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  1. Do Multiwell Plate High Throughput Assays Measure Loss of Cell Viability Following Exposure to Genotoxic Agents?

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    Razmik Mirzayans

    2017-08-01

    Full Text Available Cell-based assays in multiwell plates are widely used for radiosensitivity and chemosensitivity assessment with different mammalian cell types. Despite their relative ease of performance, such assays lack specificity as they do not distinguish between the cytostatic (reversible/sustained growth arrest and cytotoxic (loss of viability effects of genotoxic agents. We recently reported studies with solid tumor-derived cell lines demonstrating that radiosensitivity as measured by multiwell plate colorimetric (e.g., XTT and fluorimetric (e.g., CellTiter-Blue assays reflects growth arrest but not loss of viability. Herein we report similar observations with cancer cell lines expressing wild-type p53 (A549 lung carcinoma or mutant p53 (MDA–MB-231 breast carcinoma after treatment with the chemotherapeutic drug cisplatin. Importantly, we show that treatment of cancer cells with concentrations of cisplatin that result in 50% effect (i.e., IC50 in multiwell plate assays trigger the emergence of growth arrested cells that exhibit highly enlarged morphology, remain viable and adherent to the culture dish, and metabolize the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT to its formazan derivative. The emergence of markedly enlarged viable cells complicates the interpretation of chemosensitivity data obtained with multiwell plate high throughput assays. Relying solely on IC50 values could be misleading.

  2. High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.

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    Sirenko, Oksana; Mitlo, Trisha; Hesley, Jayne; Luke, Steve; Owens, Windsor; Cromwell, Evan F

    2015-09-01

    There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

  3. A comparison of assays measuring the viability of Legionella ...

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    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

  4. A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities.

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    Mirza, Haris; Teo, Joshua D W; Upcroft, Jacqui; Tan, Kevin S W

    2011-02-01

    Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

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

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    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.

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

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

    2014-01-01

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

  7. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay.

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    Silva, Filomena S G; Starostina, Irina G; Ivanova, Vilena V; Rizvanov, Albert A; Oliveira, Paulo J; Pereira, Susana P

    2016-05-04

    The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  8. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.

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    Welch, Aaron Z; Koshland, Douglas E

    2013-12-01

    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space-saving method, amenable to high-throughput screens, for accurately measuring yeast cell viability. Copyright © 2013 John Wiley & Sons, Ltd.

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

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

    2015-02-01

    Whole-organism compound sensitivity assays are a valuable strategy in infectious diseases to identify active molecules. In schistosomiasis drug discovery, larval-stage Schistosoma allows the use of a certain degree of automation in the screening of compounds. Unfortunately, the throughput is limited, as drug activity is determined by manual assessment of Schistosoma viability by microscopy. To develop a simple and quantifiable surrogate marker for viability, we targeted glucose metabolism, which is central to Schistosoma survival. Lactate is the end product of glycolysis in human Schistosoma stages and can be detected in the supernatant. We assessed lactate as a surrogate marker for viability in Schistosoma drug screening assays. We thoroughly investigated parameters of lactate measurement and performed drug sensitivity assays by applying schistosomula and adult worms to establish a proof of concept. Lactate levels clearly reflected the viability of schistosomula and correlated with schistosomulum numbers. Compounds with reported potencies were tested, and activities were determined by lactate assay and by microscopy. We conclude that lactate is a sensitive and simple surrogate marker to be measured to determine Schistosoma viability in compound screening assays. Low numbers of schistosomula and the commercial availability of lactate assay reagents make the assay particularly attractive to throughput approaches. Furthermore, standardization of procedures and quantitative evaluation of compound activities facilitate interassay comparisons of potencies and, thus, concerted drug discovery approaches. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

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    Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

    2015-06-01

    Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

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    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-10-01

    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  12. Analysis of tumor and endothelial cell viability and survival using sulforhodamine B and clonogenic assays.

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    Woolston, Caroline; Martin, Stewart

    2011-01-01

    A variety of assays, and rationales for their use, exist to monitor viability and/or survival following cellular exposure to insult. Two commonly used in vitro assays are the sulforhodamine B assay and the clonogenic survival assay which can be used to monitor the efficacy of anticancer agents, either via direct tumor cell cytotoxicity or antiangiogenic mechanisms. The techniques described are suitable for studying survival in a number of different cell types; however, this chapter describes how they may be used in the assessment of chemo-/radiosensitivity. The methods are uncomplicated and robust as long as attention is paid to key optimization steps. Except for a multiwell plate reader they do not require any specialized equipment other than that found in a typical tissue-culture laboratory.

  13. Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability.

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    Byth, H A; Mchunu, B I; Dubery, I A; Bornman, L

    2001-01-01

    The Alamar Blue (AB) assay, which incorporates a medox indicator that changes colour or fluorescence in response to metabolic activity, is commonly used to assess quantitatively the viability and/or proliferation of mammalian cells and micro-organisms. In this study the AB assay was adapted for the determination of the viability of plant cells. Cell suspension cultures of tomato, Lycopersicon esculentum, L., with differing viabilities, served as the experimental model for a comparison of the AB assay with the conventional 2,3,5-triphenyltetrazolium chloride (TTC) viability assay. The AB assay showed a sigmoidal relationship between cell viability and AB reduction (as quantified by spectrofluorometry or spectrophotometry), which was similar to that obtained using the TTC assay. Both assays detected a significant reduction in cell viability after 48 h exposure to virulent Ralstonia solanacearum (biovar III), while the TTC assay, in addition, revealed cell proliferation in control cells from 24 to 72 h. The TTC assay detected cell proliferation over a wider range of cell densities, while the AB assay was more rapid and versatile whilst being non-toxic and thus allowing subsequent cell analysis.

  14. Toxoplasma gondii detection and viability assays in ham legs and shoulders from experimentally infected pigs.

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    Gomez-Samblas, M; Vilchez, S; Racero, J C; Fuentes, M V; Osuna, A

    2016-09-01

    Epidemiological studies of toxoplasmosis show that infection in humans is mainly caused by the consumption of raw, undercooked or cured meat. Cured "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. The "Serrano" ham is prepared from pork meat and undergoes a process known as curing and a subsequent fermentation without thermal or smoking treatments. The viability of Toxoplasma gondii in hams and shoulders from experimentally infected pigs that have been subject to different curing processes has been studied in order to evaluate the best method to completely eliminate the viable protozoa. The different treatments include, i) freezing the legs and shoulders below -20 °C for 3 days before salting with marine salt, ii) salting the meat with marine salt and nitrites, iii) salting only with marine salt (traditional process) and iv) salting with marine salt and then freezing at -20 °C for 3 days after the curing period. The ham leg samples were cured for 7 months and the shoulder samples for 5 months. The presence of T. gondii in the different treatments was studied by a "magnetic-capture" method for the isolation of T. gondii DNA and a quantitative real-time PCR to estimate the T. gondii burden in the ham legs and shoulders. The infectivity capacity of T. gondii in positive samples was assayed by bioassays in mice and some physicochemical parameters, such as pH, water activity (aw) and salt content, were evaluated at the end of the curing time. In all the cases where the samples were frozen the T. gondii infectivity was eliminated. In samples in which the meat was salted in marine salt plus nitrites, the parasite viability remained for longer than in the traditional salting process. The methods described here could be useful for producers to guarantee the safety of their products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.

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    Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary

    2012-09-01

    Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

  16. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Science.gov (United States)

    Limame, Ridha; Wouters, An; Pauwels, Bea; Fransen, Erik; Peeters, Marc; Lardon, Filip; De Wever, Olivier; Pauwels, Patrick

    2012-01-01

    Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0-100 nM) correlated well with SRB (Rho>0.95) with similar IC(50) values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased

  17. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Directory of Open Access Journals (Sweden)

    Ridha Limame

    Full Text Available BACKGROUND: Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (pathobiological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer and A549 (lung cancer cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964. Cytotoxic action by paclitaxel (0-100 nM correlated well with SRB (Rho>0.95 with similar IC(50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90 and optical density (OD measurement of extracted dye (Rho>0.95. Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95. Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE: The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on

  18. A simple method to measure cell viability in proliferation and cytotoxicity assays.

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    Borra, Ricardo Carneiro; Lotufo, Mônica Andrade; Gagioti, Sonia Maria; Barros, Fabiana de Mesquita; Andrade, Priscila Maria

    2009-01-01

    Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  19. Disruption of Functional Activity of Mitochondria during MTT Assay of Viability of Cultured Neurons.

    Science.gov (United States)

    Surin, A M; Sharipov, R R; Krasil'nikova, I A; Boyarkin, D P; Lisina, O Yu; Gorbacheva, L R; Avetisyan, A V; Pinelis, V G

    2017-06-01

    The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

  20. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra

    2009-09-01

    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  1. Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays.

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    Fongaro, Gislaine; Nascimento, Mariana A do; Rigotto, Caroline; Ritterbusch, Giseli; da Silva, Alessandra D' A; Esteves, Paulo A; Barardi, Célia R M

    2013-05-28

    Human adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. Water samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization. The analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 102 HAdV genome copies per milliliter of

  2. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

    Science.gov (United States)

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

    2013-12-31

    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  3. Comparison of Meldola's Blue Staining and Hatching Assay with Potato Root Diffusate for Assessment of Globodera sp. Egg Viability.

    Science.gov (United States)

    Kroese, Duncan; Zasada, Inga A; Ingham, Russell E

    2011-09-01

    Laboratory-based methods to test egg viability include staining with Meldola's Blue and/or juvenile (J2) hatching assays using potato root diffusate (PRD). These two methods have not been tested under identical conditions to directly compare their assessments of Globodera egg viability. Using two bioassay strategies, cysts from a Globodera sp. population found in Oregon were subjected to both viability assessment methods. In strategy one, intact cysts were first stained with Meldola's Blue (primary staining) and eggs were then transferred to PRD (secondary hatching). In the second strategy, intact cysts were exposed to PRD (primary hatching) and then unhatched eggs were transferred to Meldola's Blue (secondary staining). Two different cohorts of cysts were evaluated using these experimental strategies: cohort 1 was comprised of cysts produced on potato in the greenhouse that exhibited low hatch when exposed to PRD and cohort 2 consisted of field-collected cysts whose eggs yielded significant hatch when exposed to PRD. Percentage viability was calculated and is expressed as the number of hatched J2 or unstained eggs/total number of eggs within a cyst. With field-produced cysts, primary staining with Meldola's Blue and hatching with PRD produced similar viability estimates, with averages of 74.9% and 76.3%, respectively. In contrast, with greenhouse-produced cysts the two methods yielded much lower and unequal estimates 32.4% to 2.2%, respectively for primary hatching and staining methods. In addition, J2 hatch from unstained (viable) greenhouse-produced eggs was 13.7% after secondary exposure to PRD compared to 61.5% for field-produced eggs. The majority of eggs remaining unhatched after primary exposure to PRD (> 87%) stained with Meldola's Blue regardless of cyst cohort. Staining with Meldola's Blue provided a conservative assessment of egg viability compared to hatch assay with PRD regardless of diapause.

  4. A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ

    Directory of Open Access Journals (Sweden)

    Schulze Katja

    2011-11-01

    Full Text Available Abstract Background Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating or preparation-intensive (eg. fluorescent staining. In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation. Results The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism Synechocystis sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis. Conclusions The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.

  5. Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

    Science.gov (United States)

    Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

    2017-10-15

    Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu(II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    Science.gov (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. [Influence of high molecular weight polyethylene on viability of osteoblasts and new bone formation].

    Science.gov (United States)

    Ren, Gaohong; Lin, Angru; Pei, Guoxian; Hu, Basheng

    2006-02-01

    To investigate the influence of high molecular weight polyethylene (HMWP) on the viability of osteoblasts and new bone formation in the process of fracture healing, the osteoblasts derived from adult human bone marrow were cultured in HMWP maceration extract and normal culture medium. The viability of the osteoblasts was measured by MTT assay, and the function of the osteoblasts was detected by use of alkaline phosphatase test kit. The locked double-plating (steel plate and HMWP plate) was implanted and fixed at the artificial fracture of distal femur of dogs. Specimens were gained at 3, 6, 9 and 12 weeks postoperatively, examined with macroscopy, microscope and scanning electron microscope (SEM). The results showed that HMWP did no harm to osteoblasts. There is no significant difference in activities of proliferation and alkaline phosphatase between HMWP maceration extract and normal culture medium at each observation time of at 2,4,8, and 14 dyas (P>0. 05). Bone tissue under the implanted HMWP plate manifested no absorption; the new bones formed under the HMWP plate and gradually matured as time went on. It is demonstrated in this study that HMWP has no adverse influence on the viability of osteoblasts and new bone formation and it can be used as internal fixation implant in treating fractures.

  8. Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements.

    Science.gov (United States)

    Forbes, Alaina M; Lin, Huimin; Meadows, Gary G; Meier, G Patrick

    2014-08-01

    Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

  9. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.

    Science.gov (United States)

    Stepanenko, A A; Dmitrenko, V V

    2015-12-15

    The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

    Science.gov (United States)

    Zhou, Wenhui; Leippe, Donna; Duellman, Sarah; Sobol, Mary; Vidugiriene, Jolanta; O'Brien, Martha; Shultz, John W; Kimball, Joshua J; DiBernardo, Céline; Moothart, Leonard; Bernad, Laurent; Cali, James; Klaubert, Dieter H; Meisenheimer, Poncho

    2014-03-21

    Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Assessment of Radiobiological α/β Ratio in Lung Cancer and Fibroblast Cell Lines Using Viability Assays.

    Science.gov (United States)

    Karagounis, Ilias V; Skourti, Eleni K; Liousia, Maria V; Koukourakis, Michael I

    2017-01-01

    Altered fractionation is an area of intense clinical research in radiation oncology. Estimation of the α/β ratio of individual carcinomas after establishment of primary cell cultures from tumor biopsies may prove of importance in the individualization of radiotherapy schemes. Here we proposed a simple method to estimate the α/β ratio in cultured cell lines (two lung carcinomas: A549 and H1299; one lung fibroblast cell line: MRC5), using viability assays. For the A549 cell line, the α/β ratio ranged from 14-25 Gy, for H1299 from 11-43 Gy and for the MRC5 fibroblast cell line this was far lower, ranging from 0.69 to 6 Gy. The α/β ratio decreased when extracted from comparisons of lower dose per fraction schemes. The α/β ratio of a cell line can be easily defined after simple viability/dose fractionation experiments. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  12. Viability of developmental stages of Schistosoma mansoni quantified with xCELLigence worm real-time motility assay (xWORM

    Directory of Open Access Journals (Sweden)

    Gabriel Rinaldi

    2015-12-01

    Full Text Available Infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide. Where anthelmintic drugs are available, drug resistance is a major problem in livestock parasites, and a looming threat to public health. Monitoring the efficacy of these medicines and screening for new drugs has been hindered by the lack of objective, high-throughput approaches. Several cell monitoring technologies have been adapted for parasitic worms, including video-, fluorescence-, metabolism enzyme- and impedance-based tools that minimize the screening bottleneck. Using the xCELLigence impedance-based system we previously developed a motility-viability assay that is applicable for a range of helminth parasites. Here we have improved substantially the assay by using diverse frequency settings, and have named it the xCELLigence worm real-time motility assay (xWORM. By utilizing strictly standardized mean difference analysis we compared the xWORM output measured with 10, 25 and 50 kHz frequencies to quantify the motility of schistosome adults (human blood flukes and hatching of schistosome eggs. Furthermore, we have described a novel application of xWORM to monitor movement of schistosome cercariae, the developmental stage that is infectious to humans. For all three stages, 25 kHz was either optimal or near-optimal for monitoring and quantifying schistosome motility. These improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large.

  13. Microfluidic high viability neural cell separation using viscoelastically tuned hydrodynamic spreading

    DEFF Research Database (Denmark)

    Wu, Zhigang; Hjort, Klas; Wicher, Grzegorz

    2008-01-01

    A high viability microfluidic cell separation technique of high throughput was demonstrated based on size difference continuous mode hydrodynamic spreading with viscoelastic tuning. Using water with fluorescent dye as sample fluid and in parallel introducing as elution a viscoelastic biocompatible...

  14. Microscale Electro-Hydrodynamic Cell Printing with High Viability.

    Science.gov (United States)

    He, Jiankang; Zhao, Xiang; Chang, Jinke; Li, Dichen

    2017-12-01

    Cell printing has gained extensive attentions for the controlled fabrication of living cellular constructs in vitro. Various cell printing techniques are now being explored and developed for improved cell viability and printing resolution. Here an electro-hydrodynamic cell printing strategy is developed with microscale resolution (95%). Unlike the existing electro-hydrodynamic cell jetting or printing explorations, insulating substrate is used to replace conventional semiconductive substrate as the collecting surface which significantly reduces the electrical current in the electro-hydrodynamic printing process from milliamperes (>0.5 mA) to microamperes (printed cells. The smallest width of the electro-hydrodynamically printed hydrogel filament is 82.4 ± 14.3 µm by optimizing process parameters. Multiple hydrogels or multilayer cell-laden constructs can be flexibly printed under cell-friendly conditions. The printed cells in multilayer hydrogels kept alive and gradually spread during 7-days culture in vitro. This exploration offers a novel and promising cell printing strategy which might benefit future biomedical innovations such as microscale tissue engineering, organ-on-a-chip systems, and nanomedicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Fluorescent antibody-viability staining and beta-glucuronidase assay as rapid methods for monitoring Escherichia coli viability in coastal marine waters.

    Science.gov (United States)

    Caruso, G; De Pasquale, F; Mancuso, M; Zampino, D; Crisafi, E

    2006-01-01

    A faecal pollution monitoring of coastal Messina waters was performed by comparing three (microscopic, enzyme, and culture) methods. Evidence of Escherichia coli cells (29.99 to 96.79% of the total enteropathogenic serotypes) retaining their viability into the marine environment was shown. beta-Glucuronidase activity rates suggested that living cells were also metabolically active. Heavily polluted sites were detected, where improperly treated urban wastes were discharged. Significant relationships between microscopic and enzymatic data proved both methods to be suitable alternatives to the culture method for E. coli detection, improving environmental quality assessment.

  16. A Spectrophotometric Assay for Robust Viability Testing of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model

    Directory of Open Access Journals (Sweden)

    Laura Lopez Del Egido

    2017-05-01

    Full Text Available A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic as a model. We established that 10% [w/v] trichloroacetic acid (TCA/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.

  17. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

    Directory of Open Access Journals (Sweden)

    Emily Peak

    2010-07-01

    Full Text Available Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase, praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed, relevant to industrial (384-well microtiter plate compatibility and academic (96-well microtiter plate compatibility settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species

  18. Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells.

    Science.gov (United States)

    Shenoy, Niraj; Stenson, Mary; Lawson, Joshua; Abeykoon, Jithma; Patnaik, Mrinal; Wu, Xiaosheng; Witzig, Thomas

    2017-02-27

    Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.18.

  19. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

  20. PENGHAMBATAN CAJUPUTS CANDY TERHADAP VIABILITAS KHAMIR Candida albicans SECARA IN VITRO [Inhibition of Cajuputs Candy Toward the Viability of Candida albicans by using In Vitro Assay

    Directory of Open Access Journals (Sweden)

    C. Hanny Wijaya1*

    2014-12-01

    Full Text Available The utilization of cajuput essential oil as a flavor in candy may produce a physiological active added value. Some compounds of cajuput plant (Melaleuca cajuputi L have been reported for their anti-microbial activities. Candida albicans is a normal commensal organism in human mouth. However, it may become virulent and responsible for oral diseases known as oral candidiasis. This study aimed to determine the effect of cajuput and peppermint oil in cajuputs candy in inhibiting the C. albicans biofilms formation by using in vitro biofilm assay and viability assay. Furthermore, the influence of concentration of cajuput oil on the anti-microbial activities had been analyzed. All the tested concentration of cajuput oil in cajuputs candy was effective to inhibit the viability of C. albicans. The provision of flavor components of cajuput and peppermint oil could produce synergistic effects compared to a single flavor component. The addition of cajuput oil at 0.6% was able to inhibit the viability of C. albicans. The activities of the cajuput oil showed positive correlation to the concentration. The variable of plus and minus 0.1% addition of the cajuput oil concentration, however, produced no significant difference to inhibit the growth of C. albicans in biofilm. Sensory test, hedonic test, was conducted to evaluate the flavor, aroma, and overall attributes, resulting in no significant difference between 0.6 to 0.8% additions of cajuput oil upon the sensory acceptance.

  1. Changes in subendocardial viability ratio with acute high-altitude exposure and protective role of acetazolamide.

    Science.gov (United States)

    Salvi, Paolo; Revera, Miriam; Faini, Andrea; Giuliano, Andrea; Gregorini, Francesca; Agostoni, Piergiuseppe; Becerra, Carlos G Ramos; Bilo, Grzegorz; Lombardi, Carolina; O'Rourke, Michael F; Mancia, Giuseppe; Parati, Gianfranco

    2013-04-01

    High-altitude tourism is increasingly frequent, involving also subjects with manifest or subclinical coronary artery disease. Little is known, however, on the effects of altitude exposure on factors affecting coronary perfusion. The aim of our study was to assess myocardial oxygen supply/demand ratio in healthy subjects during acute exposure at high altitude and to evaluate the effect of acetazolamide on this parameter. Forty-four subjects (21 men, age range: 24-59 years) were randomized to double-blind acetazolamide 250 mg bid or placebo. Subendocardial viability ratio and oxygen supply/demand ratio were estimated on carotid artery by means of a validated PulsePen tonometer, at sea level, before and after treatment, and after acute and more prolonged exposure to high altitude (4559 m). On arrival at high altitude, subendocardial viability ratio was reduced in both placebo (from 1.63±0.15 to 1.18±0.17; PSubendocardial viability ratio returned to sea level values (1.65±0.24) after 3 days at high altitude under acetazolamide but remained lower than at sea level under placebo (1.42±0.22; Psubendocardial oxygen supply triggered by exposure to hypobaric hypoxia. Further studies involving also subjects with known or subclinical coronary artery disease are needed to confirm a protective action of acetazolamide on myocardial viability under high-altitude exposure.

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

    Science.gov (United States)

    Chan, Grace Ka Yan; Kleinheinz, Tracy L; Peterson, David; Moffat, John G

    2013-01-01

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

  3. Low nutrient availability reduces high-irradiance–induced viability loss inoceanic phytoplankton

    NARCIS (Netherlands)

    Kulk, G.; van de Poll, W.H.; Visser, R.J.W.; Buma, A.G.J.

    2013-01-01

    In situ viability of oceanic phytoplankton may be relatively low in open oceans. This is assumed to be related to the high-irradiance and low-nutrient conditions typical for oligotrophic regions. However, experimental evidence for this phenomenon was not yet available. In the present study, the

  4. Modeling the impact of high temperatures on microalgal viability and photosynthetic activity.

    Science.gov (United States)

    Béchet, Quentin; Laviale, Martin; Arsapin, Nicolas; Bonnefond, Hubert; Bernard, Olivier

    2017-01-01

    Culture collapse due to high temperatures can significantly impact the profitability of outdoor algal cultivation systems. The objective of this study was to model for the first time the impact of high temperatures on algal activity and viability. Viability measurements on Dunaliella salina cultures were based on cytometry with two fluorescent markers (erythrosine and fluorescein di-acetate), and photosynthetic activity was measured by Pulse Amplitude Modulation (PAM) fluorometry. Kinetic studies revealed that viability and activity losses during exposure to high temperatures could be described by a Weibull model. Both mortality and activity were shown to be functions of the thermal dose received by the algae, defined as the product of duration of exposure to high temperatures and an exponential function of temperature. Simulations at five climatic locations revealed that culture collapse due to high temperatures could impact productivity of D. salina in non-temperature-controlled outdoor photobioreactors by 35 and 40% in arid and Mediterranean climates, respectively. The model developed in this study can be used to forecast the impact of high temperatures on algal biofuel productivity. When coupled with models predicting the temperature of outdoor cultivation systems, this model can also be used to select the best combination of location, system geometry, and algal species to minimize the risks of culture collapse and therefore maximize biofuel productivity.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-12

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

  6. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

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

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

    Science.gov (United States)

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

    2015-02-27

    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

  8. A comparison of assays measuring the viability of Legionella pneumophila after treatment with copper and silver ions

    Science.gov (United States)

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could p...

  9. Delayed enhancement imaging of myocardial viability: low-dose high-pitch CT versus MRI

    Energy Technology Data Exchange (ETDEWEB)

    Goetti, Robert; Feuchtner, Gudrun; Stolzmann, Paul; Donati, Olivio F.; Frauenfelder, Thomas; Leschka, Sebastian; Alkadhi, Hatem [University Hospital Zurich, Department of Radiology, Zurich (Switzerland); Wieser, Monika; Plass, Andre [University Hospital Zurich, Division of Cardiac and Vascular Surgery, Zurich (Switzerland)

    2011-10-15

    To evaluate the accuracy of high-pitch delayed enhancement (DE) CT for the assessment of myocardial viability with MRI as the reference standard. Twenty-four patients (mean age 66.9 {+-} 9.2 years) with coronary artery disease underwent DE imaging with 128-slice dual-source CT (prospective electrocardiography (ECG)-triggering) and MRI at 1.5 T. Two observers assessed DE transmurality per segment, and measured signal intensity (MRI) or attenuation (CT) in infarcted and healthy myocardium and noise in the left ventricular blood pool for calculating contrast-to-noise ratios (CNR). 75/408 (18.4%) segments in 18/24 patients (75.0%) showed DE in MRI, of which 28 segments in 10/24 (41.7%) patients were non-viable (scar tissue transmurality >50%). Sensitivity, specificity and accuracy of CT for diagnosis of non-viability were 60.7%, 96.8% and 94.4% per segment, and 90.0%, 92.9% and 91.7% per patient. CNR was significantly higher in MR (7.4 {+-} 3.0 vs. 4.6 {+-} 1.5; p = 0.018), and image noise significantly lower (11.6 {+-} 5.7 vs.15.0 {+-} 4.5; p = 0.019). Radiation dose of DECT was 0.89 {+-} 0.07 mSv. CTDE imaging in the high-pitch mode enables myocardial viability assessment at a low radiation dose and good accuracy compared with MR, although associated with a lower CNR and higher noise. (orig.)

  10. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    Science.gov (United States)

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  11. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2014-01-01

    Full Text Available Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L, constant high glucose (CHG (25 mmol/L, and IHG (rotation per 24 h in 11.1 or 25 mmol/L for 7 days. Reactive oxygen species (ROS, xanthine oxidase (XOD level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG.

  12. A high-throughput fluorescence polarization assay for inhibitors of gyrase B.

    Science.gov (United States)

    Glaser, Bryan T; Malerich, Jeremiah P; Duellman, Sarah J; Fong, Julie; Hutson, Christopher; Fine, Richard M; Keblansky, Boris; Tang, Mary J; Madrid, Peter B

    2011-02-01

    DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

  13. The relationship between sperm viability and DNA fragmentation rates.

    Science.gov (United States)

    Samplaski, Mary K; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A

    2015-05-14

    In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of >30% indicating subfertility. We postulated that in some men, the sperm viability assay could predict the sperm DNA fragmentation rates. This in turn could reduce the need for sperm DNA fragmentation assay testing, simplifying the infertility investigation and saving money for infertile couples. All men having semen analyses with both viability and DNA fragmentation testing were identified via a prospectively collected database. Viability was measured by eosin-nigrosin assay. DNA fragmentation was measured using the sperm chromosome structure assay. The relationship between DNA fragmentation and viability was assessed using Pearson's correlation coefficient. From 2008-2013, 3049 semen analyses had both viability and DNA fragmentation testing. A strong inverse relationship was seen between sperm viability and DNA fragmentation rates, with r=-0.83. If viability was ≤50% (n=301) then DNA fragmentation was ≥ 30% for 95% of the samples. If viability was ≥75% (n=1736), then the DNA fragmentation was ≤30% for 95% of the patients. Sperm viability correlates strongly with DNA fragmentation rates. In men with high levels of sperm viability≥75%, or low levels of sperm viability≤ 30%, DFI testing may be not be routinely necessary. Given that DNA fragmentation testing is substantially more expensive than vitality testing, this may represent a valuable cost-saving measure for couples undergoing a fertility evaluation.

  14. Murine High Specificity/Sensitivity Competitive Europium Insulin Autoantibody Assay

    Science.gov (United States)

    Babaya, Naru; Liu, Edwin; Miao, DongMei; Li, Marcella; Yu, Liping

    2009-01-01

    Abstract Background Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing 125I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays. Methods To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay). Results The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected. Conclusions Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format. PMID:19344197

  15. Viability of common wine spoilage organisms after exposure to high power ultrasonics.

    Science.gov (United States)

    Luo, Hua; Schmid, Frank; Grbin, Paul R; Jiranek, Vladimir

    2012-05-01

    Microbial spoilage of wine can lead to significant economic loss. At present sulfur dioxide is the main additive to juice/must/wine to prevent and control microbial spoilage. As an alternative, or complement to SO(2), high power ultrasonics (HPU) may be used to control microbes. Several wine spoilage yeasts and bacteria were treated with HPU in saline (0.9% w/v NaCl), juice and red wine to assess their susceptibility to HPU. Significant killing was seen across several yeasts and bacteria commonly associated with winemaking and wine spoilage. In general the viability of yeast was more affected than that of bacteria. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Effects of two plant growth regulators, indole-3-acetic acid and β-naphthoxyacetic acid, on genotoxicity in Drosophila SMART assay and on proliferation and viability of HEK293 cells from the perspective of carcinogenesis.

    Science.gov (United States)

    Karadeniz, Asuman; Kaya, Bülent; Savaş, Burhan; Topcuoğlu, Ş Fatih

    2011-10-01

    In this study, the mutagenic and recombinogenic effects of indole-3-acetic acid (IAA), a plant growth regulator naturally synthesized in plants but produced synthetically, and β-naphthoxyacetic acid (BNOA), a synthetic plant growth regulator widely used in agricultural regions, were investigated using the somatic mutation and recombination test (SMART) in Drosophila wings. The effect of the same plant growth regulators against the proliferation and viability of a human immortalized embryonic kidney HEK293 cells which is at the early stage of carcinogenesis were also examined with MTT and trypan-blue exclusion assays. For the SMART assay, two different crosses were used: a standard and a high-bioactivation (HB) cross, involving the flare-3 and the multiple wing hairs markers. The HB cross involved flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits the more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. The results show that IAA and BNOA are not mutagenic or recombinogenic in the wing cells of Drosophila. Furthermore, neither plant growth regulator affected the proliferation rate of HEK293 cells; however, both of them induced cell death at high concentrations.

  17. One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays.

    Science.gov (United States)

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

    2016-12-01

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

  18. Effect of high hydrostatic pressure on mycelial development, spore viability and enzyme activity of Penicillium Roqueforti.

    Science.gov (United States)

    Martínez-Rodríguez, Yamile; Acosta-Muñiz, Carlos; Olivas, Guadalupe I; Guerrero-Beltrán, José; Rodrigo-Aliaga, Dolores; Mujica-Paz, Hugo; Welti-Chanes, Jorge; Sepulveda, David R

    2014-01-03

    This study investigated the effect of high hydrostatic pressure treatments on mycelial development, spore viability, and total proteolytic and lipolytic activity of Penicillium roqueforti PV-LYO 10 D. Fungus growing in liquid medium was pressure-treated at 300, 400, and 500 MPa for 10 min at 20°C following seven days of incubation at 25°C and analyzed periodically up to day 9 after treatments to evaluate the effect on fungal growth. Mycelial mass of P. roqueforti was significantly affected at all pressure treatments evaluated, being 15.48%, 22.28%, 30.03%, and 12.53% lower than controls on day 1, 3, 6, and 9 after 300 MPa treatment, respectively. In a similar way, at 400 and 500 MPa, mycelial mass was 31.08% and 60.34% lower than controls one day after treatments and 49.74% and 80.85% lower on day 9, respectively. The viability of P. roqueforti spores decreased by 36.53% at 300 MPa, and complete inactivation took place at ≥400 MPa from an initial count of 7 log cfu/mL. Total proteolytic activity was not significantly affected at 300 MPa but was reduced by 18.22% at 400 MPa and by 43.18% at 500 MPa. Total lipolytic activity also decreased as the intensity of the pressure treatments increased. 21.69%, 39.12%, and 56.26% activity reductions were observed when treatments of 300, 400 and 500 MPa were applied, respectively. The results from this study show that pressure treatments are able to control growth, inactivate spores, and alter enzyme activity of P. roqueforti, which could be of interest in extending the shelf-life of blue-veined cheeses and other food products. © 2013.

