Toole, George A; Ausubel, Frederick M; Moskowitz, Samuel M; Joseph-McCarthy, Diane

    2017-12-01

    Bacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of P. aeruginosa persisters. Stationary-phase cultures of P. aeruginosa were used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treated P. aeruginosa to human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections in CF patients through the eradication of bacterial persisters. Copyright © 2017 American Society for Microbiology.

  16. Cell viability and PSA secretion assays in LNCaP cells: a tiered in vitro approach to screen chemicals with a prostate-mediated effect on male reproduction within the ReProTect project.

    Science.gov (United States)

    Lorenzetti, Stefano; Marcoccia, Daniele; Narciso, Laura; Mantovani, Alberto

    2010-08-01

    Prostate function is critical for male fertility; nevertheless, prostate was so far overlooked in reproductive toxicity assays. Within the EU project ReProTect, the human prostate cell line LNCaP was utilized to identify molecules targeting prostate function by the integrated assessment of cell viability (MTS assay) and prostate-specific antigen (PSA) secretion as specific marker; a training set - five (anti)androgenic chemicals - and a ReProTect feasibility set - ten chemicals - were used. Several compounds reduced PSA only at cytotoxic concentrations. Androgens (DHT, MT) markedly increased PSA as did the herbicide glufosinate ammonium, not known as androgen agonist. Anti-androgens (2OH-flutamide, linuron, vinclozolin, di-n-butyl phthalate) also increased PSA, but the effect of magnitude was much lower than for androgens. The ER-binder bisphenol A reduced PSA, while increasing cell viability. At this stage, the approach can identify chemicals able to interfere with prostate function: further refinements may allow to include prostate effects in reproductive toxicity in vitro testing. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A

    OpenAIRE

    Mazor, Ronit; Vassall, Aaron N.; Eberle, Jaime A.; Beers, Richard; Weldon, John E.; Venzon, David J.; Tsang, Kwong Y.; Benhar, Itai; Pastan, Ira

    2012-01-01

    Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4+ T-cell epitopes in PE38. We incubated p...

  18. Pseudomonas lactis sp. nov. and Pseudomonas paralactis sp. nov., isolated from bovine raw milk.

    Science.gov (United States)

    von Neubeck, Mario; Huptas, Christopher; Glück, Claudia; Krewinkel, Manuel; Stoeckel, Marina; Stressler, Timo; Fischer, Lutz; Hinrichs, Jörg; Scherer, Siegfried; Wenning, Mareike

    2017-06-01

    Five strains, designated WS 4672T, WS 4998, WS 4992T, WS 4997 and WS 5000, isolated from bovine raw milk formed two individual groups in a phylogenetic analysis. The most similar species on the basis of 16S rRNA gene sequences were Pseudomonas azotoformans IAM 1603T, Pseudomonas gessardii CIP 105469T and Pseudomonas libanensis CIP 105460T showing 99.7-99.6 % similarity. Using rpoD gene sequences Pseudomonas veronii LMG 17761T (93.3 %) was most closely related to strain WS 4672T and Pseudomonas libanensis CIP 105460T to strain WS 4992T (93.3 %). The five strains could be differentiated from their closest relatives and from each other by phenotypic and chemotaxonomic characterization and ANIb values calculated from draft genome assemblies. ANIb values of strains WS 4992T and WS4671T to the closest relatives are lower than 90 %. The major cellular polar lipids of both strains are phosphatidylethanolamine, phosphatidylglycerol, a phospholipid and diphosphatidylglycerol, and their major quinone is Q-9. The DNA G+C content of strains WS 4992T and WS 4672T were 60.0  and 59.7  mol%, respectively. Based on these genotypic and phenotypic traits two novel species of the genus Pseudomonas are proposed: Pseudomonas lactis sp. nov. [with type strain WS 4992T (=DSM 29167T=LMG 28435T) and the additional strains WS 4997 and WS 5000], and Pseudomonasparalactis sp. nov. [with type strain WS 4672T (=DSM 29164T=LMG 28439T) and additional strain WS 4998].

  19. Evaluation of biocontrol potential of epiphytic fluorescent pseudomonas as associated with healthy fruits and vegetables against root rot and root knot pathogens of mungbean

    International Nuclear Information System (INIS)

    Habiba, A.; Noreen, R.; Ali, S. A.; Sultana, V.; Ara, J.

    2016-01-01

    Endophytic and rhizospheric fluorescent Pseudomonas have widely been used as biological control agents against soilborne plant pathogens. In this study, fifteen epiphytic fluorescent Pseudomonas isolated from the surfaces of citrus (grapefruit, orange and lemon) melon and tomato fruits were characterized for their in vitro activity against root rotting fungi viz., Macrophomina phaseolina, Fusarium solan