  19. Cardiac troponin T determination by a highly sensitive assay in postmortem serum and pericardial fluid.

    Science.gov (United States)

    González-Herrera, Lucas; Valenzuela, Aurora; Ramos, Valentín; Blázquez, Antonia; Villanueva, Enrique

    2016-06-01

    The main objective of this study was to test, for the first time, a highly sensitive cardiac troponin T (cTnThs) assay in postmortem serum and pericardial fluid and to evaluate cardiac troponin T (cTnT) levels and their stability after death at different postmortem intervals, in an attempt to determine the viability of the cTnThs assay in the postmortem diagnosis of the cause of death. cTnT levels were determined in serum and pericardial fluid samples taken from 58 cadavers at known postmortem intervals, whose causes of death were categorized into the following groups: (1) sudden cardiac deaths, (2) multiple trauma, (3) mechanical asphyxia, and (4) other natural deaths. cTnT was determined by inmunoassay, using the Troponin T highly sensitive STAT assay (Roche(®)). Average cTnT levels measured by a highly sensitive assay in postmortem serum were markedly higher than clinical serum levels. Moreover, similar results, higher cTnT levels in postmortem pericardial fluid, were obtained when compared to levels found in pericardial fluid taken from two living patients during coronary artery bypass surgery. cTnT levels in both postmortem fluids remained stable for up to 34 h after death. No differences in cTnT levels in either postmortem fluid by sex and age were detected. Levels of cTnT found in pericardial fluid in the other natural deaths group were significantly lower than the cTnT levels found in that postmortem fluid from any of the other causes of death groups. It is therefore reasonable to conclude that determination of cTnT by a highly sensitive assay in pericardial fluid can provide forensic pathologists with a complementary test to the diagnosis of cause of death.

  20. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

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

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

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

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

  2. Cell viability and MRI performance of highly efficient polyol-coated magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Arteaga-Cardona, Fernando [Universidad de las Américas de Puebla, Departamento de Ciencias Químico-Biológicas (Mexico); Gutiérrez-García, Eric [Instituto Literario, Universidad Autónoma del Estado de México (Mexico); Hidalgo-Tobón, Silvia, E-mail: shid@xanum.uam.mx [Universidad Autónoma Metropolitana, Departamento de Física (Mexico); López-Vasquez, Ciro; Brito-Barrera, Yazmín A. [Universidad de las Américas de Puebla, Departamento de Ciencias Químico-Biológicas (Mexico); Flores-Tochihuitl, Julia [Benemérita Universidad Autónoma de Puebla, Facultad de Estomatología (Mexico); Angulo-Molina, Aracely [Universidad de Sonora, Departamento de Ciencias Químico-Biológicas (Mexico); Reyes-Leyva, Julio R. [Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica de Oriente (CIBIOR) (Mexico); González-Rodríguez, Roberto; Coffer, Jeffery L. [Texas Christian University, Department of Chemistry (United States); Pal, Umapada [Benemérita Universidad Autónoma de Puebla, Apdo, Instituto de Física (Mexico); and others

    2016-11-15

    This work aimed at determining conditions that would allow us to control the size of the NPs and create a system with characteristics apt for biomedical applications. We describe a comprehensive study on the synthesis and physical characterization of two highly sensitive sets of triethylene glycol (TREG) and polyethylene glycol (PEG)-coated superparamagnetic iron oxide nanoparticles (SPIONs) to be evaluated for use as magnetic resonance (MR) contrast agents. The ferrofluids demonstrated excellent colloidal stability in deionized water at pH 7.0 as indicated by dynamic light scattering (DLS) data. The magnetic relaxivities, r{sub 2}, were measured on a 1.5 T clinical MRI instrument. Values in the range from 205 to 257 mM{sup −1} s{sup −1} were obtained, varying proportionally to the SPIONs’ sizes and coating nature. Further in vitro cell viability tests and in vivo biodistribution analyses of the intravenously administered nanoparticles showed that the prepared systems have good biocompatibility and migrate to several organs, mainly the meninges, spleen, and liver. Based on these results, our findings demonstrated the potential utility of these nanosystems as clinical contrast agents for MR imaging.

  3. Viability Theory

    CERN Document Server

    Aubin, Jean-Pierre; Saint-Pierre, Patrick

    2011-01-01

    Viability theory designs and develops mathematical and algorithmic methods for investigating the adaptation to viability constraints of evolutions governed by complex systems under uncertainty that are found in many domains involving living beings, from biological evolution to economics, from environmental sciences to financial markets, from control theory and robotics to cognitive sciences. It involves interdisciplinary investigations spanning fields that have traditionally developed in isolation. The purpose of this book is to present an initiation to applications of viability theory, explai

  4. A high-throughput biliverdin assay using infrared fluorescence.

    Science.gov (United States)

    Berlec, Aleš; Štrukelj, Borut

    2014-07-01

    Biliverdin is an intermediate of heme degradation with an established role in veterinary clinical diagnostics of liver-related diseases. The need for chromatographic assays has so far prevented its wider use in diagnostic laboratories. The current report describes a simple, fast, high-throughput, and inexpensive assay, based on the interaction of biliverdin with infrared fluorescent protein (iRFP) that yields functional protein exhibiting infrared fluorescence. The assay is linear in the range of 0-10 µmol/l of biliverdin, has a limit of detection of 0.02 μmol/l, and has a limit of quantification of 0.03 µmol/l. The assay is accurate with relative error less than 0.15, and precise, with coefficient of variation less than 5% in the concentration range of 2-9 µmol/l of biliverdin. More than 95% of biliverdin was recovered from biological samples by simple dimethyl sulfoxide extraction. There was almost no interference by hemin, although bilirubin caused an increase in the biliverdin concentration, probably due to spontaneous oxidation of bilirubin to biliverdin. The newly developed biliverdin assay is appropriate for reliable quantification of large numbers of samples in veterinary medicine.

  5. Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

    Science.gov (United States)

    Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

    2017-01-01

    The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

  6. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    Science.gov (United States)

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  7. Assessment of cytotoxicity of carbon nanoparticles using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay.

    Science.gov (United States)

    Schrand, Amanda M; Lin, Jonathan B; Hussain, Saber M

    2012-01-01

    Ever since the discovery of carbon nanotubes, there has been an increasing interest in technologies that rely upon these incredibly small particles for their unique properties. However, assessment of their biological consequences has been riddled with assay limitations. Here, we describe application of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay to study cytotoxicity of various carbon-based nanomaterials on cells and discuss some pitfalls of this method.

  8. Towards a systematic assessment of assay interference: Identification of extensively tested compounds with high assay promiscuity

    National Research Council Canada - National Science Library

    Gilberg Erik; Stumpfe Dagmar; Bajorath Jürgen

    2017-01-01

    A large-scale statistical analysis of hit rates of extensively assayed compounds is presented to provide a basis for a further assessment of assay interference potential and multi-target activities...

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

    Science.gov (United States)

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

    2015-01-01

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

  10. 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.

  11. 3D Printed Vascular Networks Enhance Viability in High-Volume Perfusion Bioreactor.

    Science.gov (United States)

    Ball, Owen; Nguyen, Bao-Ngoc B; Placone, Jesse K; Fisher, John P

    2016-12-01

    There is a significant clinical need for engineered bone graft substitutes that can quickly, effectively, and safely repair large segmental bone defects. One emerging field of interest involves the growth of engineered bone tissue in vitro within bioreactors, the most promising of which are perfusion bioreactors. Using bioreactor systems, tissue engineered bone constructs can be fabricated in vitro. However, these engineered constructs lack inherent vasculature and once implanted, quickly develop a necrotic core, where no nutrient exchange occurs. Here, we utilized COMSOL modeling to predict oxygen diffusion gradients throughout aggregated alginate constructs, which allowed for the computer-aided design of printable vascular networks, compatible with any large tissue engineered construct cultured in a perfusion bioreactor. We investigated the effect of 3D printed macroscale vascular networks with various porosities on the viability of human mesenchymal stem cells in vitro, using both gas-permeable, and non-gas permeable bioreactor growth chamber walls. Through the use of 3D printed vascular structures in conjunction with a tubular perfusion system bioreactor, cell viability was found to increase by as much as 50% in the core of these constructs, with in silico modeling predicting construct viability at steady state.

  12. High throughput comet assay to study genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Naouale El Yamani

    2015-06-01

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

  13. Honey is cytotoxic towards prostate cancer cells but interacts with the MTT reagent: Considerations for the choice of cell viability assay.

    Science.gov (United States)

    Abel, Sean D A; Baird, Sarah K

    2018-02-15

    Honey is a complex biological substance, consisting mainly of sugars, phenolic compounds and enzymes. Using five quick and accessible assays for measuring honey's cytotoxicity in vitro, we found honey is cytotoxic towards prostate cancer cells PC3 and DU145. However, the level of cell death varied with assay. The MTT assay was confounded by the reduction of the MTT reagent by honey's reducing sugars and phenolic compounds, and the lactate dehydrogenase assay was invalidated by honey oxidising the enzyme cofactor NADH. The sulforhodamine B assay gave valid results, but measures only protein content, providing no information about cell death in the remaining cells. The trypan blue assay and a microscope-based propidium iodide/Hoechst staining assay assess only late stage membrane permeability. However, the propidium iodide/Hoechst assay gives morphological information about cell death mechanism. A combination of the sulforhodamine B and propidium iodide/Hoechst assays would provide the most accurate quantification of honey cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX--Viability Assay and Morphological Stability.

    Science.gov (United States)

    Meeßen, J; Wuthenow, P; Schille, P; Rabbow, E; de Vera, J-P P; Ott, S

    2015-08-01

    Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit-space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance.

  15. Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX—Viability Assay and Morphological Stability

    Science.gov (United States)

    Wuthenow, P.; Schille, P.; Rabbow, E.; de Vera, J.-P.P.; Ott, S.

    2015-01-01

    Abstract Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit–space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance. Key Words: Astrobiology—BIOMEX—EXPOSE-R2—Extremotolerance—Lichens. Astrobiology 15, 601–615. PMID:26218403

  16. The Architect Syphilis assay for antibodies to Treponema pallidum: an automated screening assay with high sensitivity in primary syphilis.

    Science.gov (United States)

    Young, H; Pryde, J; Duncan, L; Dave, J

    2009-02-01

    To determine the sensitivity and specificity of the Architect Syphilis Chemiluminescence Assay (CLIA): a new highly automated screening test for syphilis. To establish the sensitivity of the Architect Syphilis assay we tested 129 stored sera from serologically characterised cases of untreated syphilis. The sera were selected to contain a disproportionately high number of primary infections. There were 79 primary infections, 29 secondary infections, 9 early latent infections and 12 latent syphilis of unknown duration. To establish the specificity of the assay we tested 1107 sera that had been submitted for routine syphilis serology. The Architect CLIA and the Treponema pallidum particle agglutination test (TPPA) were in total agreement for all untreated infection with sensitivity of 98.4%. This was significantly higher than the sensitivity of the Murex immune capture enzyme (ICE) immunoassay (86%, pArchitect and ICE assays was entirely due to primary stage syphilis (97.5% vs 77.2%, pArchitect CLIA was very high (99.1%, 1049/1059) it was significantly lower (p = 0.016) than that of the Murex ICE assay (99.9%). The Architect CLIA is significantly more sensitive than the Murex ICE screening assay in detecting primary syphilis but it is significantly less specific. Given the relatively high levels of early syphilis, we consider a small increase in the number of confirmatory tests required to exclude false-positive results is worthwhile to increase the detection of primary syphilis by 20%.

  17. Advanced high-power pulsed light device to decontaminate food from pathogens: effects on Salmonella typhimurium viability in vitro.

    Science.gov (United States)

    Luksiene, Z; Gudelis, V; Buchovec, I; Raudeliuniene, J

    2007-11-01

    The aim of this study was to construct an advanced high-power pulsed light device for decontamination of food matrix and to evaluate its antibacterial efficiency. Key parameters of constructed device-emitted light spectrum, pulse duration, pulse power density, frequency of pulses, dependence of emitted spectrum on input voltage, irradiation homogenicity, possible thermal effects as well as antimicrobial efficiency were evaluated. Antimicrobial efficiency of high-power pulsed light technique was demonstrated and evaluated by two independent methods - spread plate and Miles-Misra method. Viability of Salmonella typhimurium as function of a given light dose (number of pulses) and pulse frequency was examined. According to the data obtained, viability of Salmonella typhimurium reduced by 7 log order after 100 light pulses with power density 133 W cm(-2). In addition, data indicate, that the pulse frequency did not influence the outcome of pathogen inactivation in the region 1-5 Hz. Moreover, no hyperthermic effect was detected during irradiation even after 500 pulses on all shelves with different distance from light source and subsequently different pulse power density (0-252 W cm(-2)). Newly constructed high-power pulsed light technique is effective nonthermal tool for inactivation of Salmonella typhimurium even by 7 log order in vitro. Novel advanced high-power pulsed light device can be a useful tool for development of nonthermal food decontamination technologies.

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

    Science.gov (United States)

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

    2016-01-10

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

  19. A High-Throughput Screening Assay to Detect ...

    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-disrupting chemicals that adversely impact neurodevelopment. The AUR-TPO assay was recently developed to screen >1,900 ToxCast chemicals for potential TPO inhibition activity. Parallel assays were used to determine which AUR-TPO actives were more selective for TPO inhibition. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO assay and an orthogonal peroxidase oxidation assay using guaiacol as substrate to confirm putative TPO inhibition profiles. Bioactivity results from the AUR-TPO assay were used to identify chemical substructures associated with in vitro TPO inhibition. Substructure profiles were generated for each chemical in the ToxCast test set using the publicly-available ToxPrint 2.0 chemotypes. Chemotypes enriched among the putative TPO inhibitors were identified using a cumulative hypergeometric probability (p assay:

  20. Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay

    Science.gov (United States)

    Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus

    2013-01-01

    There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude. PMID:23677392

  1. Antiretrovirals for low income countries: an analysis of the commercial viability of a highly competitive market.

    Science.gov (United States)

    Nakakeeto, Olive N; Elliott, Brian V

    2013-02-15

    The price of antiretroviral drugs (ARVs) in low income countries declined steadily in recent years. This raises concerns about the commercial viability of the market of ARVs in low income countries. Using 2 costing scenarios, we modeled the production cost of the most commonly used ARVs in low income countries in 2010 and 2012, and assessed whether, at the median price paid by low income countries, their manufacturers would still make profits. By interviews we consulted 11 generic manufacturers on the current state of the ARV market, and on what would be required to ensure their continued commitment to supply ARVs to low income countries. Using the lowest prices for active pharmaceutical ingredients (API) quoted to WHO, and applying published assumptions about the production cost of ARVs, our baseline estimate was that Indian generic manufacturers would have made profits on only 1 out of 13 formulations of ARVs in both 2010 and 2012, and publicly owned manufacturers would have made profits on 5 and 3 out of 13 formulations in 2010 and 2012, respectively. We needed to assume a 20% and a 40% lower API cost for our model to predict that publicly owned and Indian manufacturers, respectively, would make profits on the sale of the majority of their ARVs. Between 2010 and 2012, we estimate that--across the ARV portfolio--the gross profit on sales of ARVs to low income countries decreased with between 6% and 7% of their sales price. Generic manufacturers consider that current prices are unsustainable. They suggested amendments to the tender procedures, simplified regulatory procedures, improved forecasting, and simplification of the ARV guidelines as critical improvements to maintain a viable ARV market. While recent price decreases indicate that there is still space for price reduction, our estimate that gross profit margin on sales decreased by 6 to 7% between 2010 and 2012 lends credibility to assertions by generic manufacturers that the ARV market in low income

  2. Antiretrovirals for low income countries: an analysis of the commercial viability of a highly competitive market

    Science.gov (United States)

    2013-01-01

    Background The price of antiretroviral drugs (ARVs) in low income countries declined steadily in recent years. This raises concerns about the commercial viability of the market of ARVs in low income countries. Methods Using 2 costing scenarios, we modeled the production cost of the most commonly used ARVs in low income countries in 2010 and 2012, and assessed whether, at the median price paid by low income countries, their manufacturers would still make profits. By interviews we consulted 11 generic manufacturers on the current state of the ARV market, and on what would be required to ensure their continued commitment to supply ARVs to low income countries. Results Using the lowest prices for active pharmaceutical ingredients (API) quoted to WHO, and applying published assumptions about the production cost of ARVs, our baseline estimate was that Indian generic manufacturers would have made profits on only 1 out of 13 formulations of ARVs in both 2010 and 2012, and publicly owned manufacturers would have made profits on 5 and 3 out of 13 formulations in 2010 and 2012, respectively. We needed to assume a 20% and a 40% lower API cost for our model to predict that publicly owned and Indian manufacturers, respectively, would make profits on the sale of the majority of their ARVs. Between 2010 and 2012, we estimate that - across the ARV portfolio - the gross profit on sales of ARVs to low income countries decreased with between 6% and 7% of their sales price. Generic manufacturers consider that current prices are unsustainable. They suggested amendments to the tender procedures, simplified regulatory procedures, improved forecasting, and simplification of the ARV guidelines as critical improvements to maintain a viable ARV market. Conclusions While recent price decreases indicate that there is still space for price reduction, our estimate that gross profit margin on sales decreased by 6 to 7% between 2010 and 2012 lends credibility to assertions by generic manufacturers

  3. High throughput RNAi assay optimization using adherent cell cytometry

    Directory of Open Access Journals (Sweden)

    Pradhan Leena

    2011-04-01

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

  4. Melatonin in Arabidopsis thaliana acts as plant growth regulator at low concentrations and preserves seed viability at high concentrations.

    Science.gov (United States)

    Hernández, Ismaél Gatica; Gomez, Federico José Vicente; Cerutti, Soledad; Arana, María Verónica; Silva, María Fernanda

    2015-09-01

    Since the discovery of melatonin in plants, several roles have been described for different species, organs, and developmental stages. Arabidopsis thaliana, being a model plant species, is adequate to contribute to the elucidation of the role of melatonin in plants. In this work, melatonin was monitored daily by UHPLC-MS/MS in leaves, in order to study its diurnal accumulation as well as the effects of natural and artificial light treatments on its concentration. Furthermore, the effects of exogenous application of melatonin to assess its role in seed viability after heat stress and as a regulator of growth and development of vegetative tissues were evaluated. Our results indicate that melatonin contents in Arabidopsis were higher in plants growing under natural radiation when compared to those growing under artificial conditions, and its levels were not diurnally-regulated. Exogenous melatonin applications prolonged seed viability after heat stress conditions. In addition, melatonin applications retarded leaf senescence. Its effects as growth promoter were dose and tissue-dependent; stimulating root growth at low concentrations and decreasing leaf area at high doses. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  5. Highly Promiscuous Small Molecules from Biological Screening Assays Include Many Pan-Assay Interference Compounds but Also Candidates for Polypharmacology.

    Science.gov (United States)

    Gilberg, Erik; Jasial, Swarit; Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

    2016-11-23

    In PubChem screening assays, 466 highly promiscuous compounds were identified that were examined for known pan-assay interference compounds (PAINS) and aggregators using publicly available filters. These filters detected 210 PAINS and 67 aggregators. Compounds passing the filters included additional PAINS that were not detected, mostly due to tautomerism, and a variety of other potentially reactive compounds currently not encoded as PAINS. For a subset of compounds passing the filters, there was no evidence of potential artifacts. These compounds are considered candidates for further exploring multitarget activities and the molecular basis of polypharmacology.

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

    Science.gov (United States)

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

  7. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  8. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Lago-Cachón, D., E-mail: dlagocachon@gmail.com [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Rivas, M., E-mail: rivas@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Martínez-García, J.C., E-mail: jcmg@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Oliveira-Rodríguez, M., E-mail: oliveiramyriam@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); Blanco-López, M.C., E-mail: cblanco@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); García, J.A., E-mail: joseagd@uniovi.es [Dpto. de Física, Universidad de Oviedo, Escuela de Marina, Campus de Viesques, 33204 Gijón (Spain)

    2017-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Gisela M F Vaz

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

  10. Nutrient limitation and high irradiance acclimation reduce PAR and UV-induced viability loss in the Antarctic diatom Chaetoceros brevis (Bacillariophyceae)

    NARCIS (Netherlands)

    van de Poll, WH; van Leeuwe, MA; Roggeveld, J; Buma, AGJ

    The effects of high PAR (400-700 nm), UVA (315-400 nm), and UVB (280-315 nm) radiation on viability and photosynthesis were investigated for Chaetoceros brevis Schutt. This Antarctic marine diatom was cultivated under low, medium, and high irradiance and nitrate, phosphate, silicate, and iron

  11. Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations

    DEFF Research Database (Denmark)

    Christiansen, Torben; Michaelsen, S.; Wumpelmann, M.

    2003-01-01

    The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all....... The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable...

  12. High content cell-based assay for the inflammatory pathway

    Science.gov (United States)

    Mukherjee, Abhishek; Song, Joon Myong

    2015-07-01

    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  13. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    Science.gov (United States)

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-01-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials. PMID:28378749

  14. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    Science.gov (United States)

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-04-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

  15. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    Science.gov (United States)

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-08

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-04-01

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

  17. 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...

  18. High sensitivity PCR assay in plastic micro reactors.

    Science.gov (United States)

    Yang, Jianing; Liu, Yingjie; Rauch, Cory B; Stevens, Rauch L; Liu, Randall H; Lenigk, Robin; Grodzinski, Piotr

    2002-11-01

    Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.

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

    Directory of Open Access Journals (Sweden)

    N. Vidal

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-02-01

    Cardiac troponin (cTn) assays have quickly gained in analytical sensitivity to become what are termed 'high-sensitivity cardiac troponin' (hs-cTn) assays, bringing a flurry of dense yet incomplete literature data. The net result is that cTn assays are not yet standardized and there are still no consensus-built data on how to use and interpret cTn assay results. To address these issues, the authors take cues and clues from multiple disciplines to bring responses to frequently asked questions. In brief, the effective use of hs-cTn hinges on knowing: specific assay characteristics, particularly precision at the 99th percentile of a reference population; factors of variation at the 99th percentile value; and the high-individuality of hs-cTn assays, for which the notion of individual kinetics is more informative than straight reference to 'normal' values. The significance of patterns of change between two assay measurements has not yet been documented for every hs-cTn assay. Clinicians need to work hand-in-hand with medical biologists to better understand how to use hs-cTn assays in routine practice. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    receptors in this assay were found to be in good agreement with those from electrophysiology studies of the receptors expressed in Xenopus oocytes or mammalian cell lines. Hence, this high throughput screening assay will be of great use in future pharmacological studies of glycine receptors, particular...

  2. 20170913 - 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 mischaracteri...

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

    Science.gov (United States)

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

  4. Increased viability and resilience of haemolymph cells in blue mussels following pre-treatment with acute high-dose gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Jaeschke, B. [Stockholm University (Sweden)

    2014-07-01

    In an initial experiment, blue mussels (Mytilus edulis) were exposed to a range of acute high doses of gamma radiation in the laboratory. Haemolymph was extracted and the haemocytes (blood cells) were scored for cell viability (% living cells) under a microscope, directly after irradiation (0.04, 0.4 or 4 Gy) and again after a subsequent treatment with hydrogen peroxide in vitro (final H{sub 2}O{sub 2} conc.: 0.2 μM). Cell viability in controls (0 Gy) was approximately 100% and no cell death was observable from radiation exposure alone. When treated with H{sub 2}O{sub 2} a decrease in cell viability was seen across all treatments, however this decrease in viability was reduced with increasing radiation pre-treatment (0 Gy = 53%; 0.04 Gy = 66%; 0.4 Gy = 75%; 4 Gy = 83%). To investigate the mechanism for this therapeutic effect observed, the experiment was repeated. Using mussels from a different location, the same, but more extensive method of irradiation (0[control], 0.04, 0.4 Gy, 5 or 40 Gy) and H{sub 2}O{sub 2} treatment was used. Additional haemolymph sub-samples were taken for analysis of catalase concentration. In this second experiment, viability of cells from controls was only 62%, indicating the mussels were in a poorer condition than those of the previous experiment. The lowest level of radiation exposure (0.04 Gy) further decreased the viability (56%). However, at higher doses the viability was increased compared to control, which then gradually declined with increasing dose (0.4 Gy = 75%; 5 Gy = 72%; 40 Gy = 65%). Catalase analysis demonstrated a complimentary pattern of activity of the antioxidant in the haemolymph, directly correlating with radiation dose (0 Gy = 0.2 U; 0.04 Gy = 0.1 U; 0.4 Gy = 1.3 U; 5 Gy = 0.9 U; 40 Gy = 0.1 Gy). Treatment with H{sub 2}O{sub 2} decreased cell viability across all treatments, but no pattern between radiation treatments was discernable. The results indicate that an acute dose of radiation not only has negligible

  5. EUROarray human papillomavirus (HPV) assay is highly concordant with other commercial assays for detection of high-risk HPV genotypes in women with high grade cervical abnormalities.

    Science.gov (United States)

    Cornall, A M; Poljak, M; Garland, S M; Phillips, S; Machalek, D A; Tan, J H; Quinn, M A; Tabrizi, S N

    2016-06-01

    The purpose of this study was to evaluate the performance of the EUROIMMUN EUROArray HPV genotyping assay against the Roche Cobas 4800, Roche HPV Amplicor, Roche Linear Array and Qiagen Hybrid Capture 2 assays in the detection of high-risk HPV (HR-HPV) from liquid based cervical cytology samples collected from women undergoing follow-up for abnormal cervical cytology results. Cervical specimens from 404 women undergoing management of high-grade cytological abnormality were evaluated by EUROarray HPV for detection of HR-HPV genotypes and prediction of histologically-confirmed cervical intraepithelial neoplasia grade 2 or higher (≥CIN2). The results were compared to Hybrid Capture 2, Cobas 4800 HPV, Amplicor and Linear Array HPV. Positivity for 14 HR-HPV types was 80.0 % for EUROarray (95 % CI; 75.7-83.8 %). Agreement (κ, 95 % CI) between the EUROarray and other HPV tests for detection of HR-HPV was good to very good [Hybrid Capture κ = 0.62 (0.54-0.71); Cobas κ = 0.81 (0.74-0.88); Amplicor κ = 0.68 (0.60-0.77); Linear Array κ = 0.77 (0.70-0.85)]. For detection of HR-HPV, agreement with EUROarray was 87.90 % (Hybrid Capture), 93.58 % (Cobas), 92.84 % (Amplicor) and 92.59 % (Linear Array). Detection of HR-HPV was not significantly different between EUROarray and any other test (p detection of high-risk HPV and showed sensitivity and specificity for detection of ≥ CIN2 of 86 % and 71 %, respectively.

  6. Neuraminidase activity provides a practical read-out for a high throughput influenza antiviral screening assay

    Directory of Open Access Journals (Sweden)

    Wu Meng

    2008-09-01

    Full Text Available Abstract Background The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput. Results We have adapted a traditional virus neutralization assay to develop a practical, cell-based, high throughput screening assay. This assay uses viral neuraminidase (NA as a read-out to quantify influenza replication, thereby offering an assay that is both rapid and sensitive. In addition to identification of inhibitors that target either viral or host factors, the assay allows simultaneous evaluation of drug toxicity. Antiviral activity was demonstrated for a number of known influenza inhibitors including amantadine that targets the M2 ion channel, zanamivir that targets NA, ribavirin that targets IMP dehydrogenase, and bis-indolyl maleimide that targets protein kinase A/C. Amantadine-resistant strains were identified by comparing IC50 with that of the wild-type virus. Conclusion Antivirals with specificity for a broad range of targets are easily identified in an accelerated viral inhibition assay that uses NA as a read-out of replication. This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.

  7. An image analysis toolbox for high-throughput C. elegans assays.

    Science.gov (United States)

    Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H; Riklin-Raviv, Tammy; Conery, Annie L; O'Rourke, Eyleen J; Sokolnicki, Katherine L; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M; Carpenter, Anne E

    2012-04-22

    We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.

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

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xuejiang; Tang, Keqi

    2017-06-14

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

  9. Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

    Directory of Open Access Journals (Sweden)

    Irina Gates

    Full Text Available Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT, newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

  10. Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

    Science.gov (United States)

    Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

    2015-01-01

    Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    stimulation by endogenous or exogenous TSH is recommended by current clinical guidelines to detect occult disease with a maximum sensitivity due to the suboptimal sensitivity of older Tg assays. However, the development of new highly sensitive Tg assays with improved analytical sensitivity and precision...... at low concentrations now allows detection of very low Tg concentrations reflecting minimal amounts of thyroid tissue without the need for TSH stimulation. Use of these highly sensitive Tg assays has not yet been incorporated into clinical guidelines but they will, we believe, be used by physicians...... caring for patients with DTC. The aim of this clinical position paper is, therefore, to offer advice on the various aspects and implications of using these highly sensitive Tg assays in the clinical care of patients with DTC....

  12. A high sensitivity micro format chemiluminescence enzyme inhibition assay for determination of Hg(II).

    Science.gov (United States)

    Deshpande, Kanchanmala; Mishra, Rupesh K; Bhand, Sunil

    2010-01-01

    A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5-500 pg·mL(-1) with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL(-1) within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100-110.52%.

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

    Directory of Open Access Journals (Sweden)

    Antonios eGeorgantzoglou

    2016-01-01

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

  14. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams.

    Science.gov (United States)

    Georgantzoglou, Antonios; Merchant, Michael J; Jeynes, Jonathan C G; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E; Jena, Rajesh

    2015-01-01

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

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

    Science.gov (United States)

    Karbaschi, Mahsa; Cooke, Marcus S

    2014-11-26

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

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

    Science.gov (United States)

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

    2016-02-01

    Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. © 2015 Society for Laboratory Automation and Screening.

  17. Repeated Assessment by High-Throughput Assay Demonstrates that Sperm DNA Methylation Levels Are Highly Reproducible

    Science.gov (United States)

    Cortessis, Victoria K.; Siegmund, Kimberly; Houshdaran, Sahar; Laird, Peter W.; Sokol, Rebecca Z.

    2011-01-01

    Objective To assess reliability of high-throughput assay of sperm DNA methylation. Design Observational study comparing DNA methylation of sperm isolated from three divided and twelve longitudinally collected semen samples. Setting Academic Medical Center Patients One man undergoing screening semen analysis during evaluation of the infertile couple and two healthy fertile male volunteers. Interventions Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA-sequence specific sites. Main Outcome Measures Repeatability of sperm DNA methylation measures, estimated by correlation coefficients. Results DNA methylation levels were highly correlated within matched sets of divided samples (all r≥0.97) and longitudinal samples (average r=0.97). Conclusions The described methodology reliably assesses methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility. PMID:22035967

  18. High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

    Science.gov (United States)

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

    2013-12-01

    The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    OpenAIRE

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

    2016-01-01

    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) techniq...

  20. Effect of Diluent and Relative Humidity on Apparent Viability of Airborne Pasteurella pestis

    Science.gov (United States)

    Won, William D.; Ross, Harold

    1966-01-01

    Airborne Pasteurella pestis (A-1122) at low humidities [20 to 50% relative humidity (RH)] exhibited exponential decay when either 1% peptone or Heart Infusion Broth (HIB) was used as the diluent in the viable assay system. At higher RH values (65 and 87%), however, the 1% peptone diluent adversely affected the viability assay. In contrast, HIB as diluent was remarkably effective in demonstrating a higher number of viable cells in aerosols held at high RH values. Similarly, with HIB as diluent, aerosols were shown to contain viable cells during 90 min of observation; with 1% peptone, viability was not detectable after 20 min in the airborne state. PMID:5970462

  1. Effect of diluent and relative humidity on apparent viability of airborne Pasteurella pestis.

    Science.gov (United States)

    Won, W D; Ross, H

    1966-09-01

    Airborne Pasteurella pestis (A-1122) at low humidities [20 to 50% relative humidity (RH)] exhibited exponential decay when either 1% peptone or Heart Infusion Broth (HIB) was used as the diluent in the viable assay system. At higher RH values (65 and 87%), however, the 1% peptone diluent adversely affected the viability assay. In contrast, HIB as diluent was remarkably effective in demonstrating a higher number of viable cells in aerosols held at high RH values. Similarly, with HIB as diluent, aerosols were shown to contain viable cells during 90 min of observation; with 1% peptone, viability was not detectable after 20 min in the airborne state.

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

    Science.gov (United States)

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

  4. High-dose hook effect in six automated human chorionic gonadotrophin assays.

    Science.gov (United States)

    Al-Mahdili, Huda A; Jones, Graham R D

    2010-07-01

    The high-dose hook effect is a well-known phenomenon of two-site immunoassays including those for human chorionic gonadotrophin (hCG). We investigated the occurrence of a high-dose hook effect in six routinely available hCG assays using a sample with a total hCG concentration of approximately 3,600,000 IU/L. Dilutions of a sample with high hCG concentration were analysed using six common methods: Advia Centaur, Immulite 2000, Dimension RxL, Unicel DxI 800, Roche E170 and Abbott Architect. The measured concentrations and corresponding assay signals were obtained for each method. Performance was compared with manufacturer claims. Four of the tested platforms demonstrated a clear high-dose hook effect, while the other methods showed no hook effect at the highest level tested. Our results indicate that the hook effect may occur in some hCG assays, although the risk of reporting falsely low results was in most cases at higher concentrations than those indicated in manufacturers' product information. Assay design plays a major role in its occurrence. Laboratories should be aware of the assay limitations in this regard.

  5. Towards a systematic assessment of assay interference: Identification of extensively tested compounds with high assay promiscuity [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Erik Gilberg

    2017-08-01

    Full Text Available A large-scale statistical analysis of hit rates of extensively assayed compounds is presented to provide a basis for a further assessment of assay interference potential and multi-target activities. A special feature of this investigation has been the inclusion of compound series information in activity analysis and the characterization of analog series using different parameters derived from assay statistics. No prior knowledge of compounds or targets was taken into consideration in the data-driven study of analog series. It was anticipated that taking large volumes of activity data, assay frequency, and assay overlap information into account would lead to statistically sound and chemically meaningful results. More than 6000 unique series of analogs with high hit rates were identified, more than 5000 of which did not contain known interference candidates, hence providing ample opportunities for follow-up analyses from a medicinal chemistry perspective.

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

    Science.gov (United States)

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

    2016-08-01

    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. High Nutrient Levels and TORC1 Activity Reduce Cell Viability following Prolonged Telomere Dysfunction and Cell Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Julia Klermund

    2014-10-01

    Full Text Available Cells challenged with DNA damage activate checkpoints to arrest the cell cycle and allow time for repair. Successful repair coupled to subsequent checkpoint inactivation is referred to as recovery. When DNA damage cannot be repaired, a choice between permanent arrest and cycling in the presence of damage (checkpoint adaptation must be made. While permanent arrest jeopardizes future lineages, continued proliferation is associated with the risk of genome instability. We demonstrate that nutritional signaling through target of rapamycin complex 1 (TORC1 influences the outcome of this decision. Rapamycin-mediated TORC1 inhibition prevents checkpoint adaptation via both Cdc5 inactivation and autophagy induction. Preventing adaptation results in increased cell viability and hence proliferative potential. In accordance, the ability of rapamycin to increase longevity is dependent upon the DNA damage checkpoint. The crosstalk between TORC1 and the DNA damage checkpoint may have important implications in terms of therapeutic alternatives for diseases associated with genome instability.

  8. 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

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

    Science.gov (United States)

    Soufan, Othman; Ba-Alawi, Wail; Afeef, Moataz; 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 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. 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. 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 experimental confirmatory HTS

  10. Development of a highly reliable assay for ubiquitin-specific protease 2 inhibitors.

    Science.gov (United States)

    Wang, Zhongli; Xie, Wenjuan; Zhu, Mingyan; Zhou, Huchen

    2017-09-01

    The dynamic modification of proteins with ubiquitin plays crucial roles in major celluar functions, and is associated with a number of pathological conditions. Ubiquitin-specific proteases (USPs) cleave ubiquitin from substrate proteins, and rescue them from proteasomal degradation. Among them, USP2 is overexpressed and plays important roles in various cancers including prostate cancer. Thus, it represents an attractive target for drug discovery. In order to develop potent and selective USP2 inhibitors, a highly reliable assay is needed for in-depth structure-activity relationship study. We report the cloning, expression, and purification of USP2 and UBA52, and the development of a highly reliable assay based on readily available SDS-PAGE-Coomassie systeme using UBA52 as the substrate protein. A number of effective USP2 inhibitors were also identified using this assay. Copyright © 2017. Published by Elsevier Ltd.

  11. Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

    Science.gov (United States)

    Tian, Huan; Cao, Liching; Tan, Yuping; Williams, Stephen; Chen, Lili; Matray, Tracy; Chenna, Ahmed; Moore, Sean; Hernandez, Vincent; Xiao, Vivian; Tang, Mengxiang; Singh, Sharat

    2004-09-08

    We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.

  12. 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.

  13. High-sensitivity Troponin T Assay in Asymptomatic High Cardiovascular Risk Patients. The TUSARC Registry.

    Science.gov (United States)

    Álvarez, Isabel; Hernández, Luis; García, Héctor; Villamandos, Vicente; López, María Gracia; Palazuelos Molinero, Jorge; Martín Raymondi, Diego

    2017-04-01

    High-sensitivity troponin T assays (Hs-TnT) have been carried out in selected populations in clinical trials and in registries of the general population with low cardiovascular risk (CVR). The aim of this study was to determine the proportion of individuals with detectable Hs-TnT and the proportion of individuals with elevated Hs-TnT in a Spanish population of asymptomatic individuals with very high CVR, as well as the parameters associated with Hs-TnT elevation. The study included 690 patients. Hs-TnT detection and Hs-TnT elevation (≥99th percentile value), as well the association of elevated Hs-TnT and clinical, analytical, and treatment data were analyzed. Hs-TnT was analyzed in 646 patients and was detected in 645. Elevated TnT was detected in 212 patients (32.9%). On multivariate analysis, elevated TnT was independently associated with male sex (OR, 2.81; 95%CI, 1.67-4.73; P < .001), older age (OR, 1.06; 95%CI, 1.04-1.09; P < .001), a higher body mass index (OR, 1.07; 95%CI, 1.02-1.12; P < .002), insulin therapy (OR, 1.99; 95%CI, 1.15-3.46; P = .01), history of heart failure (OR, 3.92; 95%CI, 1.24-12.39; P = .02), and estimated glomerular filtration rate calculated by CKD-EPI (OR, 0.96; 95%CI, 0.95-0.97; P < .001). In a Spanish population of asymptomatic individuals at very high CVR, Hs-TnT was associated with older age, male sex, higher body mass index, insulin therapy, history of heart failure, and lower glomerular filtration rate. Copyright © 2016 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum

    DEFF Research Database (Denmark)

    Lloyd, Yukie M.; Ngati, Elise P.; Salanti, Ali

    2017-01-01

    Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation...

  17. 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...

  18. A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.

    Science.gov (United States)

    Tang, Lixia; Li, Yang; Wang, Xiong

    2010-06-01

    Here we have reported a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC). The assay relies upon the absorbance change at 560nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher k(cat) values, respectively, toward 1,3-dichloro-2-propanol than the wild-type HheC. In addition, a saturation mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-dichloro-2-propanol. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic analysis indicates that the resulting mutant shows 2- and 5-fold improvement in k(cat) value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, respectively, although it exhibits higher K(m) values than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants. 2010 Elsevier B.V. All rights reserved.

  19. High Throughput Assay for Bacterial Adhesion on Acellular Dermal Matrices and Synthetic Surgical Materials

    Science.gov (United States)

    Nyame, Theodore T.; Lemon, Katherine P.; Kolter, Roberto; Liao, Eric C.

    2013-01-01

    Background There has been increasing use of various synthetic and biologically derived materials in surgery. Biologic surgical materials are used in many plastic surgery procedures, ranging from breast reconstruction to hernia repairs. In particular, acellular dermal matrix (ADM) material has gained popularity in these applications. There is a paucity of data on how ADM compares to other surgical materials as a substrate for bacterial adhesion, the first step in formation biofilm, which occurs in prosthetic wound infections. We have designed a high throughput assay to evaluate Staphylococcus aureus adherence on various synthetic and biologically derived materials. Methods Clinical isolates of Staphylococcus aureus (strains SC-1 and UAMS-1) were cultured with different materials and bacterial adherence was measured using a resazurin cell vitality reporter microtiter assay. Four materials that are commonly utilized in reconstructive procedures were evaluated: prolene mesh, vicryl mesh, and two different ADM preparations (AlloDerm®, FlexHD®). We were able to develop a high throughput and reliable assay for quantifying bacterial adhesion on synthetic and biologically derived materials. Results The resazurin vitality assay can be reliably used to quantify bacterial adherence to acellular dermal matrix material, as well as synthetic material. S. aureus strains SC-1 and UAMS-1 both adhered better to ADM materials (AlloDerm® vs. FlexHD®) than to the synthetic material prolene. S. aureus also adhered better to vicryl than to prolene. Strain UAMS-1 adhered better to vicryl and ADM materials than did strain SC-1. Conclusion Our results suggest that S. aureus adheres more readily to ADM material than to synthetic material. We have developed an assay to rapidly test bacterial formation on surgical materials, using two S. aureus bacterial strains. This provides a standard method to evaluate existing and new materials with regard to bacterial adherence and potential

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

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

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

  1. High modulus biodegradable polyurethanes for applications in cardiovascular stents: Evaluation of in-vitro degradation and cell viability

    Directory of Open Access Journals (Sweden)

    Melissa eSagarito

    2015-05-01

    Full Text Available We have recently reported the mechanical properties and hydrolytic degradation behaviour of a series of NovoSorb™ [1] biodegradable polyurethanes (PUs prepared by varying the hard segment (HS weight percentage from 60-100. In this study the in-vitro degradation behaviour of the PUs with and without extracellular matrix (ECM coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C conditions to allow complete degradation. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of Human Umbilical Vein Endothelial Cells (HUVEC were examined.The results showed that for most PUs in the series the degradation medium turned acidic and the extent of this was dependent on the HS percentage. As the HS decreased, a drop in pH was observed, suggesting that the by-products of soft segment (SS degradation causing the solution to be acidic. Most of the samples were completely eroded by 18 weeks, except PU containing more than 70% HS which only showed partial degradation with no significant change in appearance. The cytotoxicity tests on HUVEC cells of the aqueous extracts obtained after 18 weeks incubation showed that toxicity was dependent on 3 factors: dose, percentage of HS and degradation time. HUVEC growth is similar to but not identical to that observed with tissue culture polystyrene (TCPS standard. The results from this in-vitro cytotoxicity study suggest that the mixture of degradation products formed during the accelerated degradation of this PU series are not toxic to cells under the experimental conditions used.

  2. Subendocardial Viability Ratio Is Impaired in Highly Proteinuric Chronic Kidney Disease Patients With Low Estimated Glomerular Filtration Rate.

    Science.gov (United States)

    Ekart, Robert; Šegula, Anja; Hartman, Tanja; Hojs, Nina; Hojs, Radovan

    2016-06-01

    Proteinuria and estimated glomerular filtration rate (eGFR) are markers of chronic kidney disease (CKD) and cardiovascular disease. With applanation tonometry, pulse wave analysis and many hemodynamic data are available. One of them is the subendocardial viability ratio (SEVR) which represents a non-invasive measure of myocardial perfusion related to the work of the heart. The aim of our study was to investigate the importance of SEVR in proteinuric CKD patients and healthy subjects. We performed a cross-sectional study in a cohort of 90 non-dialysis CKD patients and 39 healthy controls. SEVR was assessed by radial applanation tonometry (SphygmoCor, Atcor, Australia). Blood samples and urine albumin-to-creatinine ratio (UACR) were analyzed. CKD patients were divided in four groups according to the UACR and eGFR: CKD group 1: UACR > 1000 mg/g and eGFR  1000 mg/g and eGFR >30 mL/min; CKD group 3: UACR 30 mL/min. Using one-way ANOVA, we found a statistically significant difference in SEVR only between CKD group 1 and all other CKD groups and healthy control group (P < 0.022). Results of our study show that only CKD patients with UACR more than 1000 mg/g and eGFR below 30 mL/min have significantly lower SEVR. © 2016 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.

  3. 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.

  4. Evaluation of resistance development and viability recovery by toxigenic and non-toxigenic Staphylococcus aureus strains after repeated cycles of high hydrostatic pressure.

    Science.gov (United States)

    Baptista, Inês; Queirós, Rui P; Cunha, Angela; Rocha, Sílvia M; Saraiva, Jorge A; Almeida, Adelaide

    2015-04-01

    In this work, the development of resistance and the recovery of growth after several consecutive cycles of high hydrostatic pressure (HPP) were for the first time evaluated in different strains of Staphylococcus aureus. Three strains of this important and highly resilient to HPP foodborne pathogen were used: a non-enterotoxigenic ATCC 6538 strain, treated with 600 MPa for 30 min at 20 °C, and the toxigenic strains 2153 MA (with enterotoxin A) and 2065 MA (with the enterotoxins A, G and I), treated with 600 MPa for 15 min at 20 °C. After the first treatment, surviving colonies were used to produce new bacterial cultures. This procedure was repeated nine times more for each bacterium or until total inactivation occurred. The inactivation profile of non-enterotoxic strain and the two enterotoxic strains did not change after consecutive cycles, but the toxic strain with three enterotoxins was completely inactivated after the fourth cycle. The three strains did not recover their viability after 14 days. The results indicate that HPP effectively inactivates non-toxigenic and toxigenic strains of S. aureus after a single treatment. The surviving bacteria did not develop resistance after 10 cycles of pressurization and did not recover their viability after 14 days of incubation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Vascular Graft Impregnation with Antibiotics: The Influence of High Concentrations of Rifampin, Vancomycin, Daptomycin, and Bacteriophage Endolysin HY-133 on Viability of Vascular Cells.

    Science.gov (United States)

    Herten, Monika; Idelevich, Evgeny A; Sielker, Sonja; Becker, Karsten; Scherzinger, Anna S; Osada, Nani; Torsello, Giovanni B; Bisdas, Theodosios

    2017-06-27

    BACKGROUND Rifampin-soaked synthetic prosthetic grafts have been widely used for prevention or treatment of vascular graft infections (VGIs). This in vitro study investigated the effect of the antibiotics daptomycin and vancomycin and the new recombinant bacteriophage endolysin HY-133 on vascular cells, as potential alternatives compared to rifampin. MATERIAL AND METHODS Primary human ECs, vascular smooth muscle cells (vSMC), and fibroblasts were cultivated in 96-well plates and incubated with rifampin, daptomycin, vancomycin, and endolysin HY-133 for 24 h. Subsequently, after washing, cell viability was determined by measuring mitochondrial ATP concentration. Antibiotics were used in their corresponding minimum and maximum serum concentrations, in decimal multiples and in maximum soaking concentration. The experiments were performed in triplicate. RESULTS The 10-fold max serum concentrations of rifampin, daptomycin, and vancomycin did not influence viability of EC and vSMC (100 µg/ml, p>0.170). Higher concentrations of rifampin (>1 mg/ml) significantly (pEndolysin did not display any cytotoxicity towards vascular cells. CONCLUSIONS Results of this in vitro study show the high cytotoxicity of rifampin against vascular cells, and may re-initiate the discussion about the benefit of prophylactic pre-soaking in high concentrations of rifampin. Further studies are necessary to determine the influence of rifampin on the restoration of vessel functionality versus its prophylactic effect against VGIs. Future use of recombinant phage endolysins for alternative prophylactic strategies needs further investigations.

  6. Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

    Science.gov (United States)

    Arbyn, M; Snijders, P J F; Meijer, C J L M; Berkhof, J; Cuschieri, K; Kocjan, B J; Poljak, M

    2015-09-01

    Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Cell-Based Assay Design for High-Content Screening of Drug Candidates.

    Science.gov (United States)

    Nierode, Gregory; Kwon, Paul S; Dordick, Jonathan S; Kwon, Seok-Joon

    2016-02-01

    To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

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

    Science.gov (United States)

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

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  10. The viability of perilabyrinthine osteocytes

    DEFF Research Database (Denmark)

    Bloch, Sune Land; Kristensen, Søren Lund; Sørensen, Mads Sølvsten

    2012-01-01

    Bone remodeling is highly inhibited around the inner ear space, most likely by the anti-resorptive action of the inner ear cytokine osteoprotegerin (OPG) entering perilabyrinthine bone through the lacuno-canalicular porosity (LCP). This extracellular signaling pathway depends on the viability...

  11. Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates.

    Science.gov (United States)

    Rebnegger, Corinna; Vos, Tim; Graf, Alexandra B; Valli, Minoska; Pronk, Jack T; Daran-Lapujade, Pascale; Mattanovich, Diethard

    2016-08-01

    The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX /S (max)) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h(-1) to near-zero specific growth rates (μ growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes. The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an

  12. Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines

    Science.gov (United States)

    Hassanein, Mohamed; Weidow, Brandy; Koehler, Elizabeth; Bakane, Naimish; Garbett, Shawn; Shyr, Yu; Quaranta, Vito

    2013-01-01

    Purpose Metabolism, and especially glucose uptake, is a key quantitative cell trait that is closely linked to cancer initiation and progression. Therefore, developing high-throughput assays for measuring glucose uptake in cancer cells would be enviable for simultaneous comparisons of multiple cell lines and microenvironmental conditions. This study was designed with two specific aims in mind: the first was to develop and validate a high-throughput screening method for quantitative assessment of glucose uptake in “normal” and tumor cells using the fluorescent 2-deoxyglucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and the second was to develop an image-based, quantitative, single-cell assay for measuring glucose uptake using the same probe to dissect the full spectrum of metabolic variability within populations of tumor cells in vitro in higher resolution. Procedure The kinetics of population-based glucose uptake was evaluated for MCF10A mammary epithelial and CA1d breast cancer cell lines, using 2-NBDG and a fluorometric microplate reader. Glucose uptake for the same cell lines was also examined at the single-cell level using high-content automated microscopy coupled with semi-automated cell-cytometric image analysis approaches. Statistical treatments were also implemented to analyze intra-population variability. Results Our results demonstrate that the high-throughput fluorometric assay using 2-NBDG is a reliable method to assess population-level kinetics of glucose uptake in cell lines in vitro. Similarly, single-cell image-based assays and analyses of 2-NBDG fluorescence proved an effective and accurate means for assessing glucose uptake, which revealed that breast tumor cell lines display intra-population variability that is modulated by growth conditions. Conclusions These studies indicate that 2-NBDG can be used to aid in the high-throughput analysis of the influence of chemotherapeutics on glucose uptake in cancer

  13. High-throughput assay for bacterial adhesion on acellular dermal matrices and synthetic surgical materials.

    Science.gov (United States)

    Nyame, Theodore T; Lemon, Katherine P; Kolter, Roberto; Liao, Eric C

    2011-11-01

    There has been increasing use of synthetic and acellular dermal matrix materials in surgery, ranging from breast reconstruction to hernia repairs. There is a paucity of data on how acellular dermal matrix compares with other surgical materials as a substrate for bacterial adhesion, the first step in formation biofilm, which occurs in prosthetic wound infections. The authors have designed a high-throughput assay to evaluate Staphylococcus aureus adherence on various synthetic and biologically derived materials. Clinical isolates of S. aureus (strains SC-1 and UAMS-1) were cultured with different materials, and bacterial adherence was measured using a resazurin cell vitality assay. Four materials that are commonly used in surgery were evaluated: Prolene mesh, Vicryl mesh, and two different acellular dermal matrix preparations (AlloDerm and FlexHD). The authors were able to develop a high-throughput and reliable assay for quantifying bacterial adhesion on synthetic and biologically derived materials. The resazurin vitality assay can be reliably used to quantify bacterial adherence to acellular dermal matrix material and synthetic material. S. aureus strains SC-1 and UAMS-1 both adhered better to acellular dermal matrix materials (AlloDerm versus FlexHD) than to the synthetic material Prolene. S. aureus also adhered better to Vicryl than to Prolene. Strain UAMS-1 adhered better to Vicryl and acellular dermal matrix materials than did strain SC-1. The results show that S. aureus adheres more readily to acellular dermal matrix material than to synthetic material. The resazurin assay provides a standard method for evaluating surgical materials with regard to bacterial adherence and potential propensity for biofilm development.

  14. Aptamer-Based ELISA Assay for Highly Specific and Sensitive Detection of Zika NS1 Protein.

    Science.gov (United States)

    Lee, Kyung Hyun; Zeng, Huaqiang

    2017-12-05

    We report here a few Zika NS1-binding ssDNA aptamers selected using the conventional SELEX protocol, and their application in an ELISA assay for sensitive diagnosis of Zika NS1 protein. Among the aptamers identified, aptamers 2 and 10 could recognize different binding epitopes of Zika NS1 protein. This complementary in binding site, when coupled with an extraordinarily high binding affinity by 2 (41-nt, KD = 45 pM) and high specificity by 10, was used successfully to construct an ELISA-based assay where 2 and 10 serve as the capture and detection agents, respectively, giving rise to a highly specific detection of Zika NS1 with a detection limit of 100 ng/mL in buffer. Further testing of a few in-house anti-Zika NS1 antibodies show that 2 could also pair with an anti-Zika NS1 antibody. Such aptamer-antibody pairing not only lowers the detection sensitivity by 3 orders of magnitude to 0.1 ng/mL in buffer but also enable highly sensitive detection of as low as 1 and 10 ng/mL of Zika NS1 to be carried out in 10% and 100% human serum, respectively. These results suggest that the selected aptamers would be useful for medical diagnosis of Zika virus infection in various aptamer-based diagnostic devices including ELISA assay.

  15. Discovery by organism based high-throughput screening of new multi-stage compounds affecting Schistosoma mansoni viability, egg formation and production.

    Science.gov (United States)

    Guidi, Alessandra; Lalli, Cristiana; Gimmelli, Roberto; Nizi, Emanuela; Andreini, Matteo; Gennari, Nadia; Saccoccia, Fulvio; Harper, Steven; Bresciani, Alberto; Ruberti, Giovina

    2017-10-01

    Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro.

  16. Effect of Continuous Fermentation of High-Sugar Fruit Must on the Viability and Morphology of Immobilized Yeast on White Foam Glass

    Directory of Open Access Journals (Sweden)

    Joanna Skwira

    2008-01-01

    Full Text Available We studied the effect of continuous fermentation of high-sugar fruit must (containing about 320 g/L of total sugars on the viability and morphology of yeast cells. The process was carried out for 2.5 months in a 4-column bioreactor at 22 °C, using the Saccharomyces bayanus S.o./1AD wine yeast strain, which was immobilized on cubes of white foam glass. During the time of continuous fermentation, the apple wine contained 11.4–16.8 % (by volume of alcohol and a total sugar concentration of 49.2–115.4 g/L. Yeast cells isolated from the carrier at the end of continuous fermentation were bigger than the cells before immobilization and were characterized by various shapes, e.g. they were elongated, large and round or pear-shaped. Some cells were connected to other cells in the form of aggregates. Some yeast cells from the second, third and fourth columns showed a substantial number of wrinkles or folds. Moreover, it was observed that yeast from the carrier in the first column was characterized by the highest viability, 70 %. In the fourth column, the percentage of viable cells was only 11 %.

  17. Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.

    Science.gov (United States)

    Zhang, Ji-Hu; Kang, Zhao B; Ardayfio, Ophelia; Ho, Pei-i; Smith, Thomas; Wallace, Iain; Bowes, Scott; Hill, W Adam; Auld, Douglas S

    2014-06-01

    Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment. © 2013 Society for Laboratory Automation and Screening.

  18. A homogeneous, high-throughput fluorescence anisotropy-based DNA supercoiling assay.

    Science.gov (United States)

    Shapiro, Adam; Jahic, Haris; Prasad, Swati; Ehmann, David; Thresher, Jason; Gao, Ning; Hajec, Laurel

    2010-10-01

    The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

  19. 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.

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

    Science.gov (United States)

    Gantt, Richard W; Thorson, Jon S

    2012-01-01

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

  1. Assay development and high-throughput screening of caspases in microfluidic format.

    Science.gov (United States)

    Wu, Ge; Irvine, Jennifer; Luft, Chris; Pressley, David; Hodge, C Nicholas; Janzen, Bill

    2003-06-01

    Caspase proteases are familiar targets in drug discovery. A common format for screening to identify caspase inhibitors employs fluorogenic or colorimetric tetra-peptide substrates in 96, 384, or 1536 -well microtiter plates. The primary motivation for increasing the number of wells per plate is to reduce the reagent cost per test and increase the throughput of HTS operations. There are significant challenges, however, to moving into or beyond the 1536-well format, such as submicroliter liquid handling, liquid evaporation, increased surface area-to-volume ratios, and the potential for artifacts and interference from small air-borne particles such as lint. Therefore, HTS scientists remain keenly interested in technologies that offer alternatives to the ever-shrinking microtiter plate well. Microfluidic assay technology represents an attractive option that, in theory, consumes only subnanoliter volumes of reagents per test. We have successfully employed a microfluidic assay technology in fluorogenic screening assays for several caspase isoforms utilizing the Caliper Technologies Labchip platform. Caspase-3 is used as a representative case to describe microfluidic assay development and initial high-throughput screening results. In addition, microfluidic screening and plate-based screening are compared in terms of reagent consumption, data quality, and ease of operation.

  2. [Pain of high-throughput screening--pan assay interference compounds].

    Science.gov (United States)

    Xie, Tao; Du, Guan-hua

    2015-08-01

    High-throughput screening is a regular approach available for identitying new lead compounds for the growing validated drug targets in drug screening. However, it has also introduced a large number of peculiar molecules which interfere drug screening. Pan assay interference compounds (PAINS) interfere with the progress of drug screening in various ways, such as interfering with a biochemical assay, modifying the protein, aggregate-based inhibitors and so on. So it is of vital significance to remove them. This paper has consulted the concept, category of PAINS and reviewed the way of PAINS interfering and the countermeasures to cope with them to direct the approach of high through screening and improve the hits percent.

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

    KAUST Repository

    Wu, Jinbo

    2011-01-01

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

  4. High-performance liquid chromatography for assaying NAD glycohydrolase from Neurospora crassa conidia.

    Science.gov (United States)

    Pietta, P; Pace, M; Menegus, F

    1983-06-01

    A rapid and sensitive high-performance liquid chromatographic technique was developed to determinate NAD glycohydrolase (EC 3.2.2.5.) activity from Neurospora crassa conidia. The separation of the assay substrate and products was achieved by isocratic reverse-phase chromatography and the peaks were detected by the absorbance at 259 nm. Quantities of NAD+ and nicotinamide as small as 10 pmol could be measured.

  5. A SERS-based lateral flow assay biosensor for highly sensitive detection of HIV-1 DNA.

    Science.gov (United States)

    Fu, Xiuli; Cheng, Ziyi; Yu, Jimin; Choo, Priscilla; Chen, Lingxin; Choo, Jaebum

    2016-04-15

    User-friendly lateral flow (LF) strips have been extensively used for point-of-care (POC) self-diagnostics, but they have some limitations in their detection sensitivity and quantitative analysis because they only identify the high cut-off value of a biomarker by utilizing color changes that are detected with the naked eye. To resolve these problems associated with LF strips, we developed a novel surface-enhanced Raman scattering (SERS)-based LF assay for the quantitative analysis of a specific biomarker in the low concentration range. Herein, human immunodeficiency virus type 1 (HIV-1) DNA was chosen as the specific biomarker. Raman reporter-labeled gold nanoparticles (AuNPs) were employed as SERS nano tags for targeting and detecting the HIV-1 DNA marker, as opposed to using bare AuNPs in LF strips. It was possible to quantitatively analyze HIV-1 DNA with high sensitivity by monitoring the characteristic Raman peak intensity of the DNA-conjugated AuNPs. Under optimized conditions, the detection limit of our SERS-based lateral flow assay was 0.24 pg/mL, which was at least 1000 times more sensitive compared to colorimetric or fluorescent detection methods. These results demonstrate the potential feasibility of the proposed SERS-based lateral flow assay to quantitatively detect a broad range of genetic diseases with high sensitivity. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Nader Pourmand

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

  7. Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants.

    Science.gov (United States)

    Nyffeler, Johanna; Karreman, Christiaan; Leisner, Heidrun; Kim, Yong Jun; Lee, Gabsang; Waldmann, Tanja; Leist, Marcel

    2017-01-01

    Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.

  8. High-throughput cell mechanical phenotyping for label-free titration assays of cytoskeletal modifications.

    Science.gov (United States)

    Golfier, Stefan; Rosendahl, Philipp; Mietke, Alexander; Herbig, Maik; Guck, Jochen; Otto, Oliver

    2017-08-01

    The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label-free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real-time deformability cytometry (RT-DC), a novel microfluidic tool to examine the effects of biochemically modified F-actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half-maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F-actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT-DC. Drug-induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high-throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label-free dose-response assays of cytoskeletal modifications. © 2017 The Authors Cytoskeleton Published by Wiley Periodicals, Inc.

  9. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography.

    Science.gov (United States)

    Enjalbert, F; Gallion, C; Jehl, F; Monteil, H; Faulstich, H

    1992-05-15

    A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight amatoxins and phallotoxins. The method identifies both neutral toxins (alpha- and gamma-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (beta-amanitin, phallacidin and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only amatoxins. This relatively simple method should be suitable for the detection of amatoxins and phallotoxins in almost any species of mushrooms.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Lakshmi K Matukumalli

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

  12. 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

  13. A high sensitivity assay for the inflammatory marker C-Reactive protein employing acoustic biosensing

    Directory of Open Access Journals (Sweden)

    Cooper Matthew A

    2008-04-01

    Full Text Available Abstract C-Reactive Protein (CRP is an acute phase reactant routinely used as a biomarker to assess either infection or inflammatory processes such as autoimmune diseases. CRP also has demonstrated utility as a predictive marker of future risk of cardiovascular disease. A new method of immunoassay for the detection of C-Reactive Protein has been developed using Resonant Acoustic Profiling™ (RAP™ with comparable sensitivity to a high sensitivity CRP ELISA (hsCRP but with considerable time efficiency (12 minutes turnaround time to result. In one method, standard solutions of CRP (0 to 231 ng/mL or diluted spiked horse serum sample are injected through two sensor channels of a RAP™ biosensor. One contains a surface with sheep antibody to CRP, the other a control surface containing purified Sheep IgG. At the end of a 5-minute injection the initial rate of change in resonant frequency was proportional to CRP concentration. The initial rates of a second sandwich step of anti-CRP binding were also proportional to the sample CRP concentration and provided a more sensitive method for quantification of CRP. The lower limit of detection for the direct assay and the homogenous sandwich assay were both 20 ng/mL whereas for the direct sandwich assay the lower limit was 3 ng/mL. In a step towards a rapid clinical assay, diluted horse blood spiked with human CRP was passed over one sensor channel whilst a reference standard solution at the borderline cardiovascular risk level was passed over the other. A semi-quantities ratio was thus obtained indicative of sample CRP status. Overall, the present study revealed that CRP concentrations in serum that might be expected in both normal and pathological conditions can be detected in a time-efficient, label-free immunoassay with RAP™ detection technology with determined CRP concentrations in close agreement with those determined using a commercially available high sensitivity ELISA.

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

    Directory of Open Access Journals (Sweden)

    Christian E Badr

    2007-06-01

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

  15. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    Science.gov (United States)

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2015-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening. PMID:20938917

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

    Science.gov (United States)

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Use of fluorescent redox indicators to evaluate cell proliferation and viability

    DEFF Research Database (Denmark)

    Rasmussen, E.S.

    1999-01-01

    The performance of two cell viability test kits based on the use of redox indicators yielding fluorescent products, the AlamarBlue assay and a resazurin-based in vitro toxicology assay kit from Sigma, was compared in the present study. Cultures of human neonatal foreskin fibroblasts were exposed...... to equal concentrations of the two dye solutions in the cell culture media. The fluorescence intensities of the cell culture media obtained in response to cell proliferation with the two dyes showed a pronounced similarity. Both dyes were noncytotoxic to cell cultures with high initial cell densities...... components were tentatively identified as resazurin and resorufin. The AlamarBlue assay has gained wide application as a cell viability indicator that allows continuous monitoring of cell proliferation or cytotoxicity in human and animal cells, bacteria, and fungi, but no studies with the deliberate use...

  18. Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies

    Science.gov (United States)

    Tarpley, Michael; Vermeulen, Peter; Rypens, Charlotte; Van Laere, Steven; Williams, Kevin P.; Devi, Gayathri R.; Dewhirst, Mark W.

    2017-01-01

    Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy. PMID:28460441

  19. Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

    2012-01-01

    Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

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

    Directory of Open Access Journals (Sweden)

    Shannon M Clarke

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

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

    Science.gov (United States)

    Clarke, Shannon M; Henry, Hannah M; Dodds, Ken G; Jowett, Timothy W D; Manley, Tim R; Anderson, Rayna M; McEwan, John C

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project......A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...

  3. Performance of the high-sensitivity troponin assay in diagnosing acute myocardial infarction: systematic review and meta-analysis

    Science.gov (United States)

    Al-Saleh, Ayman; Alazzoni, Ashraf; Al Shalash, Saleh; Ye, Chenglin; Mbuagbaw, Lawrence; Thabane, Lehana; Jolly, Sanjit S.

    2014-01-01

    Background High-sensitivity cardiac troponin assays have been adopted by many clinical centres worldwide; however, clinicians are uncertain how to interpret the results. We sought to assess the utility of these assays in diagnosing acute myocardial infarction (MI). Methods We carried out a systematic review and meta-analysis of studies comparing high-sensitivity with conventional assays of cardiac troponin levels among adults with suspected acute MI in the emergency department. We searched MEDLINE, EMBASE and Cochrane databases up to April 2013 and used bivariable random-effects modelling to obtain summary parameters for diagnostic accuracy. Results We identified 9 studies that assessed the use of high-sensitivity troponin T assays (n = 9186 patients). The summary sensitivity of these tests in diagnosing acute MI at presentation to the emergency department was estimated to be 0.94 (95% confidence interval [CI] 0.89–0.97); for conventional tests, it was 0.72 (95% CI 0.63–0.79). The summary specificity was 0.73 (95% CI 0.64–0.81) for the high-sensitivity assay compared with 0.95 (95% CI 0.93–0.97) for the conventional assay. The differences in estimates of the summary sensitivity and specificity between the high-sensitivity and conventional assays were statistically significant (p sensitivity troponin I assays and showed similar results. Interpretation Used at presentation to the emergency department, the high-sensitivity cardiac troponin assay has improved sensitivity, but reduced specificity, compared with the conventional troponin assay. With repeated measurements over 6 hours, the area under the curve is similar for both tests, indicating that the major advantage of the high-sensitivity test is early diagnosis. PMID:25295240

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

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Synthetic Substrate for Application in both High and Low Throughput Assays for Botulinum Neurotoxin B Protease Inhibitors

    OpenAIRE

    Salzameda, Nicholas T.; Barbieri, Joseph T.; Janda, Kim D.

    2009-01-01

    A FRET peptide substrate was synthesized and evaluated for enzymatic cleavage by the BoNT/B light chain protease. The FRET substrate was found to be useful in both a high throughput assay to uncover initial “hits” and a low throughput HPLC assay to determine kinetic parameters and modes of inhibition.

  6. Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

    Science.gov (United States)

    Kováčová, Kristína; Farkaš, Vladimír

    2016-04-01

    Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

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

    Directory of Open Access Journals (Sweden)

    Jakob D Wikstrom

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

  8. High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

    Science.gov (United States)

    Li, Xiaoning; Geng, Steven B; Chiu, Mark L; Saro, Dorina; Tessier, Peter M

    2015-03-18

    Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 μg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery.

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

    DEFF Research Database (Denmark)

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

    1989-01-01

    A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme...... are common in high risk individuals seronegative by first generation ELISA. However, HIV infection do occur in subjects negative by first generation ELISA, which emphasises the need for more sensitive screening assays and/or the use of antigen detection as part of screening in high risk individuals...

  10. Cell Proliferation and Cytotoxicity Assays.

    Science.gov (United States)

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

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

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

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

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

    Science.gov (United States)

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

    2013-01-01

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

  13. High-throughput micro-plate HCI-vanillin assay for screening tannin content in sorghum grain.

    Science.gov (United States)

    Herald, Thomas J; Gadgil, Priyadarshini; Perumal, Ramasamy; Bean, Scott R; Wilson, Jeff D

    2014-08-01

    The HCI-vanillin assay is a well-accepted method for determining tannin content in sorghum but is limited to small sample sets due to the time-consuming nature of the method. The objective was to develop an accurate and repeatable high-throughput 96-well plate assay for breeders to screen large sample sets of sorghum for tannin content. Validation of the high-throughput assay was tested on 25 sorghums suspected to contain tannin. Approximately 30 measurements per day were completed using the conventional assay compared to 224 measurements using the 96-well platform. The correlation between the two tannin assays was 0.98. The coefficient of variation (CV) was 3.54% and 3.21% for the 96-well and conventional method, respectively. The 96-well assay exhibited good repeatability, with the inter-plate CV between 2.77% and 4.85%. The high-throughput 96-well HCI-vanillin assay exhibited an eightfold increase in the number of measurements completed and was as accurate as the conventional HCI-vanillin assay. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  14. Sperm viability staining in ecology and evolution: potential pitfalls

    DEFF Research Database (Denmark)

    Holman, Luke

    2009-01-01

    a number of interesting results, it has some potential pitfalls that have rarely been discussed. In the present paper, I review the major findings of ecology and evolution studies employing sperm viability staining and outline the method's principle limitations. The key problem is that the viability assay......The causes and consequences of variation in sperm quality, survival and ageing are active areas of research in ecology and evolution. In order to address these topics, many recent studies have measured sperm viability using fluorescent staining. Although sperm viability staining has produced...

  15. Economic viability of ultra high-performance fiber reinforced concrete in prestressed concrete wind towers to support a 5 MW turbine

    Directory of Open Access Journals (Sweden)

    P. V. C. N. GAMA

    Full Text Available Abstract The Ultra-High Performance Fiber-Reinforced Concrete is a material with remarkable mechanical properties and durability when compared to conventional and high performance concrete, which allows its use even without the reinforcement. This paper proposes the design of prestressed towers for a 5 MW turbine, through regulatory provisions and the limit states method, with UHPFRC and the concrete class C50, comparing the differences obtained in the design by parametric analysis, giving the advantages and disadvantages of using this new type of concrete. Important considerations, simplifications and notes are made to the calculation process, as well as in obtaining the prestressing and passive longitudinal and passive transverse reinforcement, highlighting the shear strength of annular sections comparing a model proposed here with recent experimental results present in the literature, which was obtained good agreement. In the end, it is estimated a first value within the constraints here made to ensure the economic viability of the use of UHPFRC in a 100 m prestressed wind tower with a 5 MW turbine.

  16. Viability, invariance and applications

    CERN Document Server

    Carja, Ovidiu; Vrabie, Ioan I

    2007-01-01

    The book is an almost self-contained presentation of the most important concepts and results in viability and invariance. The viability of a set K with respect to a given function (or multi-function) F, defined on it, describes the property that, for each initial data in K, the differential equation (or inclusion) driven by that function or multi-function) to have at least one solution. The invariance of a set K with respect to a function (or multi-function) F, defined on a larger set D, is that property which says that each solution of the differential equation (or inclusion) driven by F and issuing in K remains in K, at least for a short time.The book includes the most important necessary and sufficient conditions for viability starting with Nagumo's Viability Theorem for ordinary differential equations with continuous right-hand sides and continuing with the corresponding extensions either to differential inclusions or to semilinear or even fully nonlinear evolution equations, systems and inclusions. In th...

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

    Science.gov (United States)

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

    2017-02-27

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    CERN Document Server

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

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kyungmin Kim

    2017-01-01

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

  1. Stability-indicating high-performance liquid chromatographic assay method and photostability of carprofen.

    Science.gov (United States)

    Wu, A B; Chen, C Y; Chu, S D; Tsai, Y C; Chen, F A

    2001-01-01

    A rapid, sensitive, and accurate stability-indicating high-performance liquid chromatographic assay method for determining the degradation of carprofen (CPF) is developed and validated under acidic, basic, or photo-irradiated conditions. The analysis is monitored with a Cosmosil 5C18-AR column using a mobile phase of CH3CN-H2O-AcOH (50:49:1, v/v/v) at 260 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution among the parent drug and its degradation products. The results indicate that the established assay method shows good selectivity and specificity suitable for stability measurements of CPF. CPF is found to be more sensitive to exposure to light and in acidic conditions, but it is stable in a basic medium. The kinetic study of the photodegradation of CPF follows an apparent first-order reaction in a variety of solvents. The solvent effects on the rates of degradation are in the decreasing order of chloroform > dichloromethane > methanol > ethanol > 2-propanol, which is irrelevant to the dielectric constant epsilon. However, the hydrogen-donating ability of the solvents is essential to the photochemical decomposition of CPF. A plot of log k versus the Kirkwood function exhibits a linear relationship in aqueous ethanolic solutions, which implies that degradation proceeds via an ionic mechanism.

  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. Risk stratification in patients with acute chest pain using three high-sensitivity cardiac troponin assays.

    Science.gov (United States)

    Haaf, Philip; Reichlin, Tobias; Twerenbold, Raphael; Hoeller, Rebeca; Rubini Gimenez, Maria; Zellweger, Christa; Moehring, Berit; Fischer, Catherine; Meller, Bernadette; Wildi, Karin; Freese, Michael; Stelzig, Claudia; Mosimann, Tamina; Reiter, Miriam; Mueller, Mira; Hochgruber, Thomas; Sou, Seoung Mann; Murray, Karsten; Minners, Jan; Freidank, Heike; Osswald, Stefan; Mueller, Christian

    2014-02-01

    Several high-sensitivity cardiac troponin (hs-cTn) assays have recently been developed. It is unknown which hs-cTn provides the most accurate prognostic information and to what extent early changes in hs-cTn predict mortality. In a prospective, international multicentre study, cTn was simultaneously measured with three novel [high-sensitivity cardiac Troponin T (hs-cTnT), Roche Diagnostics; hs-cTnI, Beckman-Coulter; hs-cTnI, Siemens] and a conventional assay (cTnT, Roche Diagnostics) in a blinded fashion in 1117 unselected patients with acute chest pain. Patients were followed up 2 years regarding mortality. Eighty-two (7.3%) patients died during the follow-up. The 2-year prognostic accuracy of hs-cTn was most accurate for hs-cTnT [area under the receivers operating characteristic curve (AUC) 0.78 (95% CI: 0.73-0.83) and outperformed both hs-cTnI (Beckman-Coulter, 0.71 (95% CI: 0.65-0.77; P = 0.001 for comparison), hs-cTnI (Siemens) 0.70 (95% CI: 0.64-0.76; P < 0.001 for comparison)] and cTnT 0.67 (95% CI: 0.61-0.74; P < 0.001 for comparison). Absolute changes of hs-cTnT were more accurate than relative changes in predicting mortality, but inferior to presentation values of hs-cTnT. Combining changes of hs-cTnT within the first 6 h with their presentation values did not further improve prognostic accuracy. Similar results were obtained for both hs-cTnI assays regarding the incremental value of changes. Hs-cTn concentrations remained predictors of death in clinically challenging subgroups such as patients with pre-existing coronary artery disease, impaired renal function, and patients older than 75 years. High-sensitivity cardiac Troponin T is more accurate than hs-cTnI in the prediction of long-term mortality. Changes of hs-cTn do not seem to further improve risk stratification beyond initial presentation values.

  4. A high-performance, non-radioactive potency assay for measuring cytotoxicity: A full substitute of the chromium-release assay targeting the regulatory-compliance objective.

    Science.gov (United States)

    Rossignol, Alexis; Bonnaudet, Véronique; Clémenceau, Béatrice; Vié, Henri; Bretaudeau, Laurent

    2017-04-01

    Standardized and biologically relevant potency assays are required by the regulatory authorities for the characterization and quality control of therapeutic antibodies. As critical mechanisms of action (MoA) of antibodies, the antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) must be characterized by appropriate potency assays. The current reference method for measuring cytotoxicity is the 51Cr-release method. However, radioactivity handling is difficult to implement in an industrial context because of environmental and operator protection constraints. Alternative non-radioactive methods suffer from poor validation performances and surrogate assays that measure FcγR-dependent functions do not comply with the regulatory requirement of biological relevance. Starting from these observations, we developed a non-radioactive luminescent method that is specific for target cell cytolysis. In adherent and non-adherent target cell models, the ADCC (using standardized effector cells) or CDC activities of rituximab, trastuzumab and adalimumab were compared in parallel using the 51Cr or luminescent methods. We demonstrated that the latter method is highly sensitive, with validation performances similar or better than the 51Cr method. This method also detected apoptosis following induction by a chemical agent or exposure to ultraviolet light. Moreover, it is more accurate, precise and specific than the concurrent non-radioactive calcein- and TR-FRET-based methods. The method is easy to use, versatile, standardized, biologically relevant and cost effective for measuring cytotoxicity. It is an ideal candidate for developing regulatory-compliant cytotoxicity assays for the characterization of the ADCC, CDC or apoptosis activities from the early stages of development to lot release.

  5. A novel high-throughput assay for the quantitative assessment of receptor trafficking.

    Science.gov (United States)

    Grimsey, Natasha L; Narayan, Pritika J; Dragunow, Mike; Glass, Michelle

    2008-11-01

    1. Receptor transport between intracellular compartments has important consequences for receptor function and is an exciting area of current study. Existing methods for studying receptor trafficking often require labour-intensive techniques or are difficult to quantify reliably. We report a novel high-throughput method that uses automated imaging and analysis tools to accurately quantify cannabinoid CB1 receptor trafficking. 2. Haemagglutinin (HA)-tagged CB1 was stably expressed in HEK-293 cells and cell surface or total receptors were detected immunocytochemically. Images of receptor and nuclear staining were acquired with an automated fluorescent microscope (Discovery-1; Molecular Devices, Sunnyvale, CA, USA) and quantified at high throughput with MetaMorph (Molecular Devices) software. The 'Granularity' assay measured internalization by counting receptor clusters that appear during receptor endocytosis, a well-established approach. Our assay, referred to as 'Total Grey Value per Cell' (TGVC), measures the total fluorescence above background, normalized to cell count. 3. Incubation with the cannabinoid agonist HU-210 (100 nmol/L) resulted in rapid CB1 internalization, reaching a maximum within 20 min. Whether quantified by Granularity or TGVC, the time-course of endocytosis could be modelled with exponentially derived curves and with similar half-lives. We demonstrate the sensitivity of our TGVC method by measuring the concentration dependence of CB1 internalization and its versatility by measuring downregulation following chronic agonist exposure, whereby total CB1 was reduced to approximately 55% of basal after 3 h. 4. The TGVC quantification method described is efficient, accurate and versatile and is likely to provide a valuable tool in receptor trafficking studies.

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

    Directory of Open Access Journals (Sweden)

    Hansook Kim Chong

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

  7. A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

    Science.gov (United States)

    Hu, Jiaming; Yu, Yajiao; Brooks, Jessica C; Godwin, Leah A; Somasundaram, Subramaniam; Torabinejad, Ferdous; Kim, Joonyul; Shannon, Curtis; Easley, Christopher J

    2014-06-11

    Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

  8. Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

    Science.gov (United States)

    Morschett, Holger; Wiechert, Wolfgang; Oldiges, Marco

    2016-02-09

    Within the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time. Lipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells' optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded. The newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  10. High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

    Directory of Open Access Journals (Sweden)

    de Lima Bruno

    2011-04-01

    Full Text Available Abstract Background High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT. This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity ≥2%. The development of a much larger

  11. Characterisation of a highly sensitive troponin I assay and its application to a cardio-healthy population.

    Science.gov (United States)

    Koerbin, Gus; Tate, Jill; Potter, Julia M; Cavanaugh, Juleen; Glasgow, Nicholas; Hickman, Peter E

    2012-02-03

    Abbott Diagnostics have developed a new highly sensitive troponin I (hs-TnI) assay. We have assessed its analytical characteristics and applied the assay to a population of apparently cardio-healthy persons. We assessed imprecision, bias compared to the previous generation assay, matrix effects, and interferences and applied the assay to an apparently healthy population, deriving the 99th percentile limit of the distribution of values in reference populations for men and women separately. The dynamic range of the assay was ranged from 0.5-50,000 ng/L (pg/mL). The 10% CV was at a concentration of 3.9 ng/L, and the 20% CV was at a concentration of 1.8 ng/L. The new and current version of the TnI assay were highly correlated [slope: 0.98 (95%CI:0.88-1.07), y-intercept:1.20 (95%CI:-2.35-4.75) r²=0.99]. The 99th percentile limit of the distribution of values in a reference population was different for males and females: for males 14.0 ng/L and for females 11.1 ng/L and at these concentrations the assay CV was 5.0%. TnI was detectable in nearly all patient samples from the healthy reference population (98.6%). This new hs-TnI assay is able to measure to an order of magnitude lower than the current generation TnI assay from the same manufacturer. With TnI being detectable in nearly all apparently healthy subject samples this suggests that TnI presence does not always indicate cardiomyocyte necrosis.

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  13. Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88.

    Science.gov (United States)

    Decker, Ann M; Gay, Elaine A; Mathews, Kelly M; Rosa, Taylor C; Langston, Tiffany L; Maitra, Rangan; Jin, Chunyang

    2017-03-27

    GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. In this paper, we describe the development of a CHO-Gα qi5 -GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gα qi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. The CHO-Gα qi5 -GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.

  14. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay.

    Science.gov (United States)

    Wongkamchai, Sirichit; Monkong, Nuntiya; Mahannol, Pakpimom; Taweethavonsawat, Piyanan; Loymak, Sumat; Foongladda, Suporn

    2013-01-01

    Human lymphatic filariasis is caused by filarial worms such as Brugia malayi for which the major reservoir is domestic cats. However, domestic cats or dogs also carry nonhuman filaria such as Brugia pahangi and Dirofilaria immitis. We have developed a single-tube, real-time PCR with a high-resolution melting (HRM) analysis assay for detection and identification of B. malayi, B. pahangi, and D. immitis in blood samples. The designated primer pair in the PCR can amplify a 114-bp region of mitochondrial 12S rRNA genes of these filarial worms. Subsequently, the HRM assay showed a specific melting temperature for each species. The assay showed the highest sensitivity and specificity in comparison with DNA sequences after assessment with 34 cat and 14 dog blood samples. This assay could be helpful for epidemiological studies of reservoirs and vectors.

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

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

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

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

    Science.gov (United States)

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

    2010-11-18

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

  17. Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

    Science.gov (United States)

    Chan, Grace Ka Yan; Wilson, Stacy; Schmidt, Stephen; Moffat, John G

    2016-02-01

    Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines. © 2015 Society for Laboratory Automation and Screening.

  18. Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus.

    Science.gov (United States)

    Liu, Xinfeng; Guan, Yuyao; Cheng, Shiliang; Huang, Yidan; Yan, Qin; Zhang, Jun; Huang, Guanjun; Zheng, Jian; Liu, Tianqiang

    2016-12-01

    Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×103CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. Copyright © 2016. Published by Elsevier B.V.

  19. Distinct solubility and cytotoxicity regimes of paclitaxel-loaded cationic liposomes at low and high drug content revealed by kinetic phase behavior and cancer cell viability studies.

    Science.gov (United States)

    Steffes, Victoria M; Murali, Meena M; Park, Yoonsang; Fletcher, Bretton J; Ewert, Kai K; Safinya, Cyrus R

    2017-11-01

    Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the incorporation and time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for >3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥20 days for Cell viability experiments on human cancer cell lines using CL PTXL nanoparticles (NPs) in the distinct CL PTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CL PTXL NPs with ≥3

  20. Multicentre clinical evaluation of the new highly sensitive Elecsys® thyroglobulin II assay in patients with differentiated thyroid carcinoma.

    Science.gov (United States)

    Trimboli, P; Imperiali, M; Piccardo, A; CampennÌ, A; Giordani, I; Ruggeri, R M; Baldari, S; Orlandi, F; Giovanella, L

    2018-02-01

    A highly sensitive thyroglobulin assay (Elecsys® Tg II, Roche Diagnostics, Penzberg, Germany) has become available for monitoring patients with differentiated thyroid cancer (DTC). Here, we evaluated the clinical performance of Elecsys® Tg II assay in a multicentre patients series and compare it with the established Access® Tg assay (Beckman Coulter, Brea, CA, USA). Retrospective analysis on prospectively selected patients in four thyroid cancer referral centres with uniform DTC management. All DTC cases diagnosed, treated and followed up in four tertiary referral centres for thyroid cancer since January 2005 (n = 1456) were retrieved, and predefined selection criteria were applied to prevent relevant enrolment biases. A series of 204 patients was finally selected for this study. Samples had been stored at -80°C. Tg was measured by fully automated immunometric Elecsys® Tg II and Access® Tg assays in a centralized laboratory. Two hundred and four DTC were finally included. Of these, 10.8% had structural recurrence (sREC), and 81.4% showed no evidence of disease (NED) at the end of follow-up. There was a significant analytical bias between methods that cannot be used interchangeably. Using ROC curve analysis, the best basal and rhTSH-stimulated Tg cut-offs to detect sREC were 0.41 μg/L and 1.82 μg/L for Elecsys® and 0.36 μg/L and 1.62 μg/L for Access® assay, respectively. Using Cox proportional hazard regression, Tg was the only independent predictor of cancer relapse. Using appropriate assay-specific cut-offs, the clinical performance of the Elecsys® Tg II assay was comparable to that provided by the well-established Access® Tg assay. © 2017 John Wiley & Sons Ltd.

  1. Performance of mRNA- and DNA-based high-risk human papillomavirus assays in detection of high-grade cervical lesions.

    Science.gov (United States)

    Virtanen, Elina; Kalliala, Ilkka; Dyba, Tadeusz; Nieminen, Pekka; Auvinen, Eeva

    2017-01-01

    The aim was to assess the performance of two commercial assays for the detection of high-risk human papillomavirus (hrHPV): Aptima HPV Assay (Hologic, Inc., Marlborough, MA, USA) which detects mRNA of 14 different hrHPV types, and Hybrid Capture 2 HPV DNA test (HC2; Qiagen, Gaithersburg, MD, USA), which detects the DNA of 13 different hrHPV types. Test performance was compared in the settings of a standard colposcopy clinic, among the regular patient flow. Two separate cervical cell samples for Aptima and HC2 testing were collected from women referred to colposcopy or a cervical follow-up visit. Altogether, 481 paired samples were analyzed and all positive samples were also tested using the Aptima HPV 16 18/45 Genotype Assay. Results from the two assays were compared directly and with stratification by histology and cytology from the same sampling visit. The overall agreement between HC2 and Aptima assays was 92.9% (Kappa coefficient of 0.855). The sensitivity and specificity of the assays in detecting CIN2+ were 92.5 and 58.2% for HC2, and 94.0 and 59.3% for Aptima, respectively. No significant differences between the assays were found (p-values >0.5). Both assays detected all CIN3 (n = 30) and carcinoma (n = 2) cases. The mRNA-based Aptima assay and the extensively studied DNA-based HC2 test performed equally well in detecting high-grade cervical lesions. Our data contribute to the growing evidence base indicating that the mRNA-based Aptima assay could be used for the triage of patients with HPV-associated cervical disease. © 2016 Nordic Federation of Societies of Obstetrics and Gynecology.

  2. 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

  3. Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation

    Science.gov (United States)

    Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G.; Garraway, Levi A.; Struhl, Kevin

    2015-01-01

    Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment. PMID:25902495

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We...... present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well....

  5. A high throughput solubility assay for drug discovery using microscale shake-flask and rapid UHPLC-UV-CLND quantification.

    Science.gov (United States)

    Lin, Baiwei; Pease, Joseph H

    2016-04-15

    The rapid determination of key physical properties of lead compounds is essential to the drug discovery process. Solubility is one of the most important properties since good solubility is needed not only for obtaining reliable in vitro and in vivo assay results in early discovery but also to ensure sufficient concentration of the drug being in circulation to get the desired therapeutic exposure at the target of interest. In order for medicinal chemists to tune solubility of lead compounds, a rapid assay is needed to provide solubility data that is accurate and predictive so that it can be reliably used for designing the next generation of compounds with improved properties. To ensure speed and data quality, we developed a high throughput solubility assay that utilizes a single calibration UHPLC-UV-CLND method and a 24h shake-flask format for rapid quantification. A set of 46 model compounds was used to demonstrate that the method is accurate, reproducible and predictive. Here we present development of the assay, including evaluation of quantification method, filtration membranes, equilibrium times, DMSO concentrations, and buffer conditions. A comparison of thermodynamic solubility results to our high throughput 24h shake-flask solubility assay results is also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. The effect of ketorolac tromethamine, methylprednisolone, and platelet-rich plasma on human chondrocyte and tenocyte viability.

    Science.gov (United States)

    Beitzel, Knut; McCarthy, Mary Beth; Cote, Mark P; Apostolakos, John; Russell, Ryan P; Bradley, James; ElAttrache, Neal S; Romeo, Antony A; Arciero, Robert A; Mazzocca, Augustus D

    2013-07-01

    The purpose of this study was to evaluate the effect on cell viability of the isolated and combined use of allogeneic platelet-rich plasma (PRP) and ketorolac tromethamine on human chondrocytes and tenocytes in a highly controlled in vitro environment. PRP was produced from 8 subjects. Human chondrocytes (Lonza, Hopkinton, MA) and tenocytes isolated from samples of the long head of the biceps tendons were treated in culture with PRP, ketorolac tromethamine, and methylprednisolone, both alone and in combination. Control samples were treated in media containing 2% or 10% fetal bovine serum (FBS). Cells were exposed for 1 hour. Luminescence assays were obtained to examine cell viability after 24 hours and long-term effects on cell viability after 120 hours. Radioactive thymidine assay was used to measure proliferation after 120 hours. For chondrocytes, cell viability (120 hours) increased significantly with the treatment of PRP alone (43,949 ± 28,104 cells; P investigation into alternative treatment options such as combinations of PRP and ketorolac tromethamine. In vitro evaluation of their effect on cell viability might build a basis for further translational research and clinical application. Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. © The Author 2014. Published by

  8. Design of a high-throughput assay for alternative splicing using polymerase colonies.

    Science.gov (United States)

    Buhler, J D; Souvenir, R M; Zhang, W; Mitra, R D

    2004-01-01

    We propose an assay to detect and quantify alternative splicing simultaneously for numerous genes in a pool of cellular mRNA. The assay exploits polymerase colonies, a recently developed method for sampling and amplifying large numbers of individual transcript molecules into discrete spots on a gel. The proposed assay combines the advantages of microarrays for transcript quantitation with the sensitivity and precision of methods based on counting single transcript molecules. Given a collection of spots s(i), each containing an unknown splice variant of some known gene G(i), we design a series of hybridizations to short oligonucleotide probes to determine in parallel which exons of G(i) are present in every spot s(i). We give algorithms to minimize the cost of such designs.

  9. An improved high-throughput screening assay for tunicamycin sensitivity in Arabidopsis seedlings

    Directory of Open Access Journals (Sweden)

    Maggie E McCormack

    2015-08-01

    Full Text Available Tunicamycin sensitivity assays are a useful method for studies of endoplasmic reticulum stress and the unfolded protein response in eukaryotic cells. While tunicamycin sensitivity and tunicamycin recovery assays have been previously described, these existing methods are time-consuming, labor intensive and subjected to mechanical wounding. This study shows an improved method of testing tunicamycin sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates. Liquid medium bypasses the physical manipulation of seedlings, thereby eliminating the risk of potential mechanical damage and additional unwanted stress to seedlings. Seedlings were subjected to comparative treatments with various concentrations of tunicamycin on both solid and liquid media and allowed to recover. Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative tunicamycin stress assays.

  10. Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

    Science.gov (United States)

    Akimoto, Masaru; Hokazono, Eisaku; Ota, Eri; Tateishi, Takiko; Kayamori, Yuzo

    2016-01-01

    Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein. © The Author(s) 2015.

  11. Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.

    Science.gov (United States)

    van der Linden, Sander C; von Bergh, Anne R M; van Vught-Lussenburg, Barbara M A; Jonker, Lydia R A; Teunis, Marc; Krul, Cyrille A M; van der Burg, Bart

    2014-01-15

    The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. 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 ...

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

    NARCIS (Netherlands)

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

    2015-01-01

    Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess

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

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-01-01

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

  15. The Phospholipid Vesicle-Based Drug Permeability Assay: 5. Development Toward an Automated Procedure for High-Throughput Permeability Screening

    OpenAIRE

    Flaten, Gøril Eide; Awoyemi, Opeyemi Linda Ronke; Luthman, Kristina; Brandl, Martin; Massing, Ulrich

    2009-01-01

    This is the accepted manuscript version. Published version available at http://dx.doi.org/10.1016/j.jala.2008.04.002 In-vitro screening for oral absorption has become an essential part of drug discovery and development. Recently, a new phospholipid vesicle-based permeation assay was developed which has shown to satisfyingly predict passive absorption of drugs in humans. The purpose of the current study was to investigate whether the assay may be further developed into a high-th...

  16. Nucleic acid-based assays for the detection of high-risk human papillomavirus: a technical review.

    Science.gov (United States)

    Gibson, Jane S

    2014-09-01

    Nucleic acid-based high-risk human papillomavirus (hrHPV) testing is essential to contemporary cervical cancer screening. The numbers of commercially available assays approved by the US Food and Drug Administration for HPV nucleic acid detection have increased, each offering various approaches to analysis. An understanding of the methodologies associated with HPV testing is important to the practice of laboratory medicine. An overview of instruments, chemistries, laboratory workflows, and test limitations associated with current US Food and Drug Administration-approved assays is provided. © 2014 American Cancer Society.

  17. Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory.

    Science.gov (United States)

    Barnard, Marinus; Warren, Rob; Gey Van Pittius, Nico; van Helden, Paul; Bosman, Marlein; Streicher, Elizabeth; Coetzee, Gerrit; O'Brien, Richard

    2012-12-15

    Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality. To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined. A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability. The GenoType MTBDRsl LPA is a rapid and reliable DST that can be easily incorporated into the diagnostic algorithm. This assay significantly improved diagnostic yield (P < 0.001) while simultaneously

  18. Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

    Science.gov (United States)

    Hawkins, Liam J; Storey, Kenneth B

    2017-01-01

    Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.

  19. Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

    Science.gov (United States)

    Vardakou, Maria; Salmon, Melissa; Faraldos, Juan A; O'Maille, Paul E

    2014-01-01

    Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of k cat/K M among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

  20. Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; van Lenthe, H.; van Gennip, A. H.

    1999-01-01

    A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by

  1. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    Science.gov (United States)

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity.

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

    Directory of Open Access Journals (Sweden)

    Qiang Liu

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

  3. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2017-12-21

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  4. Implementation of a high-throughput ion chromatographic assay to assess glass degradation in drug product formulations.

    Science.gov (United States)

    Thirumangalathu, Renuka; Wong, Kwun Ngok; Coultas, James; Hair, Alison; Piedmonte, Deirdre Murphy

    2015-01-01

    The primary container for parenterals is usually composed of glass. Given the recent industry-wide spike in glass-related problems, assays capable of detecting glass degradation before glass-related particles are visible in solution have practical significance. A rapid, high-throughput ion chromatography method coupled with molybdate reaction is described here for detection and quantitation of silicic acid (soluble form of silica) in complex samples. The method involves ion exchange separation of the silicate anion at high pH followed by a post-column derivatization step with sodium molybdate reagent. The resulting molybdo-silicate complex is detected with high sensitivity in the visible wavelength range at 410 nm and correlates to the level of soluble silica in solution. This assay is high-throughput and amenable for implementation during the early phase of product development. The assay provides a direct measurement to assess potential incompatibility between the formulation and its glass container. The Si levels measured by this method showed a direct correlation to the vial surface morphology changes as monitored by differential interference contrast microscopy. Recently, the pharmaceutical industry has been faced with glass quality challenges that have resulted in many products being recalled from the market. Monitoring levels of soluble silica in solution is critical because silica is the primary component of glass containers used in the pharmaceutical industry. Given this recent industry-wide increase in glass-related problems, assays capable of detecting glass degradation before glass-related particles are visible in solution have practical significance. A rapid assay to detect the soluble form of silica is presented here. The method presented will enable earlier detection of a formulation and container incompatibility instead of waiting until glass-related particles are visible in solution. © PDA, Inc. 2015.

  5. Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance.

    Science.gov (United States)

    Liebman, H A; Sutherland, D; Bacon, R; McGehee, W

    1996-12-01

    Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one-stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50/117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR-based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single-stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.

  6. Advances and Challenges in Viability Detection of Foodborne Pathogens

    Directory of Open Access Journals (Sweden)

    Dexin Zeng

    2016-11-01

    Full Text Available Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR, sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology conventional and real-time PCR (qPCR is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide (EMA and propidium monoazide (PMA to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR, scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound

  7. Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA.

    Science.gov (United States)

    Stubbs, Samuel; Oura, Chris A L; Henstock, Mark; Bowden, Timothy R; King, Donald P; Tuppurainen, Eeva S M

    2012-02-01

    Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  8. Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay.

    Directory of Open Access Journals (Sweden)

    Ikechukwu Okoli

    Full Text Available Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans-C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.

  9. Understanding the Supersensitive Anti-Drug Antibody Assay: Unexpected High Anti-Drug Antibody Incidence and Its Clinical Relevance

    Directory of Open Access Journals (Sweden)

    Sam Song

    2016-01-01

    Full Text Available Numbers of biotherapeutic products in development have increased over past decade. Despite providing significant benefits to patients with unmet needs, almost all protein-based biotherapeutics could induce unwanted immunogenicity, which result in a loss of efficacy and/or increase the risk of adverse reactions, such as infusion reactions, anaphylaxis, and even life-threatening response to endogenous proteins. Recognizing these possibilities, regulatory agencies request that immunogenicity be assessed as part of the approval process for biotherapeutics. Great efforts have been made to reduce drug immunogenicity through protein engineering. Accordingly the immunogenicity incidence has been reduced from around 80% in murine derived products to 0–10% in fully human products. However, recent improvements in immunogenicity assays have led to unexpectedly high immunogenicity rates, even in fully human products, leading to new challenges in assessing immunogenicity and its clinical relevance. These new immunogenicity assays are becoming supersensitive and able to detect more of anti-drug antibodies (ADA than with earlier assays. This paper intends to review and discuss our understanding of the supersensitive ADA assay and the unexpected high ADA incidence and its potential clinical relevance.

  10. A high-throughput, homogeneous microplate assay for agents that kill mammalian tissue culture cells.

    Science.gov (United States)

    Pierce, Michael; Wang, Chunwei; Rebentisch, Matt; Endo, Mark; Stump, Mark; Kamb, Alexander

    2003-06-01

    Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.

  11. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  12. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  13. TINA, a new fully automated high-performance droplet freezing assay coupled to a customized infrared detection system

    Science.gov (United States)

    Kunert, Anna Theresa; Lamneck, Mark; Gurk, Christian; Helleis, Frank; Klimach, Thomas; Scheel, Jan Frederik; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2017-04-01

    Heterogeneous ice nucleation is frequently investigated by simultaneously cooling a defined number of droplets of equal volume in droplet freezing assays. In 1971, Gabor Vali established the quantitative assessment of ice nuclei active at specific temperatures for many droplet freezing assays. Since then, several instruments have been developed, and various modifications and improvements have been made. However, for quantitative analysis of ice nuclei, the current known droplet freezing assays are still limited by either small droplet numbers, large droplet volumes, inadequate separation of the single droplets, which can result in mutual interferences, or imprecise temperature control within the system. Here, we present the Twin Ice Nucleation Assay (TINA), which represents an improvement of the until now existing droplet freezing assays in terms of temperature range and statistics. Above all, we developed a distinct detection system for freezing events in droplet freezing assays, where the temperature gradient of each single droplet is tracked individually by infrared cameras coupled to a self-written software. In the fully automated setup, ice nucleation can be studied in two independently cooled, customized aluminum blocks run by a high-performance thermostat. We developed a cooling setup, which allows both huge and tiny temperature changes within a very short period of time, combined with an optimal insulation. Hence, measurements can be performed at temperatures down to -55 °C (218 K) and at cooling rates up to 3 K min-1. Besides that, TINA provides the analysis of nearly 1000 droplets per run with various droplet volumes between 1 µL and 50 µL. This enables a fast and more precise analysis of biological samples with complex IN composition as well as better statistics for every sample at the same time.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-03-15

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

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

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A.

    2014-03-01

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

  16. A high-throughput, modified ALS activity assay for Cyperus difformis and Schoenoplectus mucronatus seedlings.

    Science.gov (United States)

    Pedroso, Rafael M; Al-Khatib, Kassim; Hanson, Bradley D; Fischer, Albert J

    2017-01-01

    Cyperus difformis L. (CYPDI) and Schoenoplectus mucronatus (L.) Palla (SCHMU) are major weeds of California (CA) rice, where resistance to acetolactate synthase (ALS)-inhibitors was identified in several CYPDI and SCHMU populations that have also evolved resistance to photosystem II (PSII)-inhibiting herbicides. The mechanism of ALS resistance in these populations remains to be clarified but this information is crucial in a weed management program, especially in a scenario where resistance to multiple herbicides has been identified. ALS activity assays are commonly used to diagnose resistance to ALS-inhibitors, but protocols currently available are burdensome for the study of CYPDI and SCHMU, as they require large amounts of plant material from young seedlings and have low yields. Our objective was to investigate the ALS resistance mechanism in suspected ALS-resistant (R) CYPDI and SCHMU biotypes using a modified ALS activity assay that requires less plant material. ALS enzymes from suspected R biotypes were at least 10,000-fold less sensitive to bensulfuron-methyl than susceptible (S) cohorts, indicating ALS resistance that is likely due to an altered target-site. Protein concentration (mgg(-1) tissue) did not differ between R and S biotypes within each species, suggesting that R biotypes do not over produce ALS enzymes. CYPDI biotypes had up to 4-fold more protein per mg of tissue than SCHMU biotypes, but up to 7-fold more acetoin per mg(-1) protein was quantified in SCHMU, suggesting greater ALS catalytic ability in SCHMU biotypes, regardless of their herbicide resistance status. Our optimized protocol to measure ALS activity allowed for up to a 3-fold increase in the number of assays performed per g of leaf tissue. The modified assay may be useful for measuring ALS activity in other weed species that also produce small amount of foliage in early growth stages when protein in tissue is most abundant. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Conidial vigor vs. viability as predictors of virulence of entomopathogenic fungi.

    Science.gov (United States)

    Faria, Marcos; Lopes, Rogério Biaggioni; Souza, Daniela Aguiar; Wraight, Stephen P

    2015-02-01

    We tested the hypothesis that debilitated conidia exhibiting slow-germination (requiring>16h to germinate) are less virulent than vigorous conidia exhibiting fast germination (requiring⩽16h to germinate). Preparations of Beauveria bassiana s.l. strain CG 1027 with variable ratios of vigorous to debilitated conidia were assayed against third-instar larvae of Spodoptera frugiperda. As the proportion of debilitated conidia in test preparations increased, LC50 expressed in terms of total viable conidia increased, while LC50 expressed solely in terms of vigorous conidia remained constant, indicating that vigorous conidia were responsible for nearly all mortality observed in the assays. Larvae treated with conidia from low-quality batches (with high proportions of debilitated conidia) survived consistently longer than those treated with comparable doses of conidia from high-quality batches. These results confirm our previous hypotheses that inclusion of debilitated conidia in viability assessments can lead to overestimation of the quality (potency) of mycoinsecticide preparations and support our recommendation for use of short incubation periods for assessing viability whenever viability is relied upon as an indicator of product quality. Copyright © 2015. Published by Elsevier Inc.

  18. A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum.

    Science.gov (United States)

    Levy, Michael G; Poore, Matthew F; Colorni, Angelo; Noga, Edward J; Vandersea, Mark W; Litaker, R Wayne

    2007-01-18

    Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.

  19. Studies On Fermentation, Alcohol Production And Viability In ...

    African Journals Online (AJOL)

    The reverse was true in the sugarcane bagasse medium. Yeasts with high viability tended to have high alcohol production ability in the sucrose medium and vice-versa. KEY WORDS: Alcohol production; fermentation; induced mutants; Saccharomyces cerevisiae; viability. Global Journal of Pure and Applied Sciences ...

  20. Viability of Baylisascaris procyonis Eggs

    OpenAIRE

    Shira C Shafir; Sorvillo, Frank J.; Sorvillo, Teresa; Eberhard, Mark L.

    2011-01-01

    Infection with Baylisascaris procyonis roundworms is rare but often fatal and typically affects children. We attempted to determine parameters of viability and methods of inactivating the eggs of these roundworms. Loss of viability resulted when eggs were heated to 62°C or desiccated for 7 months but not when frozen at –15°C for 6 months.

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

    Science.gov (United States)

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

    2013-06-21

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

  2. Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

    OpenAIRE

    Tian, Huan; Cao, Liching; Tan, Yuping; Williams, Stephen; Chen, Lili; Matray, Tracy; Chenna, Ahmed; Moore, Sean; Hernandez, Vincent; Xiao, Vivian; Tang, Mengxiang; Singh, Sharat

    2004-01-01

    We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader® mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach w...

  3. Monitoring cell growth, viability, and apoptosis.

    Science.gov (United States)

    Butler, Michael; Spearman, Maureen; Braasch, Katrin

    2014-01-01

    The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess. Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and dieletrophoretic cytometry. These methods work most simply when a fixed volume sample can be taken from a suspension culture. Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included. Electronic particle counting is a rapid total cell count method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates. Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages replacing manual microscopic counting and viability determination. Biomass probes detect cells by their dielectric properties or their internal concentration of NADH and can be used as a continuous monitor of the progress of a culture. While the monitoring of cell growth and viability is an integral part of a bioprocess, the monitoring of apoptosis induction is also becoming more and more important in bioprocess control to increase volumetric productivity by extending bioprocess duration. Different fluorescent assays allow for the detection of apoptotic characteristics in a cell sample.Indirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity. These methods are most useful when it is difficult to obtain intact cell samples. However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture. The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used

  4. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

    Science.gov (United States)

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

    2012-01-01

    Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

  5. 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.

  6. Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase.

    Science.gov (United States)

    Katano, Hajime; Takakuwa, Masahiro; Itoh, Takafumi; Hibi, Takao

    2015-01-01

    A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na2SiO3, 600 mM Na2MoO4, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.

  7. Highly Sensitive Detection of Bisphenol A by NanoAptamer Assay with Truncated Aptamer.

    Science.gov (United States)

    Lee, Eun-Hee; Lim, Hyun Jeong; Lee, Sang-Don; Son, Ahjeong

    2017-05-03

    For the sensitive quantification of bisphenol A (BPA), we have developed NanoAptamer assay, which employs aptamer and complementary signaling DNA, a set of quantum dots (QD), and magnetic beads (MBs). Signaling DNA-QD 655 was tethered to MB-QD 565 via the aptamer. The affinity of the aptamer to BPA resulted in the release of the signaling DNA-QD 655 from the complex and hence the corresponding decrease in the QD 655 fluorescence measurement signal. Three new aptamers (23, 58, and 24-mer) were designed via truncation of the reference aptamer (73-mer). The sensitivity and selectivity of each aptamer for BPA detection via NanoAptamer assay were investigated. One of the truncated aptamers (24-mer) has shown a significantly better performance (limit of detection, LOD, 0.17 pg/mL) than the reference 73-mer aptamer (LOD, 570 pg/mL). It has also shown the best selectivity for BPA detection over BPA analogues (i.e., bisphenol B, bisphenol C, and diethylstilbestrol). It corresponded to a normalized fluorescence change of 33.7% at the environmentally relevant concentration of 1 ng/mL (1 ppb) BPA; however, the analogues remained unchanged (2.3-3.9%).

  8. Identification of low level gamma-irradiation of meats by high sensitivity comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Miyahara, Makoto E-mail: mmiyaha@nihs.go.jp; Saito, Akiko; Ito, Hitoshi; Toyoda, Masatake

    2002-03-01

    The detection of low levels of irradiation in meats (pork, beef, and chicken) using the new comet assay was investigated in order to assess the capability of the procedure. The new assay includes a process that improves its sensitivity to irradiation and a novel evaluation system for each slide (influence score and comet-type distribution). Samples used were purchased at retailers and were irradiated at 0.5 and 2 kGy at 0 deg. C. The samples were processed to obtain comets. Slides were evaluated by typing comets, calculating the influence score and analyzing the comet-type distribution chart of shown on the slide. Influence scores of beef, pork, and chicken at 0 kGy were 287(SD=8.0), 305 (SD=12.9), and 320 (SD=21.0), respectively. Those at 500 Gy, were 305 (SD=5.3), 347 (SD=10.6), and 364 (12.6), respectively. Irradiation levels in food were successfully determined. Sensitivity to irradiation differed among samples (chicken>pork>beef)

  9. A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

    Directory of Open Access Journals (Sweden)

    Sylvie Kemleu

    Full Text Available Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT, and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL. Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002, and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523. Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay

  10. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  11. Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay.

    Directory of Open Access Journals (Sweden)

    Aruna D Balgi

    Full Text Available The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-24

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

  13. Polyphenolic Extracts of Edible Flowers Incorporated onto Atelocollagen Matrices and Their Effect on Cell Viability

    Directory of Open Access Journals (Sweden)

    Jorge López-García

    2013-10-01

    Full Text Available The phenolic extract of chives flowers (Allium schoenoprasum, Liliaceae, introduced Sage (Salvia pratensis, Lamiaceae, European elderberry (Sambucus nigra, Caprifoliaceae and common dandelion (Taraxacum officinale, Asteraceae were characterised by High Performance Liquid Chromatography and incorporated in different concentrations onto atelocollagen thin films. In order to assess the biological impact of these phenolic compounds on cell viability, human immortalised non-tumorigenic keratinocyte cell line was seeded on the thin films and cell proliferation was determined by using an MTT assay. In addition, their antimicrobial activity was estimated by using an agar diffusion test. Data indicated the concomitance between cell viability and concentration of polyphenols. These findings suggest that these phenolic-endowed atelocollagen films might be suitable for tissue engineering applications, on account of the combined activity of polyphenols and collagen.

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

    DEFF Research Database (Denmark)

    Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper

    2009-01-01

    alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E......-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately....... coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats...

  15. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    Science.gov (United States)

    Guo, Jianjun; Morrell-Falvey, Jennifer L.; Labbé, Jessy L.; Muchero, Wellington; Kalluri, Udaya C.; Tuskan, Gerald A.; Chen, Jin-Gui

    2012-01-01

    Background Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways. PMID:23028673

  16. High-throughput, dual probe biological assays based on single molecule detection

    Science.gov (United States)

    Hollars, Christopher W [Brentwood, CA; Huser, Thomas R [Livermore, CA; Lane, Stephen M [Oakland, CA; Balhorn, Rodney L [Livermore, CA; Bakajin, Olgica [San Leandro, CA; Darrow, Christopher [Pleasanton, CA; Satcher, Jr., Joe H.

    2006-07-11

    A method and apparatus with the sensitivity to detect and identify single target molecules through the localization of dual, fluorescently labeled probe molecules. This can be accomplished through specific attachment of the taget to a surface or in a two-dimensional (2D) flowing fluid sheet having approximate dimensions of 0.5 .mu.m.times.100 .mu.m.times.100 .mu.m. A device using these methods would have 10.sup.3 10.sup.4 greater throughput than previous one-dimensional (1D) micro-stream devices having 1 .mu.m.sup.3 interrogation volumes and would for the first time allow immuno- and DNA assays at ultra-low (femtomolar) concentrations to be performed in short time periods (.about.10 minutes). The use of novel labels (such as metal or semiconductor nanoparticles) may be incorporated to further extend the sensitivity possibly into the attomolar range.

  17. Testing the genotoxicity of some perfumes with high diethylphthalate (DEP levels using comet assay

    Directory of Open Access Journals (Sweden)

    Iman Al-Saleh

    2015-05-01

    Full Text Available The presence of phthalates in perfumes has gained recently some attention since these chemicals are added sometimes intentionally as a fixative. Our previous study tested 47 branded perfumes sold in Saudi market and found 68% of tested samples had diethylphthalate (DEP, above reported threshold limit of 1 ppm. Of these phthalates, DEP was found to have the highest mean value (1621.63 ppm. These results enticed us to test the potential genotoxicity of 7 brands in which their DEP contents were in the range of 1.06 ppm to 23649.3 ppm in human TK-6 cells using single cell gel electrophoresis assay (comet assay. Cells were exposed to 400 µl perfume for 2 hrs, at room temperature. Tail moment was (TM used as a metric measure for DNA damage and 25 cells per sample were determined by image analysis software. Four perfumes with DEP above 40 ppm produced significant DNA damage in TK-6 cells with TM of 54.27 ± 1.04, n=500 compare to the other 3 perfumes with DEP < 2 ppm (21.94 ± 1.09, n=300, untreated cells (2.99 ± 0.17, n=250 and cells induced with ethanol (33.76 ± 1.4, n=75 or methanol (23.82 ± 1.84, n=100 which are the vehicles used in perfume's preparations. The results suggest that DEP in perfumes might be one of the ingredients that provoked DNA damage; however, further investigation is required confirming our observation.

  18. A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

    Science.gov (United States)

    Puri, Aastha; Quan, Yong; Narang, Ajit S; Adams, Monica; Gandhi, Rajesh; Nashine, Vishal C

    2017-04-01

    Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

  19. Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals.

    Science.gov (United States)

    Wong, Chin Lin; Lam, Ai-Leen; Smith, Maree T; Ghassabian, Sussan

    2016-01-01

    The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight

  20. A highly sensitive TaqMan real-time PCR assay for early detection of Schistosoma species.

    Science.gov (United States)

    Zhou, Li; Tang, Jingfeng; Zhao, Youyun; Gong, Rui; Lu, Xuan; Gong, Lulu; Wang, Yefu

    2011-01-01

    Schistosomiasis is a major infectious disease and a public health concern in many areas in China and other countries. Sensitive method for detection of the parasite is critical for early diagnosis and for monitoring of effective treatment of the disease. In this study, we developed a highly sensitive TaqMan real-time PCR assay for the detection of Schistosoma japonicum DNA in mouse feces and serum samples. This assay was based on the DNA sequence of the S. japonicum 18S rRNA gene and was able to detect 10 fg of S. japonicum genomic DNA, which is 100 times more sensitive than conventional PCR. We were able to detect the S. japonicum DNA one week post-infection in mouse sera and 4 weeks post-infection in feces, which was one week earlier than egg detection by microscopy in feces. This assay was also highly specific for Asian Schistosomes which are causative species of human Schistosomiasis. In single sex male cercariae infected mice, parasite DNA was only detected in the first 4 weeks post-infection, suggesting that the DNA was derived from decaying worms' corpse in the first 4 weeks whereas the DNA was mainly from decaying parasite eggs afterwards. Therefore we conclude that the established TaqMan real-time PCR assay is a sensitive, specific and convenient method that could be used for the early diagnostic evaluation of S. japonicum infection in humans and for monitoring outbreaks in endemic areas with low prevalence. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

    Science.gov (United States)

    Kwon, Keehwan; Nagarajan, Rajesh; Stivers, James T

    2004-11-30

    Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.

  2. Hesperidin inhibits ovarian cancer cell viability through endoplasmic reticulum stress signaling pathways

    OpenAIRE

    Zhao, Jun; Li, Yali; Gao, Jinfang; De, Yinshan

    2017-01-01

    Hesperidin is a vitamin P flavonoid compound primarily present in citrus fruits. The aim of the present study was to investigate whether hesperidin inhibits ovarian cancer cell viability via endoplasmic reticulum stress signaling pathways. A2780 cells were treated with various doses of hesperidin for 6, 12 or 24 h, and the viability of A2780 cells was assessed using the MTT assay. Hesperidin decreased the viability of A2780 cells and increased cytotoxicity in a dose- and time-dependent manner...

  3. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen; Nakano, Hiroyasu

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  4. [Presence and viability of Giardia spp. and Cryptosporidium spp. in drinking water and wastewater in the high basin of Bogotá river].

    Science.gov (United States)

    Alarcón, Marlén Andrea; Beltrán, Milena; Cárdenas, Martha Liliana; Campos, María Claudia

    2005-09-01

    Faecal contamination in wastewater and drinking water is linked to the dissemination of water related diseases. The bacteria, virus and parasites present in drinking water are responsible for substantial morbidity and mortality, especially among infants. Giardia spp. and Cryptosporidium spp. were the organisms selected as parasite contamination indicators. Their presence serves as a useful tool for evaluating water quality and determining sanitary risk. At present, in Colombia, concentration and occurrence of these parasites is unknown and an immediate assessment was considered necessary. Protozoan presence was determined in five sampling stations in the Bogotá river upper basin and in two drinking water plants near the same area. The techniques applied for counting encysted forms consisted of inorganic flocculation for wastewater or filtration for drinking water. Fluorogenic vital dyes tested for viability. The presence of Cryptosporidium spp. was confirmed in two of the sampled stations and at two of the drinking water plants. Giardia spp. was found at two of the drinking water plants but not at the sampled stations. Viable cysts were found for Cryptosporidium spp. in one of the samples from the Bogotá river, but only inviable exemplars were obtained from the drinking water plants. The results revealed protozoan presence in drinking and residual water implying the presence of a potential sanitary hazard.

  5. Viability of mesenchymal stem cells during electrospinning

    Directory of Open Access Journals (Sweden)

    G. Zanatta

    2012-02-01

    Full Text Available Tissue engineering is a technique by which a live tissue can be re-constructed and one of its main goals is to associate cells with biomaterials. Electrospinning is a technique that facilitates the production of nanofibers and is commonly used to develop fibrous scaffolds to be used in tissue engineering. In the present study, a different approach for cell incorporation into fibrous scaffolds was tested. Mesenchymal stem cells were extracted from the wall of the umbilical cord and mononuclear cells from umbilical cord blood. Cells were re-suspended in a 10% polyvinyl alcohol solution and subjected to electrospinning for 30 min under a voltage of 21 kV. Cell viability was assessed before and after the procedure by exclusion of dead cells using trypan blue staining. Fiber diameter was observed by scanning electron microscopy and the presence of cells within the scaffolds was analyzed by confocal laser scanning microscopy. After electrospinning, the viability of mesenchymal stem cells was reduced from 88 to 19.6% and the viability of mononuclear cells from 99 to 8.38%. The loss of viability was possibly due to the high viscosity of the polymer solution, which reduced the access to nutrients associated with electric and mechanical stress during electrospinning. These results suggest that the incorporation of cells during fiber formation by electrospinning is a viable process that needs more investigation in order to find ways to protect cells from damage.

  6. Viability of smallholder dairying in Wedza, Zimbabwe.

    Science.gov (United States)

    Zvinorova, Plaxedis Ivy; Halimani, Tinyiko Edward; Mano, Renneth T; Ngongoni, Nobbert Takarwirwa

    2013-04-01

    Viability differences in smallholder dairy farming are a result of differences in access to markets and services. It is hypothesized that innovations that improve productivity and market linkages also improve returns and viability. The viability of smallholder dairying in Wedza was characterised by interviewing 52 households using semi-structured questionnaires. Information on demographics, production, marketing, livestock numbers, assets and constraints was obtained. Farmers were resource-constrained with differences in access to resources. The highly resourced farmers had higher milk output and numbers of livestock. Almost 40 % of the households were female-headed, and these dominated the poor category. Household sizes ranged from 4 to 13 persons. Milk off-take was low (3.7 ± 0.53 l/cow/day), due to various constraints. Only rich farmers had viable enterprises in purely financial terms. Per litre cost of milk was more than selling price (US$0.96) for most farmers except the relatively rich. Operating ratios were 1.7, 0.6, 1.4 and 1.1 for the poor, rich, sub-centre and milk collection centre farmers, respectively. This means incomes from the dairy activities did not cover costs. Sensitivity analysis indicated that increases in total variable costs and labour reduced returns. Milk production and viability were influenced by access to resources and markets.

  7. High throughput image cytometry micronucleus assay to investigate the presence or absence of mutagenic effects of cold physical plasma.

    Science.gov (United States)

    Bekeschus, Sander; Schmidt, Anke; Kramer, Axel; Metelmann, Hans-Robert; Adler, Frank; von Woedtke, Thomas; Niessner, Felix; Weltmann, Klaus-Dieter; Wende, Kristian

    2018-02-08

    Promising cold physical plasma sources have been developed in the field of plasma medicine. An important prerequisite to their clinical use is lack of genotoxic effects in cells. During optimization of one or even different plasma sources for a specific application, large numbers of samples need to be analyzed. There are soft and easy-to-assess markers for genotoxic stress such as phosphorylation of histone H2AX (γH2AX) but only few tests are accredited by the OECD with regard to mutagenicity detection. The micronucleus (MN) assay is among them but often requires manual counting of many thousands of cells per sample under the microscope. A high-throughput MN assay is presented using image flow cytometry and image analysis software. A human lymphocyte cell line was treated with plasma generated with ten different feed gas conditions corresponding to distinct reactive species patterns that were investigated for their genotoxic potential. Several millions of cells were automatically analyzed by a MN quantification strategy outlined in detail in this work. Our data demonstrates the absence of newly formed MN in any feed gas condition using the atmospheric pressure plasma jet kINPen. As positive control, ionizing radiation gave a significant 5-fold increase in micronucleus frequency. Thus, this assay is suitable to assess the genotoxic potential in large sample sets of cells exposed chemical or physical agents including plasmas in an efficient, reliable, and semiautomated manner. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  8. Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

    Science.gov (United States)

    Yaoi, Takuro; Chamnongpol, Sangpen; Jiang, Xin; Li, Xianqiang

    2006-05-01

    Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

  9. PubChem BioAssay: A Decade's Development toward Open High-Throughput Screening Data Sharing.

    Science.gov (United States)

    Wang, Yanli; Cheng, Tiejun; Bryant, Stephen H

    2017-07-01

    High-throughput screening (HTS) is now routinely conducted for drug discovery by both pharmaceutical companies and screening centers at academic institutions and universities. Rapid advance in assay development, robot automation, and computer technology has led to the generation of terabytes of data in screening laboratories. Despite the technology development toward HTS productivity, fewer efforts were devoted to HTS data integration and sharing. As a result, the huge amount of HTS data was rarely made available to the public. To fill this gap, the PubChem BioAssay database ( https://www.ncbi.nlm.nih.gov/pcassay/ ) was set up in 2004 to provide open access to the screening results tested on chemicals and RNAi reagents. With more than 10 years' development and contributions from the community, PubChem has now become the largest public repository for chemical structures and biological data, which provides an information platform to worldwide researchers supporting drug development, medicinal chemistry study, and chemical biology research. This work presents a review of the HTS data content in the PubChem BioAssay database and the progress of data deposition to stimulate knowledge discovery and data sharing. It also provides a description of the database's data standard and basic utilities facilitating information access and use for new users.

  10. High-sensitivity troponin assays in the evaluation of patients with acute chest pain in the emergency department.

    Science.gov (United States)

    Christ, Michael; Bertsch, Thomas; Popp, Steffen; Bahrmann, Philipp; Heppner, Hans-Jürgen; Müller, Christian

    2011-09-06

    Evaluating patients with acute chest pain presenting to the emergency department remains an ongoing challenge. The spectrum of etiologies in acute chest pain ranges from minor disease entities to life-threatening diseases, such as pulmonary embolism, acute aortic dissection or acute myocardial infarction (MI). The diagnosis of acute MI is usually made integrating the triad of patient history and clinical presentation, readings of 12-lead ECG and measurement of cardiac troponins (cTn). Introduction of high-sensitivity cTn assays substantially increases sensitivity to identify patients with acute MI even at the time of presentation to the emergency department at the cost of specificity. However, the proportion of patients presenting with cTn positive, non-vascular cardiac chest pain triples with the implementation of new sensitive cTn assays increasing the difficulty for the emergency physician to identify those patients who are at need for invasive diagnostics. The main objectives of this mini-review are 1) to discuss elements of disposition decision made by the emergency physician for the evaluation of chest pain patients, 2) to summarize recent advances in assay technology and relate these findings into the clinical context, and 3) to discuss possible consequences for the clinical work and suggest an algorithm for the clinical evaluation of chest pain patients in the emergency department.

  11. Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting.

    Science.gov (United States)

    Strydom, K; Ismail, F; Matabane, M M Z; Onwuegbuna, O; Omar, S V; Ismail, N

    2015-09-01

    In a head-to-head comparison of the MTBDRplus version 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting

    OpenAIRE

    Strydom, K.; Ismail, F.; Matabane, M.M.Z.; O. Onwuegbuna; S V Omar; N. Ismail

    2015-01-01

    In a head-to-head comparison of the MTBDRplus version 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity.

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

    Science.gov (United States)

    Guha, Rajarshi; Schürer, Stephan C.

    2008-06-01

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

  14. A high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay.

    Science.gov (United States)

    Gutmann, Daniel A P; Mizohata, Eiichi; Newstead, Simon; Ferrandon, Sebastian; Postis, Vincent; Xia, Xiaobing; Henderson, Peter J F; van Veen, Hendrik W; Byrne, Bernadette

    2007-07-01

    One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time- and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 microL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.

  15. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

    Directory of Open Access Journals (Sweden)

    Lötzerich Mark

    2010-10-01

    Full Text Available Abstract Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2 detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens

  16. Comparison of Abbott RealTime High-Risk HPV and Hybrid Capture 2 Assays for Detection of HPV Infection.

    Science.gov (United States)

    Ko, Kiwoong; Yu, Shinae; Lee, Eun Hee; Park, Hyosoon; Woo, Hee-Yeon; Kwon, Min-Jung

    2016-09-01

    Various assays for detecting high-risk human papillomavirus (HR HPV) have been introduced recently, including the Abbott RealTime High-Risk HPV assay. We sought to compare the performance of Abbott PCR to Hybrid Capture 2 for the detection of HR HPV. A total of 941 cervical swab specimens were obtained. We submitted all specimens for HR HPV detection with HC2 and Abbott PCR, and then additionally analyzed discordant and concordant positive results using restriction fragment mass polymorphism (RFMP) genotyping analysis. HC2 detected one of 13 HR HPV types in 12.3% (116/941) of cases, while Abbott PCR detected one of 14 detectable HR HPV types in 12.9% (121/941) of cases. The overall agreement rate was 97.3% with a kappa coefficient of 0.879. Discordant results between these two assays were observed in 25 cases. HC2 showed a sensitivity of 90.0% and specificity of 95.9%, while Abbott PCR showed a sensitivity of 98.0% and specificity of 96.8% when using RFMP results as the gold standard. For HPV 16/18 detection, Abbott PCR showed 95.8%/88.9% sensitivity and 99.2%/99.8% specificity, respectively. The overall coinfection rate between HPV 16, 18 and non-16/18 was 9.9% (12/121) in Abbott PCR analysis. Considering its high agreement rate with HC2, higher sensitivity/specificity compared to HC2, and ability to differentiate HPV 16/18 from other HPV types, Abbott PCR could be a reliable laboratory testing method for the screening of HPV infections. © 2016 by the Association of Clinical Scientists, Inc.

  17. Clinical Use of Ultrasensitive Cardiac Troponin I Assay in Intermediate- and High-Risk Surgery Patients

    Directory of Open Access Journals (Sweden)

    Flávia Kessler Borges

    2013-01-01

    Full Text Available Background. Cardiac troponin levels have been reported to add value in the detection of cardiovascular complications in noncardiac surgery. A sensitive cardiac troponin I (cTnI assay could provide more accurate prognostic information. Methods. This study prospectively enrolled 142 patients with at least one Revised Cardiac Risk Index risk factor who underwent noncardiac surgery. cTnI levels were measured postoperatively. Short-term cardiac outcome predictors were evaluated. Results. cTnI elevation was observed in 47 patients, among whom 14 were diagnosed as having myocardial infarction (MI. After 30 days, 16 patients had major adverse cardiac events (MACE. Excluding patients with a final diagnosis of MI, predictors of cTnI elevation included dialysis, history of heart failure, transoperative major bleeding, and elevated levels of pre- and postoperative N-terminal pro-B-type natriuretic peptide (NT-proBNP. Maximal cTnI values showed the highest sensitivity (94%, specificity (75%, and overall accuracy (AUC 0.89; 95% CI 0.80–0.98 for postoperative MACE. Postoperative cTnI peak level (OR 9.4; 95% CI 2.3–39.2 and a preoperative NT-proBNP level ≥917 pg/mL (OR 3.47; 95% CI 1.05–11.6 were independent risk factors for MACE. Conclusions. cTnI was shown to be an independent prognostic factor for cardiac outcomes and should be considered as a component of perioperative risk assessment.

  18. Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior

    DEFF Research Database (Denmark)

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Arvidson, Sandra Marie Neumann

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim...... was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses...... in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans....

  19. A universal indicator dye pH assay for crystallization solutions and other high-throughput applications.

    Science.gov (United States)

    Newman, Janet; Sayle, Roger A; Fazio, Vincent J

    2012-08-01

    In protein crystallization, as well as in many other fields, it is known that the pH at which experiments are performed is often the key factor in the success or failure of the trials. With the trend towards plate-based high-throughput experimental techniques, measuring the pH values of solutions one by one becomes prohibitively time- and reagent-expensive. As part of an HT crystallization facility, a colour-based pH assay that is rapid, uses very little reagent and is suitable for 96-well or higher density plates has been developed.

  20. Inhibitory effects of brain-derived neurotrophic factor precursor on viability and neurite growth of murine hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Jia CHEN

    2014-10-01

    Full Text Available Objective To explore the mediation effect of p75 neurotrophin receptor (p75NTR in the effect of brainderived neurotrophic factor precursor (proBDNF on viability and neurite growth of murine hippocampal neurons. Methods  Hippocampal neurons were obtained from p75NTR+/+ and p75NTR-/- 18-day mice and primarily cultured. For p75NTR+/+ neurons, three experimental groups were set, i.e. control, proBDNF (30ng/ml, and proBDNF (30ng/ml+p75/Fc (30µg/ml groups. For p75NTR-/- neurons, two experimental groups were set, i.e. control and proBDNF (30ng/ml groups. MTT assays were performed after 24h to examine the viability of neonatal primary neurons. Immunofluorescent staining was conducted after 72h to investigate the neurite length. Results With MAP2 and DAPI double fluorescent staining it was identified that the neonatal hippocampal neurons were successfully cultured in vitro with high purity. For viability assay of p75NTR+/+ neurons, it was found that the absorbance value at 570nm (A570 in proBDNF group was significantly lower than that in control group (P0.05. With neurite growth assay of p75NTR+/+ neurons, it was found that the neurite length in proBDNF group was significantly shorter than that in control group (P0.05. With neurite growth assay of p75NTR-/- neurons, no difference in neurite length was observed between proBDNF group and control group. Conclusion proBDNF may inhibit the neuronal viability and neurite growth via p75NTR. DOI: 10.11855/j.issn.0577-7402.2014.09.03

  1. Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

    Science.gov (United States)

    Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

    Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The design of a high-efficiency neutron counter for waste drums to provide optimized sensitivity for plutonium assay

    Energy Technology Data Exchange (ETDEWEB)

    Menlove, H.O.; Beddingfield, D.H.; Pickrell, M.M. [Los Alamos National Lab., NM (United States)] [and others

    1997-11-01

    An advanced passive neutron counter has been designed to improve the accuracy and sensitivity for the nondestructive assay of plutonium in scrap and waste containers. The High-Efficiency Neutron Counter (HENC) was developed under a Cooperative Research Development Agreement between the Los Alamos National Laboratory and Canberra Industries. The primary goal of the development was to produce a passive assay system for 200-L drums that has detectability limits and multiplicity counting features that are superior to previous systems. A detectability limit figure of merit (FOM) was defined that included the detector efficiency, the neutron die-away time, and the detector`s active volume and density that determine the cosmic-ray background. Monte Carlo neutron calculations were performed to determine the parameters to provide an optimum FOM. The system includes the {sup 252}Cf {open_quotes}add-a-source{close_quotes} feature to improve the accuracy as well as statistical filters to reduce the cosmic-ray spallation neutron background. The final decision gave an efficiency of 32% for plutonium with a detector {sup 3}He tube volume that is significantly smaller than for previous high-efficiency systems for 200-L drums. Because of the high efficiency of the HENC, we have incorporated neutron multiplicity counting for matrix corrections for those cases where the plutonium is localized in nonuniform hydrogenous materials. The paper describes the design and performance testing of the advanced system. 5 refs., 8 figs., 3 tabs.

  3. Clinical Validation of the Abbott RealTime High Risk HPV Assay According to the Guidelines for Human Papillomavirus DNA Test Requirements for Cervical Screening

    OpenAIRE

    Hesselink, A. T.; Meijer, C J L M; Poljak, M.; Berkhof, J; van Kemenade, F. J.; van der Salm, M. L.; Bogaarts, M.; Snijders, P J F; Heideman, D. A. M.

    2013-01-01

    This study showed that the Abbott RealTime High Risk HPV assay fulfilled cross-sectional clinical equivalence and reproducibility criteria of international consensus guidelines, which indicates that this assay can be considered clinically validated for cervical cancer screening purposes.

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

    Science.gov (United States)

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

    2014-03-01

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

  5. Genetic Correlation with the DNA Repair Assay in Mice Exposed to High-LET

    Science.gov (United States)

    Penninckx, Sebastien; Ray, Shayoni; Degorre, Charlotte; Guiet, Elodie; Viger, Louise; Pluth, Janice; Snijders, Antoine; Mao, Jian-Hua; Costes, Sylvain V.

    2017-01-01

    We hypothesize that DNA damage induced by high local energy deposition, occurring when cells are traversed by high-LET (Linear Energy Transfer) particles, can be experimentally modeled by exposing cells to high doses of low-LET. In this work, we validate such hypothesis by characterizing and correlating the time dependence of 53BP1 radiation-induced foci (RIF) for various doses and LET across 72 primary skin fibroblast from mice. This genetically diverse population allows us to understand how genetic may modulate the dose and LET relationship. The cohort was made on average from 3 males and 3 females belonging to 15 different strains of mice with various genetic backgrounds, including the collaborative cross (CC) genetic model (10 strains) and 5 reference mice strains. Cells were exposed to two fluences of three HZE (High Atomic Energy) particles (Si 350 megaelectronvolts per nucleon, Ar 350 megaelectronvolts per nucleon and Fe 600 megaelectronvolts per nucleon) and to 0.1, 1 and 4 grays from a 160 kilovolt X-ray. Individual radiation sensitivity was investigated by high throughput measurements of DNA repair kinetics for different doses of each radiation type. The 53BP1 RIF dose response to high-LET particles showed a linear dependency that matched the expected number of tracks per cell, clearly illustrating the fact that close-by DNA double strand breaks along tracks cluster within one single RIF. By comparing the slope of the high-LET dose curve to the expected number of tracks per cell we computed the number of remaining unrepaired tracks as a function of time post-irradiation. Results show that the percentage of unrepaired track over a 48 hours follow-up is higher as the LET increases across all strains. We also observe a strong correlation between the high dose repair kinetics following exposure to 160 kilovolts X-ray and the repair kinetics of high-LET tracks, with higher correlation with higher LET. At the in-vivo level for the 10-CC strains, we observe that

  6. High-throughput clone screening followed by protein expression cross-check: A visual assay platform.

    Science.gov (United States)

    Bose, Partha Pratim; Kumar, Prakash

    2017-01-01

    In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-PCR based visual detection technique, which can be used as simultaneous high throughput clone screening followed by the determination of expression of desired protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. A highly sensitive resonance Rayleigh scattering and colorimetric assay for the recognition of propranolol in β-adrenergic blocker.

    Science.gov (United States)

    Tan, Xuanping; Yang, Jidong; Yang, Qiong; Li, Qin

    2017-11-01

    In this work, a highly sensitive, citrate anion-capped gold nanoparticles (AuNPs)-based assay for the determination of propranolol in real samples with resonance Rayleigh scattering (RRS) and colorimetry was developed. When AuNPs were prepared by the sodium citrate reduction method, citrate anions self-assembled on the surface of AuNPs to form supramolecular complex anions. In BR 4.6 buffer solution, propranolol was positively charged and could bind with AuNPs to form larger aggregates through electrostatic force and hydrophobic effects. This results in remarkable enhancement of the RRS intensity and a color change in the AuNPs solution from red to blue via purple. Thus, a highly sensitive RRS and colorimetric assay the for detection of propranolol was developed with a linear range of 0.2-5.2 and 8-112 ng/ml, respectively. In addition, no difference was seen when comparing R-propranolol with S-propranolol, therefore, this method could not be used in the recognition of chiral propranolol. However, upon addition of other β-adrenergic blockers, no phenomenon like that seen with propranolol was observed, meaning that this method can be used for determining the presence of propranolol in a mixture β-adrenergic blockers. Finally, the optimum conditions, factors influencing the reaction, its mechanism and the reasons for enhancement of the RRS were discussed. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus.

    Science.gov (United States)

    Aljofan, Mohamad; Porotto, Matteo; Moscona, Anne; Mungall, Bruce A

    2008-04-01

    There are currently no antiviral drugs approved for the highly lethal Biosafety Level 4 pathogens Nipah and Hendra virus. A number of researchers are developing surrogate assays amenable to Biosafety Level 2 biocontainment but ultimately, the development of a high throughput screening method for directly quantifying these viruses in a Biosafety Level 4 environment will be critical for final evaluation of antiviral drugs identified in surrogate assays, in addition to reducing the time required for effective antiviral drug development. By adapting an existing immunoplaque assay and using enzyme linked immunodetection in a microtitre plate format, the current experiments describe a simple two step assay protocol involving an overnight virus inoculation of Vero cell monolayers (with or without antiviral drug treatment) at Biosafety Level 4, followed by cell fixation and virus inactivation enabling removal of plates from the Biosafety Level 4 laboratory and a subsequent immunodetection assay using a chemiluminescent horse radish peroxidase substrate to be performed at Biosafety Level 2. The analytical sensitivity (limit of detection) of this assay is 100 tissue culture infectious dose50/ml of either Nipah or Hendra virus. In addition this assay enables linear quantitation of virus over three orders of magnitude and is unaffected by dimethyl sulfoxide concentrations of 1% or less. Intra-assay coefficients of variation are acceptable (less than 20%) when detecting a minimum of 1000 tissue culture infectious dose50/ml of either virus although inter-assay variation is considerably greater. By an assessment of efficacies of the broad spectrum antiviral Ribavirin and an experimental fusion inhibitory peptide, this assay reveals a good correlation with previously published fluorescent immunodetection assays. The current experiments describe for the first time, a high throughput screening method amenable for direct assessment of live henipavirus antiviral drug activity.

  9. Device and method for enhanced collection and assay of chemicals with high surface area ceramic

    Energy Technology Data Exchange (ETDEWEB)

    Addleman, Raymond S.; Li, Xiaohong Shari; Chouyyok, Wilaiwan; Cinson, Anthony D.; Bays, John T.; Wallace, Krys

    2016-02-16

    A method and device for enhanced capture of target analytes is disclosed. This invention relates to collection of chemicals for separations and analysis. More specifically, this invention relates to a solid phase microextraction (SPME) device having better capability for chemical collection and analysis. This includes better physical stability, capacity for chemical collection, flexible surface chemistry and high affinity for target analyte.

  10. Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals

    Directory of Open Access Journals (Sweden)

    Chin Lin eWong

    2016-03-01

    Full Text Available The direct peptide reactivity assay (DPRA is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium and high concentrations and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme and non-sensitizers with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF, cysteine- (Ac-RFAACAA and lysine- (Ac-RFAAKAA containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7% and glass (47.3% vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2,4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further

  11. Highly specific detection of myostatin prodomain by an immunoradiometric sandwich assay in serum of healthy individuals and patients.

    Directory of Open Access Journals (Sweden)

    Astrid Breitbart

    Full Text Available BACKGROUND: Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker. METHODS: We established an immunoradiometric sandwich assay (IRMA, which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease. RESULTS: The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25(th-75(th percentiles, 2-7ng/ml and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25(th-75(th percentiles, 7-100ng/ml in the serum of underweight patients with chronic pulmonary disease. CONCLUSIONS: We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease

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

    Science.gov (United States)

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

    2014-03-01

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

  13. High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential.

    Science.gov (United States)

    Garcia, G; Santos, C Nunes do; Menezes, R

    2016-01-01

    The association between altered proteostasis and inflammatory responses has been increasingly recognized, therefore the identification and characterization of novel compounds with anti-inflammatory potential will certainly have a great impact in the therapeutics of protein-misfolding diseases such as degenerative disorders. Although cell-based screens are powerful approaches to identify potential therapeutic compounds, establishing robust inflammation models amenable to high-throughput screening remains a challenge. To bridge this gap, we have exploited the use of yeasts as a platform to identify lead compounds with anti-inflammatory properties. The yeast cell model described here relies on the high-degree homology between mammalian and yeast Ca(2+)/calcineurin pathways converging into the activation of NFAT and Crz1 orthologous proteins, respectively. It consists of a recombinant yeast strain encoding the lacZ gene under the control of Crz1-recongition elements to facilitate the identification of compounds interfering with Crz1 activation through the easy monitoring of β-galactosidase activity. Here, we describe in detail a protocol optimized for high-throughput screening of compounds with potential anti-inflammatory activity as well as a protocol to validate the positive hits using an alternative β-galactosidase substrate.

  14. Determination of dynamic doxorubicin-EC50 value in an automated high-content workstation for cellular assays.

    Science.gov (United States)

    Wolf, Peter; Hartl, Florian; Brischwein, Martin; Wolf, Bernhard

    2011-12-01

    To overcome the problems of endpoint tests routinely required for EC50 determination, we utilized a novel automated high-content workstation and calculated a time-resolved EC50 value of MCF-7 mamma carcinoma cells treated with a pharmacologic agent. Measuring parameters were the cellular oxygen consumption and the extracellular acidification. These parameters were detected in real-time and label free with optochemical sensor spots in a modified 24-well sensor plate. In particular, the objective was to compare the measuring data of the workstation with a classical standard resazurin cell assay and to transfer the benefit of continuously recorded metabolic data of the workstation to practical time-resolved information about the effect of the applied active reagent (doxorubicin). MCF-7 cells were treated with a broad range of doxorubicin concentrations (100 μM to 1 nM) over 24 h and cellular activities were investigated with both, the resazurin assay and the automated workstation. Twenty-four hours after treatment, the resazurin assay showed an EC50 value (6.3 μM) which was about one decade above the value obtained from oxygen consumption rate (0.37 μM) and extracellular acidification rate (0.72 μM), measured with the workstation. Presumably, the differences are due to the different metabolic nature and regulation behind these measuring parameters. By polynomial fitting of continuously recorded metabolic data, we were able to point out a dynamic, time-resolved EC50 characteristic for different time points. The workstation is a powerful tool to record in vitro kinetic data of pharmacologic effects in vital cells in an automated experimental run. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.

    2007-01-01

    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery...

  16. Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance.

    Science.gov (United States)

    Takae, Seiji; Akiyama, Yoshitsugu; Yamasaki, Yuichi; Nagasaki, Yukio; Kataoka, Kazunori

    2007-01-01

    A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.

  17. High content imaging-based assay to classify estrogen receptor-α ligands based on defined mechanistic outcomes.

    Science.gov (United States)

    Ashcroft, F J; Newberg, J Y; Jones, E D; Mikic, I; Mancini, M A

    2011-05-15

    Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Viability of encapsulated Lactobacillus sp. Mar 8

    Directory of Open Access Journals (Sweden)

    EVI TRIANA

    2006-04-01

    Full Text Available Lactobacillus sp. Mar 8 had advantages as probiotic digestive system cholesterol lowering Lactobacillus. Applying in industry, particular processing technique is necessary for gaining product that ready for marketing and consuming. Spray drying is common technique using in various food processing. High processing temperature, 100-200oC, for 3-10 second become the barrier because cells were under extreme temperature stress. Therefore, encapsulate was needed to protect the cells from those extreme conditions. Viability and survival rate of encapsulated Lactobacillus sp. Mar 8 have been investigated. The result showed that Lactobacillus sp. Mar 8 that was encapsulated by 10% skim milk has higher viability than those by 5% skim milk, namely 72.37% and 51.69% respectively. Survival rate of encapsulated Lactobacillus cells will come to zero in 41.28 years. Therefore, encapsulated Lactobacillus sp. Mar 8 may use as probiotic agent.

  19. A multilocus assay reveals high nucleotide diversity and limited differentiation among Scandinavian willow grouse (Lagopus lagopus

    Directory of Open Access Journals (Sweden)

    Quintela Maria

    2008-12-01

    Full Text Available Abstract Background There is so far very little data on autosomal nucleotide diversity in birds, except for data from the domesticated chicken and some passerines species. Estimates of nucleotide diversity reported so far in birds have been high (~10-3 and a likely explanation for this is the generally higher effective population sizes compared to mammals. In this study, the level of nucleotide diversity has been examined in the willow grouse, a non-domesticated bird species from the order Galliformes, which also holds the chicken. The willow grouse (Lagopus lagopus has an almost circumpolar distribution but is absent from Greenland and the north Atlantic islands. It primarily inhabits tundra, forest edge habitats and sub-alpine vegetation. Willow grouse are hunted throughout its range, and regionally it is a game bird of great cultural and economical importance. Results We sequenced 18 autosomal protein coding loci from approximately 15–18 individuals per population. We found a total of 127 SNP's, which corresponds to 1 SNP every 51 bp. 26 SNP's were amino acid replacement substitutions. Total nucleotide diversity (πt was between 1.30 × 10-4 and 7.66 × 10-3 (average πt = 2.72 × 10-3 ± 2.06 × 10-3 and silent nucleotide diversity varied between 4.20 × 10-4and 2.76 × 10-2 (average πS = 9.22 × 10-3 ± 7.43 × 10-4. The synonymous diversity is approximately 20 times higher than in humans and two times higher than in chicken. Non-synonymous diversity was on average 18 times lower than the synonymous diversity and varied between 0 and 4.90 × 10-3 (average πa = 5.08 × 10-4 ± 7.43 × 103, which suggest that purifying selection is strong in these genes. FST values based on synonymous SNP's varied between -5.60 × 10-4 and 0.20 among loci and revealed low levels of differentiation among the four localities, with an overall value of FST = 0.03 (95% CI: 0.006 – 0.057 over 60 unlinked loci. Non-synonymous SNP's gave similar results. Low

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

    Directory of Open Access Journals (Sweden)

    Natalia Piotrzkowski

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

  1. A 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.

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

    Science.gov (United States)

    Piotrzkowski, Natalia; Schillberg, Stefan; Rasche, Stefan

    2012-01-01

    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.

  3. A high-throughput assay for quantifying appetite and digestive dynamics.

    Science.gov (United States)

    Jordi, Josua; Guggiana-Nilo, Drago; Soucy, Edward; Song, Erin Yue; Lei Wee, Caroline; Engert, Florian

    2015-08-15

    Food intake and digestion are vital functions, and their dysregulation is fundamental for many human diseases. Current methods do not support their dynamic quantification on large scales in unrestrained vertebrates. Here, we combine an infrared macroscope with fluorescently labeled food to quantify feeding behavior and intestinal nutrient metabolism with high temporal resolution, sensitivity, and throughput in naturally behaving zebrafish larvae. Using this method and rate-based modeling, we demonstrate that zebrafish larvae match nutrient intake to their bodily demand and that larvae adjust their digestion rate, according to the ingested meal size. Such adaptive feedback mechanisms make this model system amenable to identify potential chemical modulators. As proof of concept, we demonstrate that nicotine, l-lysine, ghrelin, and insulin have analogous impact on food intake as in mammals. Consequently, the method presented here will promote large-scale translational research of food intake and digestive function in a naturally behaving vertebrate. Copyright © 2015 the American Physiological Society.

  4. A high-throughput assay for quantifying appetite and digestive dynamics

    Science.gov (United States)

    Guggiana-Nilo, Drago; Soucy, Edward; Song, Erin Yue; Lei Wee, Caroline; Engert, Florian

    2015-01-01

    Food intake and digestion are vital functions, and their dysregulation is fundamental for many human diseases. Current methods do not support their dynamic quantification on large scales in unrestrained vertebrates. Here, we combine an infrared macroscope with fluorescently labeled food to quantify feeding behavior and intestinal nutrient metabolism with high temporal resolution, sensitivity, and throughput in naturally behaving zebrafish larvae. Using this method and rate-based modeling, we demonstrate that zebrafish larvae match nutrient intake to their bodily demand and that larvae adjust their digestion rate, according to the ingested meal size. Such adaptive feedback mechanisms make this model system amenable to identify potential chemical modulators. As proof of concept, we demonstrate that nicotine, l-lysine, ghrelin, and insulin have analogous impact on food intake as in mammals. Consequently, the method presented here will promote large-scale translational research of food intake and digestive function in a naturally behaving vertebrate. PMID:26108871

  5. Screening for latent TB in patients with rheumatic disorders prior to biologic agents in a 'high-risk' TB population: comparison of two interferon gamma release assays.

    Science.gov (United States)

    Melath, Sunil; Ismajli, Mediola; Smith, Robin; Patel, Ishita; Steuer, Alan

    2014-01-01

    Patients with rheumatic disorders treated with TNF inhibitors are at increased risk of developing TB. There is no 'gold-standard' for the diagnosis of latent TB prior to initiation of biologic agents. We report our own experience of comparing two interferon gamma release assays (IGRAs) in screening for latent TB in a 'high-risk' TB area in patients with rheumatic disorders. The study demonstrated good concordance between the two tests. We believe the additional cost of these assays is justified in high-risk populations prior to biologic agents, with 16% of the current study population with at least one positive IGRA assay.

  6. A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

    Directory of Open Access Journals (Sweden)

    Aldred Shelley F

    2009-10-01

    Full Text Available Abstract Background Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells. Results In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1. Conclusion These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.

  7. Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

    Science.gov (United States)

    Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

    2016-01-01

    To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

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

    Science.gov (United States)

    Lee, Linda G.; Nordman, Eric S.; Johnson, Martin D.; Oldham, Mark F.

    2013-01-01

    We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE) to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA) as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG) as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting. PMID:25586412

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

    Directory of Open Access Journals (Sweden)

    Linda G. Lee

    2013-10-01

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

  10. 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.

  11. Detection of high-risk human papillomavirus infection in tonsillar specimens using 2 commercially available assays.

    Science.gov (United States)

    Cockerill, Cara C; Orvidas, Laura J; Moore, Eric J; Binnicker, Matthew J; Duresko, Brian J; Espy, Mark J; Cockerill, Franklin R; Tombers, Nicole M; Pritt, Bobbi S

    2016-12-01

    THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

    Science.gov (United States)

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

    Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

  13. A high throughput in vivo assay for taste quality and palatability.

    Directory of Open Access Journals (Sweden)

    R Kyle Palmer

    Full Text Available Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli. Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity. Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints. We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm. Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor. As a rat's tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser. The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever. Each disruption of the laser was counted as a lick. Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy. Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness. All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue. The technology is capable of generating multiple concentration-response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.

  14. Thromboxane Formation Assay to Identify High On-Treatment Platelet Reactivity to Aspirin.

    Science.gov (United States)

    Mohring, Annemarie; Piayda, Kerstin; Dannenberg, Lisa; Zako, Saif; Schneider, Theresa; Bartkowski, Kirsten; Levkau, Bodo; Zeus, Tobias; Kelm, Malte; Hohlfeld, Thomas; Polzin, Amin

    2017-01-01

    Platelet inhibition by aspirin is indispensable in the secondary prevention of cardiovascular events. Nevertheless, impaired aspirin antiplatelet effects (high on-treatment platelet reactivity [HTPR]) are frequent. This is associated with an enhanced risk of cardiovascular events. The current gold standard to evaluate platelet hyper-reactivity despite aspirin intake is the light-transmittance aggregometry (LTA). However, pharmacologically, the most specific test is the measurement of arachidonic acid (AA)-induced thromboxane (TX) B2 formation. Currently, the optimal cut-off to define HTPR to aspirin by inhibition of TX formation is not known. Therefore, in this pilot study, we aimed to calculate a TX formation cut-off value to detect HTPR defined by the current gold standard LTA. We measured platelet function in 2,507 samples. AA-induced TX formation by ELISA and AA-induced LTA were used to measure aspirin antiplatelet effects. TX formation correlated nonlinearly with the maximum of aggregation in the AA-induced LTA (Spearman's rho R = 0.7396; 95% CI 0.7208-0.7573, p < 0.0001). Receiver operating characteristic analysis and Youden's J statistics revealed 209.8 ng/mL as the optimal cut-off value to detect HTPR to aspirin with the TX ELISA (area under the curve: 0.92, p < 0.0001, sensitivity of 82.7%, specificity of 90.3%). In summary, TX formation ELISA is reliable in detecting HTPR to aspirin. The calculated cut-off level needs to be tested in trials with clinical end points. © 2017 S. Karger AG, Basel.

  15. Triple Bottom Line Thinking for High Arsenic Bearing Copper-Gold Project in Northern Peru: Assessing the Viability of an Integrated Mine, Mill, and Hydrometallurgical Refinery

    OpenAIRE

    Jonathan Robert Mean

    2011-01-01

    Growth in global urbanization, industrialization and infrastructure expansions will require the development of non-traditional copper resources such as those containing high arsenic to meet the demand for future mine supply. Arsenic levels are rising in current and future mines in resource rich countries such as Peru. Conventional smelting operations cannot accept concentrates high in arsenic (>0.5%) due to process limitations and tightening environmental restrictions. Alternative refining...

  16. Economic viability of ultra high-performance fiber reinforced concrete in prestressed concrete wind towers to support a 5 MW turbine

    OpenAIRE

    GAMA, P. V. C. N.; BITTENCOURT, T. N.

    2017-01-01

    Abstract The Ultra-High Performance Fiber-Reinforced Concrete is a material with remarkable mechanical properties and durability when compared to conventional and high performance concrete, which allows its use even without the reinforcement. This paper proposes the design of prestressed towers for a 5 MW turbine, through regulatory provisions and the limit states method, with UHPFRC and the concrete class C50, comparing the differences obtained in the design by parametric analysis, giving th...

  17. A Fluorescence-Based High-Throughput Assay for the Identification of Anticancer Reagents Targeting Fructose-1,6-Bisphosphate Aldolase.

    Science.gov (United States)

    Cho, Eun Jeong; Devkota, Ashwini K; Stancu, Gabriel; Edupunganti, Ramakrishna; Powis, Garth; Dalby, Kevin N

    2017-08-01

    A high rate of glycolysis, which supplies energy and materials for anabolism, is observed in a wide range of tumor cells, making it a potential pathway to control cancer growth. ALDOA is a multifunctional enzyme in the glycolytic pathway and also promotes HIF-1α, which is of importance in hypoxic solid tumors. The current method for assaying ALDOA activity involves monitoring the consumption of NADH in vitro using absorbance or intrinsic fluorescence via a coupled enzymatic reaction. Here, we report the development of a homogeneous biochemical assay that can overcome limitations of current methods, in particular for the application of high-throughput drug screening. The assay utilizes the commercially available Elite NADH Assay Kit, which incorporates an enzymatic reaction to measure the level of NADH using a fluorescent probe. Assay optimization and validation are discussed. Its feasibility for high-throughput screening (HTS) was demonstrated by screening 65,000 compounds for the identification of small molecules that inhibit ALDOA. Through a validation screen and dose-response evaluation, four inhibitors with IC50 below 10 µM were identified. In conclusion, we demonstrate that a traditional ALDOA assay can be transformed readily into a fluorescence-based assay utilizing a commercial NADH detection kit that is rapid, sensitive, inexpensive, and HTS friendly.

  18. Simultaneous high-performance liquid chromatography assay of acetylsalicylic acid and salicylic acid in film-coated aspirin tablets.

    Science.gov (United States)

    Fogel, J; Epstein, P; Chen, P

    1984-12-28

    A reversed-phase high-performance liquid chromatography (HPLC) method has been developed for the simultaneous assay of acetylsalicylic acid (I) and salicylic acid (II) in film-coated aspirin tablets. As little as 0.1% II (relative to I) can be quantitatively determined. Using a 5-microns octadecylsilane column with water-acetonitrile-phosphoric acid (76:24:0.5) as the mobile phase enabled the chromatographic separation to be completed in 4 min. Due to the slow rate of decomposition of I to II in the extraction solvent, acetonitrile-methanol-phosphoric acid (92:8:0.5), the analysis of many samples was routinely performed by means of automated HPLC equipment. Other compounds (non-aspirin salicylates, caffeine and acetaminophen) were also separated by the chromatographic system.

  19. 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.

  20. Effect of Allium sativum (garlic) methanol extract on viability and ...

    African Journals Online (AJOL)

    Effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cell lines. ... bromide (MTT) assay at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ug/mL of Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of cell ...

  1. Pleomorphism and Viability of the Lyme Disease Pathogen Borrelia burgdorferi Exposed to Physiological Stress Conditions: A Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy Study.

    Science.gov (United States)

    Vancová, Marie; Rudenko, Nataliia; Vaněček, Jiří; Golovchenko, Maryna; Strnad, Martin; Rego, Ryan O M; Tichá, Lucie; Grubhoffer, Libor; Nebesářová, Jana

    2017-01-01

    To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

  2. Enzyme assays

    OpenAIRE

    Bisswanger, Hans

    2014-01-01

    The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Neverthele...

  3. Evaluation of high pressure processing, freezing, and fermentation/drying on viability of Trichinella spiralis larvae in raw pork and in Genoa salami

    Science.gov (United States)

    We evaluated high pressure processing (HPP), freezing, and fermentation/drying to inactivate Trichinella spiralis larvae in both infected pig muscle and in Genoa salami produced with trichinae infected pork. In part A, in each of two trials 10 gram portions (2 replicates per treatment) of fresh pig ...

  4. Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

    Science.gov (United States)

    Toots, Mart; Ustav, Mart; Männik, Andres; Mumm, Karl; Tämm, Kaido; Tamm, Tarmo; Ustav, Mart

    2017-01-01

    Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers. PMID:28182794

  5. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2018-01-01

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  6. A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

    Science.gov (United States)

    Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

    2016-01-01

    ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

  7. Viability of sublethally injured coliform bacteria on fresh-cut cabbage stored in high CO2atmospheres following rinsing with electrolyzed water.

    Science.gov (United States)

    Izumi, Hidemi; Inoue, Ayano

    2018-02-02

    The extent of sublethally injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, was evaluated during storage in air and high CO 2 controlled atmospheres (5%, 10%, and 15%) at 5°C and 10°C using the thin agar layer (TAL) method. Sublethally injured coliform bacteria on nonrinsed shredded cabbage were either absent or they were injured at a 64-65% level when present. Rinsing of shredded cabbage with electrolyzed water containing 25ppm available chlorine reduced the coliform counts by 0.4 to 1.1 log and caused sublethal injury ranging from 42 to 77%. Pantoea ananatis was one of the species injured by chlorine stress. When shredded cabbage, nonrinsed or rinsed with electrolyzed water, was stored in air and high CO 2 atmospheres at 5°C for 7days and 10°C for 5days, coliform counts on TAL plates increased from 3.3-4.5 to 6.5-9.0 log CFU/g during storage, with the increase being greater at 10°C than at 5°C. High CO 2 of 10% and 15% reduced the bacterial growth on shredded cabbage during storage at 5°C. Although injured coliform bacteria were not found on nonrinsed shredded cabbage on the initial day, injured coliforms at a range of 49-84% were detected on samples stored in air and high CO 2 atmospheres at 5°C and 10°C. Injured cells were detected more frequently during storage at both temperatures irrespective of the CO 2 atmosphere when shredded cabbage was rinsed with electrolyzed water. These results indicated that injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, exhibited different degrees of injury during storage regardless of the CO 2 atmosphere and temperature tested. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The reliability of high-risk human papillomavirus detection by Aptima HPV assay in women with ASC-US cytology.

    Science.gov (United States)

    Castle, Philip E; Reid, Jennifer; Dockter, Janel; Getman, Damon

    2015-08-01

    The Aptima HPV assay (AHPV) for high-risk human papillomavirus (hrHPV), and the Aptima HPV 16 18/45 Genotype assay (AHPV GT) for HPV16 and for HPV18 and/or HPV45 (HPV18/45) genotypes are approved for cervical cancer screening by the U.S. Food and Drug Administration. There are limited data on the reliability of these tests for detection of hrHPV, HPV16, and HPV18/45. To assess the reliability of AHPV and AHPV GT on paired specimens from women with atypical squamous cells of undetermined significance (ASC-US) cytology. In a population of women with ASC-US cytology (n=988), cervical specimens were collected at a routine screening baseline visit and at the colposcopy visit that occurred a median of 29 days later. Specimens were tested by AHPV and if positive, by AHPV GT. There was no significant difference in the percent AHPV positive between the colposcopy and baseline specimens (41.9% vs. 43.0%, respectively, p=0.3). The percent agreement, percent positive agreement, and the kappa value were 88.6%, 76.3%, and 0.766, respectively. There were no significant differences between AHPV testing of the colposcopy and baseline specimen in the sensitivity (95.2% vs. 92.9%, respectively, p=1) and specificity (60.5% vs. 59.2%, respectively, p=0.3) for CIN3+. Comparing the hierarchical AHPV and AHPV GT results on the two specimens, the percent exact agreement was 86.2%, the percent positive agreement was 68.4%, and the kappa value was 0.746. AHPV and AHPV GT demonstrated good reliability for hrHPV detection and risk stratification. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  10. Performance and reliability of five commercial enzyme-linked immunosorbent assay kits in screening for anti-human immunodeficiency virus antibody in high-risk subjects.

    OpenAIRE

    Ozanne, G; Fauvel, M

    1988-01-01

    Anti-human immunodeficiency virus enzyme-linked immunosorbent assay kits marketed by Electro-Nucleonics Inc. (ENI), Genetic Systems Corp. (GSC), Organon Teknika Inc. (OTI), Ortho Diagnostic Systems Inc. (ODSI), and Wellcome Diagnostics (WD) were evaluated by using 289 randomly selected serum samples from a high-risk population and 53 serum samples likely to produce false-positive results. The radioimmunoprecipitation assay was used as the reference test. Sensitivities ranged from 96.51% (ODSI...

  11. Peri-operative troponin monitoring using a prototype high-sensitivity cardiac troponin I (hs-cTnI) assay: comparisons with hs-cTnT and contemporary cTnI assays.

    LENUS (Irish Health Repository)

    Lee, Graham R

    2013-09-18

    Non-cardiac surgery is associated with major vascular complications and higher incidences of elevated plasma troponin (cTn) concentration. Goal-directed therapy (GDT) is a stroke volume (SV)-guided approach to intravenous (IV) fluid therapy that improves tissue perfusion, oxygenation and reduces post-operative complications. In patients undergoing major gastro-intestinal surgery, we compared high sensitive and contemporary troponin assays and correlated results with patient outcome.

  12. A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

    Science.gov (United States)

    Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

    2017-10-01

    Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Hugo Alves

    Full Text Available Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

  14. Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors.

    Science.gov (United States)

    Zhu, Mao-Rong; Du, Dao-Hai; Hu, Jun-Chi; Li, Lian-Chun; Liu, Jing-Qiu; Ding, Hong; Kong, Xiang-Qian; Jiang, Hua-Liang; Chen, Kai-Xian; Luo, Cheng

    2017-08-31

    Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.

  15. High-performance liquid chromatographic assay of parabens in wash-off cosmetic products and foods using chemiluminescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Qunlin [Department of Chemistry, University of Science and Technology of China, Jinzai Road 96, Hefei, Anhui 230026 (China); Lian Mei [Department of Chemistry, University of Science and Technology of China, Jinzai Road 96, Hefei, Anhui 230026 (China); Liu Lijuan [Department of Chemistry, University of Science and Technology of China, Jinzai Road 96, Hefei, Anhui 230026 (China); Cui Hua [Department of Chemistry, University of Science and Technology of China, Jinzai Road 96, Hefei, Anhui 230026 (China)]. E-mail: hcui@ustc.edu.cn

    2005-04-29

    A new method for the simultaneous determination of parabens including methylparaben, ethylparaben, propylparaben, and butylparaben by high-performance liquid chromatography (HPLC) coupled with chemiluminescence detection was developed. The procedure was based on the chemiluminescent enhancement by parabens of the cerium(IV)-rhodamine 6G system in the strong sulfuric acid medium. The good separation of parabens was carried out with an isocratic elution using a mixture of methanol and water (60:40, v/v) within 8.5 min. Under the optimized conditions, a linear working range extends three orders of magnitude with the relative standard deviations of intra- and inter-day precision below 4.5%, and the detection limits were 1.9 x 10{sup -9}, 2.7 x 10{sup -9}, 3.9 x 10{sup -9}, and 5.3 x 10{sup -9} g ml{sup -1} for methylparaben, ethylparaben, propylparaben, and butylparaben, respectively. The chemiluminescence reaction was well compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the assay of parabens in wash-off cosmetic products and foods with the minimal sample preparation.

  16. Acute effects of TiO2 nanomaterials on the viability and taxonomic composition of aquatic bacterial communities assessed via high-throughput screening and next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Chu Thi Thanh Binh

    Full Text Available The nanotechnology industry is growing rapidly, leading to concerns about the potential ecological consequences of the release of engineered nanomaterials (ENMs to the environment. One challenge of assessing the ecological risks of ENMs is the incredible diversity of ENMs currently available and the rapid pace at which new ENMs are being developed. High-throughput screening (HTS is a popular approach to assessing ENM cytotoxicity that offers the opportunity to rapidly test in parallel a wide range of ENMs at multiple concentrations. However, current HTS approaches generally test one cell type at a time, which limits their ability to predict responses of complex microbial communities. In this study toxicity screening via a HTS platform was used in combination with next generation sequencing (NGS to assess responses of bacterial communities from two aquatic habitats, Lake Michigan (LM and the Chicago River (CR, to short-term exposure in their native waters to several commercial TiO2 nanomaterials under simulated solar irradiation. Results demonstrate that bacterial communities from LM and CR differed in their sensitivity to nano-TiO2, with the community from CR being more resistant. NGS analysis revealed that the composition of the bacterial communities from LM and CR were significantly altered by exposure to nano-TiO2, including decreases in overall bacterial diversity, decreases in the relative abundance of Actinomycetales, Sphingobacteriales, Limnohabitans, and Flavobacterium, and a significant increase in Limnobacter. These results suggest that the release of nano-TiO2 to the environment has the potential to alter the composition of aquatic bacterial communities, which could have implications for the stability and function of aquatic ecosystems. The novel combination of HTS and NGS described in this study represents a major advance over current methods for assessing ENM ecotoxicity because the relative toxicities of multiple ENMs to thousands

  17. Abundance, viability and culturability of Antarctic bacteria

    Digital Repository Service at National Institute of Oceanography (India)

    LokaBharathi, P.A.; DeSouza, M.J.B.D.; Nair, S.; Chandramohan, D.

    The viability of total number of bacteria decide the mineralisation rate in any ecosystem and ultimately the fertility of the region. This study aims at establishing the extent of viability in the standing stock of the Antarctic bacterial population...

  18. STUDY ON POLLEN VIABILITY AS BIOINDICATOR OF AIR QUALITY

    Directory of Open Access Journals (Sweden)

    Florentina ŞTEFLEA

    2012-01-01

    Full Text Available The aim of this research is to estimate the relationship between pollen viability and atmospheric pollution (in polluted and non-polluted conditions. The study was carried out in the city of Timisoara. Two areas, with different intensity of road traffic (very high and absent but all characterized by the presence of the same plant species, were selected. The pollen of herbaceous spontaneous species, arboreal species and a shrub species was used (Robinia pseudacacia, Aesculus x carnea, Catalpa bignonioides, Albizzia julibrissin, Rosa canina, Sambucus nigra, Malva neglecta, Ranunculus acer, Trifolium repens, Cichorium intybus. The pollen of these species was treated with TTC (2, 3, 5 Tryphenil-Tetrazolium-Chloride staining solution and viability was then estimated by light microscopy. The results of the mean pollen viability percentage of the examined species are reported. Pollen viability of herbaceous plants is significantly different between the two environments.

  19. Comparison of two high-throughput assays for quantification of adenovirus type 5 neutralizing antibodies in a population of donors in China.

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    Full Text Available BACKGROUND: The presence of various levels of Adenovirus serotype 5 neutralizing antibodies (Ad5NAb is thought to contribute to the inconsistent clinical results obtained from vaccination and gene therapy studies. Currently, two platforms based on high-throughput technology are available for Ad5NAb quantification, chemiluminescence- and fluorescence-based assays. The aim of this study was to compare the results of two assays in the seroepidemiology of Ad5NAb in a local population of donors. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based neutralizing antibody detection test (FRNT using recombinant Ad5-EGFP virus and the chemiluminescence-based neutralizing antibody test (CLNT using Ad5-Fluc were developed and standardized for detecting the presence of Ad5NAb in serum samples from the population of donors in Beijing and Anhui provinces, China. First, the overall percentage of people positive for Ad5NAb performed by CLNT was higher than that obtained by FRNT (85.4 vs 69.9%, p<0.001. There was an 84.5% concordance between the two assays for the 206 samples tested (144 positive in both assays and 30 negative in both assays. All 32 discordant sera were CLNT-positive/FRNT-negative and were confirmed positive by western blot. Secondly, for all 144 sera positive by both assays, the two assays showed high correlation (r = 0.94, p<0.001 and close agreement (mean difference: 0.395 log(10, 95% CI: -0.054 log(10 to 0.845 log(10. Finally, it was found by both assays that there was no significant difference observed for titer or prevalence by gender (p = 0.503 vs 0.818, for two assays; however, age range (p = 0.049 vs 0.010 and geographic origin (p = 0.007 vs 0.011 were correlated with Ad5NAb prevalence in northern regions of China. CONCLUSION: The CLNT assay was relatively more simple and had higher sensitivity than the FRNT assay for determining Ad5NAb titers. It is strongly suggested that the CLNT assay be used for future

  20. Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

    Science.gov (United States)

    Cook, James L; Stannard, James P; Stoker, Aaron M; Bozynski, Chantelle C; Kuroki, Keiichi; Cook, Cristi R; Pfeiffer, Ferris M

    2016-05-01

    chondrocytes with infiltration of fibroblasts from the surrounding recipient tissues. In situ polymerase chain reaction (fluorescence in situ hybridization [FISH]) assays were performed in an attempt to distinguish donor (male) cells from recipient (female) cells. Unfortunately, this technique was exceptionally difficult to perform on intact articular cartilage sections, and consistent, repeatable data could not be obtained from this testing. However, the data did support histologic and live-dead data, which strongly suggested that successful grafts retained viable donor (male) chondrocytes and unsuccessful grafts degraded and were replaced by fibrous tissue populated with recipient (female) fibroblasts. Viable chondrocytes in OCAs at the time of transplantation are primarily responsible for maintenance of donor articular cartilage health in the long term. Optimizing chondrocyte viability in all aspects of OCA transplantation-including procurement, processing, storage, transportation, and surgical implantation-needs to be a primary focus for OCA clinical use. © 2016 The Author(s).

  1. Photo- and Bio-physical Studies of Lectin-Conjugated Fluorescent Nanoparticles: Reduced Sensitivity in High Density Assays

    Science.gov (United States)

    Wang, Yaqi; Gildersleeve, Jeffrey C.; Basu, Amit

    2010-01-01

    Lectin conjugated, fluorescent silica nanoparticles (fNP) have been developed for carbohydrate based histopathology evaluations of epithelial tissue biopsies. The fNP platform was selected for its enhanced emissive brightness compared to direct dye labeling. Carbohydrate microarray studies were performed to compare the carbohydrate selectivity of the mannose recognizing lectin Concanavalin A (ConA) before and after conjugation to fluorescent silica nanoparticles (ConA-fNP). These studies revealed surprisingly low emission intensities upon staining with ConA-fNP compared to biotin-ConA / Cy3-strepavidin staining. A series of photophysical and biophysical characterizations of the fNP and ConA-fNP conjugates were performed to probe the low sensitivity from fNP in the microarray assays. Up to 1200 fluorescein (FL) and 80 tetramethylrhodamine (TR) dye molecules were incorporated into 46 nm diameter fNP, yielding emissive brightness’ 400 and 35 times larger than the individual dye molecules, respectively. ConA lectin conjugated to carboxylic acid surface modified nanoparticles covers 15–30% of the fNP surface. The CD spectra and mannose substrate selectivity of ConA conjugated to the fNP differed slightly compared to soluble ConA. Although, the high emissive brightness of fNP enhances detection sensitivity for samples with low analyte densities, large fNP diameters limit fNP recruitment and binding to samples with high analyte densities. The high analyte density and nearly two-dimensional target format of carbohydrate microarrays make probe size a critical parameter. In this application, fNP labels afford minimal sensitivity advantage compared to direct dye labeling. PMID:20496897

  2. Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human corneal endothelial cells.

    Science.gov (United States)

    Wang, Jiong; Stachon, Tanja; Eppig, Timo; Langenbucher, Achim; Seitz, Berthold; Szentmáry, Nóra

    2013-04-01

    Photodynamic inactivation (PDI) may be a potential alternative in case of therapy-resistant infectious keratitis. PDI using the photosensitizer chlorin e6 (Ce6) with high photosensitizing efficacy offers a valuable option, also for keratitis. The purpose of our study was to determine the impact of PDI with the photosensitizer Ce6 on viability, apoptosis, and proliferation of human corneal endothelial cells (HCECs), in vitro. Human corneal endothelial cell line was cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. HCECs cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50-500 nM concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit. Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Following PDI, viability and total DNA content of HCECs decreased significantly above 150 nM Ce6 concentration (P proliferation of endothelial cells decreased significantly (P proliferation, and also triggers apoptosis of HCECs in vitro. PDI using the photosensitizer Ce6 may be a potential treatment alternative in infectious keratitis. However, to avoid endothelial cell damage, the photosensitizer must not penetrate the endothelium.

  3. Validation of a Multiparametric, High-Content-Screening Assay for Predictive/Investigative Cytotoxicity: Evidence from Technology Transfer Studies and Literature Review.

    Science.gov (United States)

    O'Brien, Peter James; Edvardsson, Anna

    2017-03-20

    A multiparametric, live-cell, high-content-screening (HCS) cytotoxicity assay was first demonstrated in 2006 ( Arch. Toxicol. 2006 , 80 , 580 - 604 ) to be highly concordant with human hepatotoxicity, including idiosyncratic hepatotoxicities and other target organ toxicities in contrast to historical assays. The success of the assay was attributed to its simultaneous measurement of multiple appropriate "cytobiomarkers": use of human cells with xenometabolic competence for toxicities mediated by metabolites, 72 h exposure to enable expression of slower-acting toxicants, exposure to a wide-range of concentrations from 30- to 100-fold the efficacious concentration, and normalizing the in vitro cytotoxic concentration to an estimate of the in vivo concentration of exposure. An overwhelming volume of evidence has accumulated over the last 10 years to support this approach as necessary in predictive toxicology. Equivalent assays have now been successfully applied in ∼50 studies across a wide variety of toxicants, toxicities, cell types, and disciplines. Review herein of the wider literature on cytotoxicity since the first assay was reported 100 years ago supports the selection of key cytobiomarkers along a final common pathway of cell injury, including cell proliferation, mitochondrial activity, apoptosis, lysosomal mass, oxidative stress, and cell membrane permeability. HCS studies without inclusion of such key cytobiomarkers or without testing to sufficiently high concentration have not been as successful. Furthermore, a subset of the original toxicants has been reanalyzed herein using the original HCS assay and has confirmed their high sensitivities and specificities across locations, HCS technologies, staff, laboratories, and time. A protocol is demonstrated for operational validation of the assay within laboratories to demonstrate proficiency and quality management.

  4. Tychastic measure of viability risk

    CERN Document Server

    Aubin, Jean-Pierre; Dordan, Olivier

    2014-01-01

    This book presents a forecasting mechanism of the price intervals for deriving the SCR (solvency capital requirement) eradicating the risk during the exercise period on one hand, and measuring the risk by computing the hedging exit time function associating with smaller investments the date until which the value of the portfolio hedges the liabilities on the other. This information, summarized under the term “tychastic viability measure of risk” is an evolutionary alternative to statistical measures, when dealing with evolutions under uncertainty. The book is written by experts in the field and the target audience primarily comprises research experts and practitioners.

  5. Separation and Quantification of Eight Antidiabetic Drugs on A High-Performance Liquid Chromatography: Its Application to Human Plasma Assay

    Science.gov (United States)

    Lakshmi, Karunanidhi S.; Rajesh, Tirumala

    2011-01-01

    An analytical method based on isocratic reverse phase high-performance liquid chromatography was developed and validated for the separation and quantification of eight antidiabetic drugs: rosiglitazone, pioglitazone, glipizide, gliclazide, repaglinide, nateglinide, glibenclamide, and glimepiride for their application in human plasma assay. Metformin is used as internal standard. Analysis was done on Onyx monolithic C18 column (100 × 4.6 mm, i.d., 5 μm) using a mixture of 0.05% formic acid in water and methanol in the ratio of 42 : 58 (v/v) fixed at a flow rate of 0.5 mL/min, and they were monitored at 234 nm. Separation was achieved in less than 20 min. The calibration curves were linear in the range of 50–2000 ng/mL. The method was validated for its recovery, intra- and interday precision, stability, specificity, and selectivity. Plasma samples were prepared using solid-phase extraction of analytes. Hence, the developed method was found to be suitable for the routine analysis of selected antidiabetic drugs in biological matrices. PMID:22389851

  6. High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

    Science.gov (United States)

    Waldron, Anna M.; Galea, Francesca; Whittington, Ann-Michele; Saunders, Vanessa F.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

    2014-01-01

    Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and

  7. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

    Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses

  8. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Science.gov (United States)

    Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

    2017-01-01

    Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

  9. Viability of pollen grains of tetraploid banana

    Directory of Open Access Journals (Sweden)

    Taliane Leila Soares

    2016-01-01

    Full Text Available ABSTRACT Obtaining banana tetraploid cultivars from triploid strains results in total or partial reestablishment of fertility, allowing the occurrence of some fruits with seeds, a feature that is undesirable from a marketing perspective. The objective of this study was to assess the viability of pollen of 12 banana tetraploid hybrids (AAAB by means of in vitro germination and two histochemical tests (acetocarmine and 2,3,5-triphenyltetrazolium chloride. The pollen tube growth was evaluated by germinating grains in three culture media — M1: 0.03% Ca(NO3∙4H2O, 0.02% Mg(SO4∙7H2O, 0.01% KNO3, 0.01% H3BO3 and 15% sucrose; M2: 0.03% Ca(NO3∙4H2O, 0.01% KNO3, 0.01% H3BO3 and 10% sucrose; and M3: 0.015% H3BO3, 0.045% Ca3(PO42 and 25% sucrose. The acetocarmine staining indicated high viability (above 80%, except for the genotypes YB42-17 and Caprichosa, which were 76 and 70%, respectively. However, the in vitro germination rate was lower than 50% for all the genotypes, except for the hybrids YB42-17 (M1 and YB42-47 (M1. The medium M1 provided the greatest germination percentage and pollen tube growth. Among the genotypes assessed, YB42-47 presented the highest germination rate (61.5% and tube length (5.0 mm. On the other hand, the Vitória cultivar had the lowest germination percentage (8.2% in medium M1. Studies of meiosis can shed more light on the differences observed in the evaluated tetraploids, since meiotic irregularities can affect pollen viability.

  10. A high-throughput assay for evaluating state dependence and subtype selectivity of Cav2 calcium channel inhibitors.

    Science.gov (United States)

    Dai, Ge; Haedo, Rodolfo J; Warren, Vivien A; Ratliff, Kevin S; Bugianesi, Randal M; Rush, Alison; Williams, Mark E; Herrington, James; Smith, McHardy M; McManus, Owen B; Swensen, Andrew M

    2008-04-01

    Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.

  11. A fit-for-purpose approach to analytical sensitivity applied to a cardiac troponin assay: time to escape the 'highly-sensitive' trap.

    Science.gov (United States)

    Ungerer, Jacobus P J; Pretorius, Carel J

    2014-04-01

    Highly-sensitive cardiac troponin (cTn) assays are being introduced into the market. In this study we argue that the classification of cTn assays into sensitive and highly-sensitive is flawed and recommend a more appropriate way to characterize analytical sensitivity of cTn assays. The raw data of 2252 cardiac troponin I (cTnI) tests done in duplicate with a 'sensitive' assay was extracted and used to calculate the cTnI levels in all, including those below the 'limit of detection' (LoD) that were censored. Duplicate results were used to determine analytical imprecision. We show that cTnI can be quantified in all samples including those with levels below the LoD and that the actual margins of error decrease as concentrations approach zero. The dichotomous classification of cTn assays into sensitive and highly-sensitive is theoretically flawed and characterizing analytical sensitivity as a continuous variable based on imprecision at 0 and the 99th percentile cut-off would be more appropriate.

  12. A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

    Science.gov (United States)

    Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun

    2018-03-01

    The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA.

    Science.gov (United States)

    Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Dai, Mingyan; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-01-01

    Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer. Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer. The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests. Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.

  14. Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

    OpenAIRE

    Chitarra, L.G.

    2001-01-01

    Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on serological techniques. Plating methods provide information about cell viability, but are generally laborious and time-consuming. Serological techniques, such as immunofluorescence microscopy (IF) and enzym...

  15. Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO2 laser (Tru-Pulse) in pig skin.

    Science.gov (United States)

    Smith, K J; Skelton, H G; Graham, J S; Hamilton, T A; Hackley, B E; Hurst, C G

    1997-08-01

    CO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage. This study was performed to evaluate thermal damage produced by a CO2 laser. Routine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser. At a pulse energy of 300 mJ, with a pulse duration of 60 microseconds, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis. Hyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis.

  16. Moringa oleifera's Nutritious Aqueous Leaf Extract Has Anticancerous Effects by Compromising Mitochondrial Viability in an ROS-Dependent Manner.

    Science.gov (United States)

    Madi, Niveen; Dany, Mohammed; Abdoun, Salah; Usta, Julnar

    2016-01-01

    Moringa oleifera (MO) is an important dietary component for many populations in West Africa and the Indian subcontinent. In addition to its highly nutritious value, almost all parts of this plant have been widely used in folk medicine in curing infectious, cardiovascular, gastrointestinal, hepatic, and other diseases. Evidence-based research supported its versatile medicinal properties; however, more rigorous research is required to establish it in cancer therapy. As such, in this study we aim to investigate the in vitro anticancerous effect of Moringa oleifera's aqueous leaf extract. Moringa extract was prepared by soaking pulverized leaves in hot water mimicking the people's mode of the leaf drink preparation. Several assays were used to study the effect of different percentage concentrations of the extract on viability of A549 cells; levels of adenosine triphosphate (ATP), reactive oxygen species (ROS), and glutathione (GSH) generated; as well as percentage of lactate dehydrogenase (LDH) released at different time points. In addition to mitochondrial membrane potential, apoptotic events were assessed using western blotting for apoptotic markers and immunoflourescent flourescent labeled inhibitor of caspases (FLICA) assay. MO extract treatment resulted in a significant decrease in mitochondrial membrane potential (1 hour) and ATP levels (3 hours), followed by an increase in (6 hours) ROS, caspase activation, proapoptotic proteins expression (p53, SMAC/Diablo, AIF), and PARP-1 cleavage. This eventually resulted in decreased GSH levels and a decrease in viability. The cytotoxic effect was prevented upon pretreatment with antioxidant N-acetyl-cysteine. MO decreased as well the viability of HepG2, CaCo2, Jurkat, and HEK293 cells. Our findings identify a plant extract with an anticancerous effect on cancer cell lines. MO extract exerts its cytotoxic effect in A549 cancer cells by affecting mitochondrial viability and inducing apoptosis in an ROS-dependent manner.

  17. Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Directory of Open Access Journals (Sweden)

    Ball Amanda

    2007-08-01

    Full Text Available Abstract Background Knockdown resistance (kdr is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1 TaqMan probes and 2 high resolution melt (HRM analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR, Heated Oligonucleotide Ligation Assay (HOLA, Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost, and safety (requirement for hazardous chemicals. Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions and the most specific (with the lowest number of incorrect scores. Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS

  18. Cell motility, morphology, viability and proliferation in response to nanotopography on silicon black

    DEFF Research Database (Denmark)

    Lopacinska, Joanna M.; Gradinaru, Cristian; Wierzbicki, Rafal

    2012-01-01

    standard measurements of cell viability, proliferation, and morphology on various surfaces. We also analyzed the motility of cells on the same surfaces, as recorded in time lapse movies of sparsely populated cell cultures. We find that motility and morphology vary strongly with nano-patterns, while...... viability and proliferation show little dependence on substrate type. We conclude that motility analysis can show a wide range of cell responses e. g. over a factor of two in cell speed to different nano-topographies, where standard assays, such as viability or proliferation, in the tested cases show much...

  19. Development of a high-throughput cell-based reporter assay for screening of JAK3 inhibitors.

    Science.gov (United States)

    Yin, Chang-Hong; Bach, Erika A; Baeg, Gyeong-Hun

    2011-04-01

    JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rβ/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.

  20. Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.

    Science.gov (United States)

    Jensen, Nickels A; Gerth, Klaus; Grotjohann, Tim; Kapp, Dieter; Keck, Matthias; Niehaus, Karsten

    2009-03-10

    High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts.

  1. Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients

    Directory of Open Access Journals (Sweden)

    Elena L. Enache

    2015-09-01

    Full Text Available Chronic hepatitis C (CHC is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3 gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724–1 accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism. This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

  2. Evaluation of goat milk as storage media to preserve viability of human periodontal ligament cells in vitro.

    Science.gov (United States)

    Ulusoy, Ayça Tuba; Kalyoncuoglu, Elif; Kaya, Senay; Cehreli, Zafer Cavit

    2016-08-01

    The purpose of this study was to evaluate the effectiveness of goat milk as a storage media for maintenance of periodontal ligament (PDL) cell viability of avulsed teeth and compare it with commonly used and/or investigated storage media. PDL cells were obtained from the root surface of healthy premolars and were cultured in Eagle's maintenance medium (EMM). Cell cultures were treated with the following storage media: tap water (negative control); EMM (positive control); Hank's balanced salt solution; ultra high temperature (UHT) long-shelf-life lactose-free cow milk; UHT long-shelf-life whole cow milk; UHT long-shelf-life skimmed cow milk; UHT long-shelf-life soy milk; UHT long-shelf-life goat milk, UHT long-shelf-life follow on milk with probiotic, 20% propolis, and egg white. Culture plates were incubated with experimental media at 20°C for 1, 3, 6, 12, and 24 h. PDL cell viability was assessed by tetrazolium salt-based colorimetric (MTT) assay at each test period. One-way anova was used to evaluate the effects of storage solutions at each time point, followed by post hoc Duncan's multiple comparison test (P = 0.05). A dendrogram was constructed to show the arrangement of hierarchical clustering. Goat milk displayed the highest capacity to maintain cell viability at all test intervals (P milk with the probiotic showed the lowest time-dependent PDL cell viability among all test media (P milks, HBSS performed significantly less effectively in maintaining PDL cell viability during the entire test period (P milk can be recommended as a suitable storage medium for avulsed teeth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Low Concentration of Sodium Nitroprusside Promotes Mesenchymal Stem Cell Viability and Proliferation Through Elevation of Metabolic Activity

    Directory of Open Access Journals (Sweden)

    A.Mohammadi

    2017-06-01

    Full Text Available Background: Sodium nitroprusside (SNP releases nitric oxide which has signaling role. Objectives: This study was conducted to understand the role of low concentration of SNP on viability, proliferation and biochemical properties of rat bone marrow mesenchymal stem cells (MSCs. Materials and Methods: MSCs were used to evaluate the viability and morphology in presence of SNP (1 to 100 µM at 12, 24 and 36 hours. Then 10, 50 and 100 µM of SNP as well as 24 hours were selected for further study. Cell proliferation was investigated by colony forming assay and population doubling number (PDN. Calcium (Ca2+ potassium (K+ and sodium (Na+ level as well as activity of alanine transaminase (ALT, aspartate transaminase (AST, alkaline phosphatase (ALP and lactate dehydrogenase (LDH were measured. Results: The MSCs viability increased when treatment with 1 and 10 µM at all the treatment periods while 90 and 100 µM caused significant reduction after 24 and 36 hours. Also 10 µM caused elevation whereas 50 and 100 µM showed reduction of proliferation ability. We observed morphological changes and significant reduction of all the investigated enzymes with 100 µM. Activity of ALT and AST were elevated with 10 µM after 24 hours, whereas LDH and ALP activities were not changed. Na+, K+ and Ca2+ was not changed due to 10 and 50 µM treatments, whereas 100 µM only elevated the level of calcium and sodium ions. Conclusions: Low concentration of SNP caused increase of viability and proliferation due to metabolic activity elevation. But the high concentration of SNP induced cell viability and proliferation reduction caused by metabolic and ionic imbalance as well as infrastructure alteration.

  4. Wildlife Tunnel Enhances Population Viability

    Directory of Open Access Journals (Sweden)

    Rodney van der Ree

    2009-12-01

    Full Text Available Roads and traffic are pervasive components of landscapes throughout the world: they cause wildlife mortality, disrupt animal movements, and increase the risk of extinction. Expensive engineering solutions, such as overpasses and tunnels, are increasingly being adopted to mitigate these effects. Although some species readily use such structures, their success in preventing population extinction remains unknown. Here, we use population viability modeling to assess the effectiveness of tunnels for the endangered Mountain Pygmy-possum (Burramys parvus in Australia. The underpasses reduced, but did not completely remove, the negative effects of a road. The expected minimum population size of a "reconnected" population remained 15% lower than that of a comparable "undivided" population. We propose that the extent to which the risk of extinction decreases should be adopted as a measure of effectiveness of mitigation measures and that the use of population modeling become routine in these evaluations.

  5. Malthus, Boserup and population viability.

    Science.gov (United States)

    Bonneuil, N

    1994-01-01

    The Malthus-Boserup explanatory framework is revisited from the point of view of viability theory. Instead of imposing a univocal relationship between population pressure and level of knowledge, the way technology will change is not determined, it is only constrained. This leads to regard any situation as associated to a set of reachable futures. When no possibility is left for systems to avoid extinction, systems are no longer viable. Hence, the control-phase space can be divided into regions corresponding to gradual danger or security. This point of view allows the introduction of ideas such as incentives to create or to use new knowledge, gives a role to the threatening power of Malthusian checks, and leaves space for a specific variety of behaviors. The Boserupian theme then appears indirectly, emerging from the constraints imposed by the inertia of technological change.

  6. Viability of telework at PROCEMPA.

    Science.gov (United States)

    Fetzner, Maria Amelia de Mesquita

    2003-02-01

    At the end of the 20th century, telework appears as one of the modalities of flexible work, which is related to new organizational structures as well as to increasing use of technology. It revolutionizes the traditional ways of performing work. Its implementation creates a number of questions to be answered by the organizations and the individuals involved. This article presents a case study on the viability of implementing telework at Procempa (The Data Processing Company of the City of Porto Alegre). The case study analyzes the technical, organizational, psychological, legal, and labor union dimensions. As a result of this study, we can identify the organization's stage of readiness for telework, the conditions under which it would be implemented, and the specific issues of an implementation.

  7. Understanding the Effects of Roasting on Antioxidant Components of Coffee Brews by Coupling On‐line ABTS Assay to High Performance Size Exclusion Chromatography

    Science.gov (United States)

    Opitz, Sebastian E.W.; Goodman, Bernard A.; Keller, Marco; Smrke, Samo; Wellinger, Marco; Schenker, Stefan

    2016-01-01

    Abstract Introduction Coffee is a widely consumed beverage containing antioxidant active compounds. During roasting the phytochemical composition of the coffee bean changes dramatically and highly polymeric substances are produced. Besides chlorogenic acids that are already present in green coffee beans, melanoidins show antioxidant capacity as well. Objective To employ post‐column derivatisation by coupling high performance size exclusion chromatography (HPSEC) to an antioxidant assay to investigate the effect of roasting on the properties of antioxidant active compounds in coffee brews. Methodology We have investigated the antioxidant capacity of Coffea arabica (Arabica) and C. canephora (Robusta) beans that were roasted over the full spectrum of roast conditions (four roasting speeds to three roast degrees) by comparing the results from HPSEC coupled on‐line to the ABTS assay with those from two batch assays, Folin Ciocalteu (FC) and oxygen radical absorbance capacity (ORAC) assay. Results The antioxidant capacity showed a general decrease towards slower and darker roasted coffee for all three assays, indicative of heat degradation of active compounds. Hence, low molecular weight (LMW) compounds such as chlorogenic acids (CGAs) decreased progressively already from relatively mild roasting conditions. In contrast, high molecular weight (HMW) compounds (e.g. melanoidins) increased from light to dark roast degrees with lowering magnitude towards slower roasting profiles. Conclusion By coupling HPSEC on‐line to the ABTS assay we were able to separately quantify the contribution of HMW and LMW compounds to the total antioxidant capacity, increasing our understanding of the roast process. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. PMID:28008674

  8. Understanding the Effects of Roasting on Antioxidant Components of Coffee Brews by Coupling On-line ABTS Assay to High Performance Size Exclusion Chromatography.

    Science.gov (United States)

    Opitz, Sebastian E W; Goodman, Bernard A; Keller, Marco; Smrke, Samo; Wellinger, Marco; Schenker, Stefan; Yeretzian, Chahan

    2017-03-01

    Coffee is a widely consumed beverage containing antioxidant active compounds. During roasting the phytochemical composition of the coffee bean changes dramatically and highly polymeric substances are produced. Besides chlorogenic acids that are already present in green coffee beans, melanoidins show antioxidant capacity as well. To employ post-column derivatisation by coupling high performance size exclusion chromatography (HPSEC) to an antioxidant assay to investigate the effect of roasting on the properties of antioxidant active compounds in coffee brews. We have investigated the antioxidant capacity of Coffea arabica (Arabica) and C. canephora (Robusta) beans that were roasted over the full spectrum of roast conditions (four roasting speeds to three roast degrees) by comparing the results from HPSEC coupled on-line to the ABTS assay with those from two batch assays, Folin Ciocalteu (FC) and oxygen radical absorbance capacity (ORAC) assay. The antioxidant capacity showed a general decrease towards slower and darker roasted coffee for all three assays, indicative of heat degradation of active compounds. Hence, low molecular weight (LMW) compounds such as chlorogenic acids (CGAs) decreased progressively already from relatively mild roasting conditions. In contrast, high molecular weight (HMW) compounds (e.g. melanoidins) increased from light to dark roast degrees with lowering magnitude towards slower roasting profiles. By coupling HPSEC on-line to the ABTS assay we were able to separately quantify the contribution of HMW and LMW compounds to the total antioxidant capacity, increasing our understanding of the roast process. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd.

  9. Comparison of the analytical and clinical performances of Abbott RealTime High Risk HPV, Hybrid Capture 2, and DNA Chip assays in gynecology patients.

    Science.gov (United States)

    Park, Seungman; Kang, Youjin; Kim, Dong Geun; Kim, Eui-Chong; Park, Sung Sup; Seong, Moon-Woo

    2013-08-01

    The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots.

    NARCIS (Netherlands)

    Meesters, R.J.; Kampen, J.J. van; Reedijk, M.L.; Scheuer, R.D.; Dekker, L.J.; Burger, D.M.; Hartwig, N.G.; Osterhaus, A.D.; Luider, T.M.; Gruters, R.A.

    2010-01-01

    Kaletra (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for

  11. Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

    NARCIS (Netherlands)

    R.J.W. Meesters (Roland); J.J.A. van Kampen (Jeroen); M.L. Reedijk (Mariska); R.D. Scheuer (Rachel); L.J.M. Dekker (Lennard); D.M. Burger (David); N.G. Hartwig (Nico); A.D.M.E. Osterhaus (Albert); T.M. Luider (Theo); R.A. Gruters (Rob)

    2010-01-01

    textabstractKaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used

  12. Assessment of constituents in Allium by multivariate data analysis, high-resolution α-glucosidase inhibition assay and HPLC-SPE-NMR

    DEFF Research Database (Denmark)

    Schmidt, Jeppe Secher; Nyberg, Nils; Stærk, Dan

    2014-01-01

    Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010–2012 were investigated with multivariate data analysis, high-resolution α-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management...

  13. A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins.

    Science.gov (United States)

    Stefanova, Miglena; Bobba, Sudheer; Gutheil, William G

    2010-01-01

    High-molecular-mass penicillin-binding proteins (HMM PBPs) are essential for bacterial cell wall biosynthesis and are the lethal targets of beta-lactam antibiotics. When purified, HMM PBPs give undetectable or weak enzyme activity. This has impeded efforts to develop assays for HMM PBPs and to develop new inhibitors for HMM PBPs as HMM PBP targeted antibacterial agents. However, even when purified, HMM PBPs retain their ability to bind beta-lactams. Here we describe a fluorescently detected microtiter plate-based assay for inhibitor binding to HMM PBPs based on competition with biotin-ampicillin conjugate (BIO-AMP) binding.

  14. A Microtiter Plate-Based β-Lactam Binding Assay for Inhibitors of the High Molecular Mass Penicillin-Binding Proteins†

    Science.gov (United States)

    Stefanova, Miglena; Bobba, Sudheer; Gutheil, William G.

    2009-01-01

    The high molecular mass (HMM) PBPs are essential for bacterial cell wall biosynthesis, and are the lethal targets of the β–lactam antibiotics. When purified, the HMM PBPs give undetectable or weak enzyme activity. This has impeded efforts to develop assays for the HMM PBPs, and to develop new inhibitors for the HMM PBPs as HMM PBP targeted antibacterial agents. However, even when purified the HMM PBPs retain their ability to bind β-lactams. We describe here a fluorescently detected microtiter plate-based assay for inhibitor binding to the HMM PBPs based on competition with biotin-ampicillin (BIO-AMP) conjugate binding. PMID:19748471

  15. Viability Study for an Unattended UF6 Cylinder Verification Station: Phase I Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Leon E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Miller, Karen A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Garner, James R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Branney, Sean [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); McDonald, Benjamin S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Webster, Jennifer B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Zalavadia, Mital A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Todd, Lindsay C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kulisek, Jonathan A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Nordquist, Heather [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Deshmukh, Nikhil S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Stewart, Scott [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2016-05-31

    In recent years, the International Atomic Energy Agency (IAEA) has pursued innovative techniques and an integrated suite of safeguards measures to address the verification challenges posed by the front end of the nuclear fuel cycle. Among the unattended instruments currently being explored by the IAEA is an Unattended Cylinder Verification Station (UCVS) that could provide automated, independent verification of the declared relative enrichment, 235U mass, total uranium mass and identification for all declared UF6 cylinders in a facility (e.g., uranium enrichment plants and fuel fabrication plants). Under the auspices of the United States and European Commission Support Programs to the IAEA, a project was undertaken to assess the technical and practical viability of the UCVS concept. The US Support Program team consisted of Pacific Northwest National Laboratory (PNNL, lead), Los Alamos National Laboratory (LANL), Oak Ridge National Laboratory (ORNL) and Savanah River National Laboratory (SRNL). At the core of the viability study is a long-term field trial of a prototype UCVS system at a Westinghouse fuel fabrication facility. A key outcome of the study is a quantitative performance evaluation of two nondestructive assay (NDA) methods being considered for inclusion in a UCVS: Hybrid Enrichment Verification Array (HEVA), and Passive Neutron Enrichment Meter (PNEM). This report provides context for the UCVS concept and the field trial: potential UCVS implementation concepts at an enrichment facility; an overview of UCVS prototype design; field trial objectives and activities. Field trial results and interpretation are presented, with a focus on the performance of PNEM and HEVA for the assay of over 200 “typical” Type 30B cylinders, and the viability of an “NDA Fingerprint” concept as a high-fidelity means to periodically verify that the contents of a given cylinder are consistent with previous scans. A modeling study, combined with field

  16. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

    Science.gov (United States)

    Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

    2014-12-11

    The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

  17. A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

    Science.gov (United States)

    Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

    2012-03-01

    A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

  18. A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

    Science.gov (United States)

    Gagnon, David; Archambault, Jacques

    2015-01-01

    A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

  19. A Rapid and High-Throughput Screening Approach for Methicillin-Resistant Staphylococcus aureus Based on the Combination of Two Different Real-Time PCR Assays

    Science.gov (United States)

    van Maarseveen, Noortje M.; van Hannen, Erik J.; van Zwet, Anton A.; Mascini, Ellen M.

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that has been responsible for major nosocomial epidemics worldwide. For infection control programs, rapid and adequate detection of MRSA is of great importance. We developed a rapid and high-throughput molecular screening approach that consists of an overnight selective broth enrichment, followed by mecA, mecC, and S. aureus-specific (SA442 gene) real-time PCR assays, with subsequent confirmation using a staphylococcal cassette chromosome mec element (SCCmec)-orfX-based real-time PCR assay (GeneOhm MRSA assay) and culture. Here, the results of the screening approach over a 2-year period are presented. During this period, a total of 13,387 samples were analyzed for the presence of MRSA, 2.6% of which were reported as MRSA positive. No MRSA isolates carrying the mecC gene were detected during this study. Based on the results of the real-time PCR assays only, 95.2% of the samples could be reported as negative within 24 h. Furthermore, the performance of these real-time PCR assays was evaluated using a set of 104 assorted MRSA isolates, which demonstrated high sensitivity for both the combination of mecA and mecC with SA442 and the BD GeneOhm MRSA assay (98.1% and 97.1%, respectively). This molecular screening approach proved to be an accurate method for obtaining reliable negative results within 24 h after arrival at the laboratory and contributes to improvement of infection control programs, especially in areas with a low MRSA prevalence. PMID:24871220

  20. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc- Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

    Directory of Open Access Journals (Sweden)

    Ajit G Thomas

    Full Text Available The cystine-glutamate antiporter (system xc- is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the

  1. Relationship between humidity and influenza A viability in droplets and implications for influenza's seasonality.

    Directory of Open Access Journals (Sweden)

    Wan Yang

    Full Text Available Humidity has been associated with influenza's seasonality, but the mechanisms underlying the relationship remain unclear. There is no consistent explanation for influenza's transmission patterns that applies to both temperate and tropical regions. This study aimed to determine the relationship between ambient humidity and viability of the influenza A virus (IAV during transmission between hosts and to explain the mechanisms underlying it. We measured the viability of IAV in droplets consisting of various model media, chosen to isolate effects of salts and proteins found in respiratory fluid, and in human mucus, at relative humidities (RH ranging from 17% to 100%. In all media and mucus, viability was highest when RH was either close to 100% or below ∼50%. When RH decreased from 84% to 50%, the relationship between viability and RH depended on droplet composition: viability decreased in saline solutions, did not change significantly in solutions supplemented with proteins, and increased dramatically in mucus. Additionally, viral decay increased linearly with salt concentration in saline solutions but not when they were supplemented with proteins. There appear to be three regimes of IAV viability in droplets, defined by humidity: physiological conditions (∼100% RH with high viability, concentrated conditions (50% to near 100% RH with lower viability depending on the composition of media, and dry conditions (<50% RH with high viability. This paradigm could help resolve conflicting findings in the literature on the relationship between IAV viability in aerosols and humidity, and results in human mucus could help explain influenza's seasonality in different regions.

  2. Economic viability of anaerobic digestion

    Energy Technology Data Exchange (ETDEWEB)

    Wellinger, A. [INFOENERGIE, Ettenhausen (Switzerland)

    1996-01-01

    The industrial application of anaerobic digestion is a relatively new, yet proven waste treatment technology. Anaerobic digestion reduces and upgrades organic waste, and is a good way to control air pollution as it reduces methane and nitrous gas emissions. For environmental and energy considerations, anaerobic digestion is a nearly perfect waste treatment process. However, its economic viability is still in question. A number of parameters - type of waste (solid or liquid), digester system, facility size, product quality and end use, environmental requirements, cost of alternative treatments (including labor), and interest rates - define the investment and operating costs of an anaerobic digestion facility. Therefore, identical facilities that treat the same amount and type of waste may, depending on location, legislation, and end product characteristics, reveal radically different costs. A good approach for evaluating the economics of anaerobic digestion is to compare it to treatment techniques such as aeration or conventional sewage treatment (for industrial wastewater), or composting and incineration (for solid organic waste). For example, the cost (per ton of waste) of in-vessel composting with biofilters is somewhat higher than that of anaerobic digestion, but the investment costs 1 1/2 to 2 times more than either composting or anaerobic digestion. Two distinct advantages of anaerobic digestion are: (1) it requires less land than either composting or incinerating, which translates into lower costs and milder environmental and community impacts (especially in densely populated areas); and (2) it produces net energy, which can be used to operate the facility or sold to nearby industries.

  3. Evaluation of the Xpert MTB/RIF assay at a tertiary care referral hospital in a setting where tuberculosis and HIV infection are highly endemic.

    Science.gov (United States)

    O'Grady, Justin; Bates, Matthew; Chilukutu, Lophina; Mzyece, Judith; Cheelo, Busiku; Chilufya, Moses; Mukonda, Lukundo; Mumba, Maxwell; Tembo, John; Chomba, Mumba; Kapata, Nathan; Maeurer, Markus; Rachow, Andrea; Clowes, Petra; Hoelscher, Michael; Mwaba, Peter; Zumla, Alimuddin

    2012-11-01

    BACKGROUND. There were 1.45 million deaths from tuberculosis in 2011. A substantial proportion of active pulmonary tuberculosis cases in countries where tuberculosis, human immunodeficiency virus (HIV) infection, and AIDS are highly endemic remain undiagnosed because of the reliance on sputum-smear microscopy. This study evaluated the performance of the Xpert MTB/RIF assay at a tertiary care referral center in Zambia, a country where the burden of tuberculosis and HIV infection is high. METHODS. A total of 881 adult inpatients admitted to University Teaching Hospital in Lusaka who were able to produce sputum were enrolled and analyzed in the study, irrespective of admission diagnosis. Sputum specimens were analyzed by fluorescence smear microscopy, the Xpert MTB/RIF assay, mycobacterial growth indicator tube (MGIT) culture,and MGIT drug-susceptibility testing. The sensitivity and specificity of the Xpert MTB/RIF assay were evaluated using culture as the gold standard. RESULTS. Culture-confirmed tuberculosis was found in 201 of 881 patients (22.8%). The specificity of the Xpert MTB/RIF assay was 95.0% (95% confidence interval [CI], 92.4%–96.8%),and the sensitivity was 86.1% (95% CI, 80.3%–90.4%). In sputum smear–negative, culture-positive cases, the assay was 74.7% sensitive (95% CI, 64.6%–82.8%), identifying 71 additional tuberculosis cases that were not detected by smear microscopy.A total of 18 of 111 patients with tuberculosis who were tested (16.2%) had multidrug-resistant (MDR) tuberculosis.The sensitivity and specificity of the Xpert MTB/RIF assay for detecting culture-confirmed, rifampicin-resistant tuberculosis was 81.3% (95% CI, 53.7%–95.0%) and 97.5% (95% CI,90.4%–99.6%), respectively. CONCLUSIONS. The Xpert MTB/RIF assay performs better than smear microscopy in an inpatient setting in a country where tuberculosis and HIV infection are highly endemic. Assessment of its usefulness and cost-effectiveness for increased detection of tuberculosis

  4. Quantitative RT-PCR assay for high-throughput screening (HTS of drugs against the growth of Cryptosporidium parvum in vitro

    Directory of Open Access Journals (Sweden)

    Haili eZhang

    2015-09-01

    Full Text Available Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent. Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively. This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

  5. Evaluation of Fermentation, Drying, and/or High Pressure Processing on Viability of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in Raw Pork and Genoa Salami

    Science.gov (United States)

    In the present study, we evaluated the effect of fermentation/drying on viability of T. spiralis in Genoa salami. We also evaluated HPP for efficacy towards T. spiralis larvae in trichinae-infected pig masseter muscle as an alternate to curing for trichinae control. We also validated the integrated ...

  6. TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

    Directory of Open Access Journals (Sweden)

    C.A. Dalla Torre

    2002-01-01

    Full Text Available Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

  7. DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

    OpenAIRE

    Xian, J; Harrington, M G; Davidson, E H

    1996-01-01

    A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method ...

  8. 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

  9. Optimized automated data analysis for the cytokinesis‐block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry

    Science.gov (United States)

    Rodrigues, M. A.; Probst, C. E.; Beaton‐Green, L. A.

    2016-01-01

    Abstract The cytokinesis‐block micronucleus (CBMN) assay is a well‐established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope‐based methods and most recently has been modified for application on the ImageStreamX (ISX) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide‐based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS® data analysis software for the ISX that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the ISX‐based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large‐scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC PMID:27272602

  10. A Method for Quantitative Determination of Biofilm Viability

    Directory of Open Access Journals (Sweden)

    Maria Strømme

    2012-06-01

    Full Text Available In this study we present a scheme for quantitative determination of biofilm viability offering significant improvement over existing methods with metabolic assays. Existing metabolic assays for quantifying viable bacteria in biofilms usually utilize calibration curves derived from planktonic bacteria, which can introduce large errors due to significant differences in the metabolic and/or growth rates of biofilm bacteria in the assay media compared to their planktonic counterparts. In the presented method we derive the specific growth rate of Streptococcus mutans bacteria biofilm from a series of metabolic assays using the pH indicator phenol red, and show that this information could be used to more accurately quantify the relative number of viable bacteria in a biofilm. We found that the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h−1, compared to 1.09 h−1 in planktonic growth. This method should be applicable to other bacteria types, as well as other metabolic assays, and, for example, to quantify the effect of antibacterial treatments or the performance of bactericidal implant surfaces.

  11. A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

    Directory of Open Access Journals (Sweden)

    Brandon J. Biesiadecki

    2011-01-01

    Full Text Available To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

  12. Multimodality imaging in the assessment of myocardial viability

    Science.gov (United States)

    Partington, Sara L.; Kwong, Raymond Y.

    2014-01-01

    The prevalence of heart failure due to coronary artery disease continues to increase, and it portends a worse prognosis than non-ischemic cardiomyopathy. Revascularization improves prognosis in these high-risk patients who have evidence of viability; therefore, optimal assessment of myocardial viability remains essential. Multiple imaging modalities exist for differentiating viable myocardium from scar in territories with contractile dysfunction. Given the multiple modalities available, choosing the best modality for a specific patient can be a daunting task. In this review, the physiology of myocardial hibernation and stunning will be reviewed. All the current methods available for assessing viability including echocardiography, cardiac magnetic resonance imaging, nuclear imaging with single photon emission tomography and positron emission tomography imaging and cardiac computed tomography will be reviewed. The effectiveness of the various techniques will be compared, and the limitations of the current literature will be discussed. PMID:21069458

  13. Does the balanced scorecard support organizational viability?

    NARCIS (Netherlands)

    Achterbergh, J.M.I.M.; Beeres, R.J.M.; Vriens, D.J.

    2003-01-01

    In this paper we assess whether the balanced scorecard (BSC) supports the necessary functions for organizational viability. To this purpose, we use the viable system model (VSM) as a means to describe the functions required for organizational viability. Then we use the VSM as a template to assess

  14. Pollen viability and membrane lipid composition

    NARCIS (Netherlands)

    Bilsen, van D.G.J.L.

    1993-01-01

    In this thesis membrane lipid composition is studied in relation to pollen viability during storage. Chapter 1 reviews pollen viability, membranes in the dry state and membrane changes associated with cellular aging. This chapter is followed by a study of age-related changes in phospholipid

  15. IN VITRO ASSESSMENT OF EXPOSURE TO NONYLPHENOL ON VIABILITY OF BOVINE SPERMATOZOA

    Directory of Open Access Journals (Sweden)

    Jana Lukáčová

    2014-02-01

    Full Text Available Nonylphenol (NP is a toxic xenobiotic compound classified as an endocrine disruptor that bioaccumulates in the body and causes endocrine disruption. NP can result in male reproductive dysfunction, altered testicular development and decreased male fertility. The target of this in vitro study was to determine the effect of NP as an endocrine disruptor on the viability of spermatozoa. We examined the dose- and time-dependent effect of nonylphenol (1, 10, 100 and 200 µg/mL dissolved either in 0.1% dimethyl sulfoxide (DMSO and 0.1% ethanol on the viability of bovine spermatozoa after 6 h of in vitro cultivation. The viability of bovine spermatozoa was detected by the MTT cytotoxicity assay. The viability in groups with NP dissolved in 0.1% DMSO was significantly (P 10 µg/mL of NP and was decreased significantly (P<0.001 in all experimental groups with NP dissolved in 0.1% ethanol. After 6 h of culture the MTT assay proved a negative effect of all NP doses on the cell viability. The lowest survival of spermatozoa was determined after the addition of 200 µg/mL of NP. The obtained data indicate that the negative effect of NP on the viability must be seriously considered in the case of exposure to NP in animals and humans.

  16. Banana nectar as a medium for testing pollen viability and ...

    African Journals Online (AJOL)

    Administrator

    2007-05-16

    May 16, 2007 ... A quick and reliable method for evaluating pollen quality is essential in a breeding program, especially in a crop such as banana that is characterized by high male and female sterility. In this study the germination and viability of banana pollen was evaluated in a sucrose solution and diluted banana nectar.

  17. Banana nectar as a medium for testing pollen viability and ...

    African Journals Online (AJOL)

    A quick and reliable method for evaluating pollen quality is essential in a breeding program, especially in a crop such as banana that is characterized by high male and female sterility. In this study the germination and viability of banana pollen was evaluated in a sucrose solution and diluted banana nectar. Twenty banana ...

  18. Measurement of cardiac troponin I in serum with a new high-sensitivity assay in a large multi-ethnic Asian cohort and the impact of gender.

    Science.gov (United States)

    Aw, Tar-Choon; Phua, Soon-Kieng; Tan, Shiow-Pin

    2013-06-25

    As new high-sensitivity (hs) cardiac troponin (cTn) assays are introduced decision limits, based on the 99th percentile upper reference limit (99 percentile URL), for each method must be determined and from a sufficiently large cohort to mitigate against distortionary effects of high-end outliers. There is a paucity of studies with large multi-ethnic cohorts. We determined the 99 percentile URL in 1120 (mean age: 50.4±8.2 y) apparently healthy (no history of diabetes, hypertension, heart, lung, or renal disease) Asians (597 men aged 35-65 y, 523 women aged 40-65 y) in a pre-market hs-cTnI assay (Abbott Diagnostics). Hs-cTnI performance was: limit of blank - 0.6 ng/l, limit of detection (LoD) - 1.5 ng/l; cTnI inter-assay coefficient of variation of 20% and 10% were 1.5 and 6.0 ng/l respectively. Hs-cTnI concentrations (range: 0-49.3 ng/l) were detectable (>assay LoD) in 92.3% of participants, and higher in men and individuals >50 y. All-subject, male, and female 99 percentile URL (90% CI) were 25.6 (19.6-32.6), 32.7 (21.1-47.9) and 17.9 (10.7-26.3) ng/l respectively. This hs-cTnI assay exhibits hs performance besides gender and age differences. The all-subject 99 percentile URL values are similar to those reported from some groups but not in others. Users need to establish their own decision limits. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2011-11-01

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

  20. Performance of a Branch Chain RNA In Situ Hybridization Assay for the Detection of High-risk Human Papillomavirus in Head and Neck Squamous Cell Carcinoma.

    Science.gov (United States)

    Kerr, Darcy A; Arora, Kshitij S; Mahadevan, Krishnan K; Hornick, Jason L; Krane, Jeffrey F; Rivera, Miguel N; Ting, David T; Deshpande, Vikram; Faquin, William C

    2015-12-01

    High-risk human papillomavirus (HR-HPV) is a major etiologic agent in a subset of head and neck squamous cell carcinomas (HNSCCs), and its recognition has prognostic and predictive implications. The availability of a sensitive and specific test to assess HR-HPV status is limited. We evaluate an RNA in situ hybridization (ISH) method using branch chain technology to detect HR-HPV and compare its results with DNA ISH, p16 immunohistochemistry, and polymerase chain reaction (PCR). Tissue sections from 54 patients were stained with a manual RNA ISH assay (ViewRNA), which detects 14 HR-HPV types, an automated DNA ISH assay, and p16 immunohistochemistry. Most cases (83%, n=45) were also tested on an automated platform for 14 HR-HPV types and 1 limited to HPV 16/18. PCR was performed in all cases and was successful in 93% (n=50). The RNA ISH assay produced results in 96% of the cases with strong signals and was easily interpreted. HR-HPV was detected in more cases (63%, n=34) by RNA ISH than by DNA ISH (39%, n=21). Compared with PCR, both ISH platforms were 94% specific. RNA ISH was more sensitive (91%) than DNA ISH (65%), and RNA ISH correlated more strongly with p16 immunostaining. HPV 16 represented 89% of HR-HPV detected. The cocktail HPV 16/18 platform was concordant with the pooled HR-HPV assay in all expected cases. The automated assay demonstrated high concordance (96%) with the manual version, showed decreased background, and should allow for easy implementation into the workflow of the diagnostic pathology laboratory.

  1. Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.

    Directory of Open Access Journals (Sweden)

    Nao Nishida

    Full Text Available The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM and specific (concentration of unexpected amplicons <2 nM amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

  2. A high-throughput absorbance-based assay for methionine produced by methionine aminopeptidase using S-adenosyl-L-methionine synthetase.

    Science.gov (United States)

    Shapiro, Adam B; Gao, Ning; Thresher, Jason; Walkup, Grant K; Whiteaker, James

    2011-06-01

    Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.

  3. 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 Gi......-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors...... 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....

  4. Comparison of the Roche Cobas® 4800 HPV assay to Roche Amplicor for detection of high-risk human papillomavirus.

    Science.gov (United States)

    Phillips, S; Cornall, A M; Machalek, D A; Garland, S M; Bateson, D; Garefalakis, M; Tabrizi, S N

    2016-08-01

    Roche Amplicor HPV (AMP) had previously been used for detection of high-risk human papillomavirus (HR-HPV) in epidemiological and clinical studies. As this assay is no longer available, we compared its performance using PreservCyt samples from women aged of 18-24 years attending for routine cervical cytology screening to Roche Cobas® 4800 (Cobas) to determine if subsequent studies could continue using the Cobas assay. Overall 507 samples were tested on Cobas and compared to previous AMP results, with discrepant samples tested on Roche Linear Array. Overall, agreement between the Cobas and AMP for the presence of HR HPV types was very high (κ = 0.81) (95 % CI: 0.76 - 0.87) with percentage agreement of 91.57 %. Cobas is comparable to AMP for the detection of HR-HPV types in a community recruited cohort of healthy women.

  5. High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms.

    Science.gov (United States)

    Sutherland, D Robert; Ortiz, Fernando; Quest, Graeme; Illingworth, Andrea; Benko, Miroslav; Nayyar, Rakesh; Marinov, Iuri

    2018-01-30

    Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired Hematopoietic Stem Cell disorder characterized by an inability to make glyco-phosphatidyl-inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 =1) and monocytes (R2 =0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6- and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. This article is protected by copyright. All rights reserved. © 2018 International Clinical Cytometry Society.

  6. 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

    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...

  7. Comparison of Clinical Performance of Abbott RealTime High Risk HPV Test with That of Hybrid Capture 2 Assay in a Screening Setting▿

    OpenAIRE

    Carozzi, F. M.; Burroni, E.; Bisanzi, S.; Puliti, D; Confortini, M.; Giorgi Rossi, P; Sani, C; Scalisi, A.; Chini, F.

    2011-01-01

    Randomized trials have produced sound evidence about the efficacy of screening with human papillomavirus (HPV) DNA tests in reducing cervical cancer incidence and mortality. We evaluated the clinical performance and reproducibility of the Abbott RealTime High Risk (HR) HPV test compared with that of the HR hybrid capture 2 (HC2) assay as assessed by a noninferiority score test. A random sample of 998 cervical specimens (914 specimens of cervical intraepithelial neoplasia less severe than grad...

  8. Population-specific life histories contribute to metapopulation viability

    Science.gov (United States)

    Halsey, Samniqueka J.; Bell, Timothy J.; McEachern, A. Kathryn; Pavlovic, Noel B.

    2016-01-01

    Restoration efforts can be improved by understanding how variations in life-history traits occur within populations of the same species living in different environments. This can be done by first understanding the demographic responses of natural occurring populations. Population viability analysis continues to be useful to species management and conservation with sensitivity analysis aiding in the understanding of population dynamics. In this study, using life-table response experiments and elasticity analyses, we investigated how population-specific life-history demographic responses contributed to the metapopulation viability of the Federally threatened Pitcher's thistle (Cirsium pitcheri). Specifically, we tested the following hypotheses: (1) Subpopulations occupying different environments within a metapopulation have independent demographic responses and (2) advancing succession results in a shift from a demographic response focused on growth and fecundity to one dominated by stasis. Our results showed that reintroductions had a positive contribution to the metapopulation growth rate as compared to native populations which had a negative contribution. We found no difference in succession on the contribution to metapopulation viability. In addition, we identified distinct population-specific contributions to metapopulation viability and were able to associate specific life-history demographic responses. For example, the positive impact of Miller High Dunes population on the metapopulation growth rate resulted from high growth contributions, whereas increased time of plant in stasis for the State Park Big Blowout population resulted in negative contributions. A greater understanding of how separate populations respond in their corresponding environment may ultimately lead to more effective management strategies aimed at reducing extinction risk. We propose the continued use of sensitivity analyses to evaluate population-specific demographic influences on

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

    Science.gov (United States)

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

    2013-10-01

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

  10. Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay.

    Science.gov (United States)

    Wille, T; Thiermann, H; Worek, F

    2010-04-01

    Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  11. Effects of drinking desalinated seawater on cell viability and proliferation.

    Science.gov (United States)

    Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

    2017-06-01

    Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

  12. High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay

    Directory of Open Access Journals (Sweden)

    Robert W. Yucha

    2017-06-01

    Full Text Available Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent infection. We therefore developed a microfluidic single-cell-in-droplet (scdPCR assay to directly measure the number of CD4+ T cells that produce unspliced (usRNA and multiply spliced (msRNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin or T cell receptor (TCR stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

  13. A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate Efflux.

    Science.gov (United States)

    Perez, Dominique; Simons, Peter C; Smagley, Yelena; Sklar, Larry A; Chigaev, Alexandre

    2016-01-01

    Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).

  14. Development of a dose assay for a Clostridium difficile vaccine using a tandem ion exchange high performance liquid chromatography method.

    Science.gov (United States)

    Wang, Feng; Ha, Sha; Rustandi, Richard R

    2017-05-19

    Clostridium difficile is a gram-positive intestine bacterium that causes a severe diarrhea and could eventually be lethal. The main virulence factor is related to the release of two major exotoxins, toxin A (TcdA) and toxin B (TcdB). Recent C. difficile-associated disease (CDAD) outbreaks have been caused by hypervirulent strains which secrete an additional binary toxin (CDTa/CDTb). Vaccination against these toxins is considered the best way to combat the CDAD. Recently, a novel tetravalent C. difficile vaccine candidate containing all four toxins produced from a baculovirus expression system has been developed. A dose assay to release this tetravalent C. difficile vaccine was developed using tandem ion-exchange HPLC chromatography. A sequential weak cation exchange (carboxyl group) and weak anion exchange (tertiary amine group) columns were employed. The four C. difficile vaccine antigen pIs range from 4.4 to 8.6. The final optimized separation employs salt gradient elution at two different pHs. The standard analytical parameters such as LOD, LOQ, linearity, accuracy, precision and repeatability were evaluated for this method and it was deemed acceptable as a quantitative assay for vaccine release. Furthermore, the developed method was utilized for monitoring the stability of the tetravalent C. difficile vaccine in final container. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry

    Science.gov (United States)

    Li, Xingnan; Franke, Adrian A.

    2015-01-01

    An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2′-deoxycytidine (5MedC) to 2′-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC–ESI-MS/MS. The cost-effective internal standards 15N3-dC and 15N5-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4 femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring. PMID:21843675

  16. Highly porous acrylonitrile-based submicron particles for UO2(2+) absorption in an immunosensor assay.

    Science.gov (United States)

    Sahiner, Nurettin; Yu, Haini; Tan, Grace; He, Jibao; John, Vijay T; Blake, Diane A

    2012-01-01

    Our laboratory has previously reported an antibody-based assay for hexavalent uranium (UO(2)(2+)) that could be used on-site to rapidly assess uranium contamination in environmental water samples (Melton, S. J.; et al. Environ. Sci. Technol. 2009, 43, 6703-6709). To extend the utility of this assay to less-characterized sites of uranium contamination, we required a uranium-specific adsorbent that would rapidly remove the uranium from groundwater samples, while leaving the concentrations of other ions in the groundwater relatively unaltered. This study describes the development of hydrogel particles containing amidoxime groups that can rapidly and selectively facilitate the uptake of uranyl ions. A miniemulsion polymerization technique using SDS micelles was employed for the preparation of the hydrogel as linked submicrometer particles. In polymerization, acrylonitrile was used as the initial monomer, ethylene glycol dimethacrylate as the crosslinker and 2-hydroxymethacrylate, 1-vinyl-2-pyrrolidone, acrylic acid, or methacrylic acid were added as co-monomers after the initial seed polymerization of acrylonitrle. The particles were characterized by transmission electron spectroscopy, scanning electron microscopy (SEM) and cryo-SEM. The amidoximated particles were superior to a commercially available resin in their ability to rapidly remove dissolved UO(2)(2+) from spiked groundwater samples. © 2011 American Chemical Society

  17. Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

    NARCIS (Netherlands)

    Chitarra, L.G.

    2001-01-01

    Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on

  18. Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

    Science.gov (United States)

    Catros, Sylvain; Guillotin, Bertrand; Bačáková, Markéta; Fricain, Jean-Christophe; Guillemot, Fabien

    2011-04-01

    Biofabrication of three dimensional tissues by Laser-Assisted Bioprinting (LAB) implies to develop specific strategies for assembling the extracellular matrix (ECM) and cells. Possible strategies consist in (i) printing cells onto or in the depth of ECM layer and/or (ii) printing bioinks containing both cells and ECM-like printable biomaterial. The aim of this article was to evaluate combinatorial effects of laser pulse energy, ECM thickness and viscosity of the bioink on cell viability. A LAB workstation was used to print Ea.hy926 endothelial cells onto a quartz substrate covered with a film of ECM mimicking Matrigel™. Hence, effect of laser energy, Matrigel™ film thickness and bioink viscosity was addressed for different experimental conditions (8-24 μJ, 20-100 μm and 40-110 mPa s, respectively). Cell viability was assessed by live/dead assay performed 24 h post-printing. Results show that increasing the laser energy tends to augment the cell mortality while increasing the thickness of the Matrigel™ film and the viscosity of the bioink support cell viability. Hence, critical printing parameters influencing high cell viability have been related to the cell landing conditions and more specifically to the intensity of the cell impacts occurring at the air-ECM interface and at the ECM-glass interface.

  19. High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

    Science.gov (United States)

    Yapa, Udeni; Prusakiewicz, Jeffery J; Wrightstone, Ann D; Christine, Lori J; Palandra, Joe; Groeber, Elizabeth; Wittwer, Arthur J

    2012-02-15

    Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Use of a highly-sensitive cardiac troponin I assay in a screening population for hypertrophic cardiomyopathy: a case-referent study.

    Science.gov (United States)

    McGorrian, Catherine M; Lyster, Sarah; Roy, Andrew; Tarrant, Heloise; Codd, Mary; Doran, Peter; Fitzgibbon, Maria; Galvin, Joseph; Mahon, Niall G

    2013-09-11

    Hypertrophic cardiomyopathy (HCM) is a genetic condition, and relatives of affected persons may be at risk. Cardiac troponin biomarkers have previously been shown to be elevated in HCM. This study examines the new highly-sensitive cardiac troponin I (hsTnI) assay in a HCM screening population. Nested case-control study of consecutive HCM sufferers and their relatives recruited from May 2010 to September 2011. After informed consent, participants provided venous blood samples and clinical and echocardiographic features were recorded. Associations between the natural log (ln) of the contemporary troponin I (cTnI) and hsTnI assays and markers of cardiac hypertrophy were examined. Multiple regression models were fitted to examine the predictive ability of hsTnI for borderline or definite HCM. Of 107 patients, 24 had borderline and 19 had definite changes of HCM. Both TnI assays showed significant, positive correlations with measures of cardiac muscle mass. After age and sex adjustment, the area under the receiver operator characteristic (AUROC) curve for the outcome of HCM was 0.78, 95% CI [0.65, 0.90], for ln(hsTnI), and 0.66, 95% CI [0.51, 0.82], for ln(cTnI) (p=0.11). Including the hsTnI assay in a multiple-adjusted "screening" model for HCM resulted in a non-significant improvement in both the AUROC and integrated discrimination index. Both cTnI and hsTnI show a graded, positive association with measures of cardiac muscle mass in persons at risk of HCM. Further studies will be required to evaluate the utility of these assays in ECG- and symptom-based identification of HCM in at-risk families.

  1. Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1.

    Science.gov (United States)

    Rong, Juan; Pass, Ian; Diaz, Paul W; Ngo, Tram A; Sauer, Michelle; Magnuson, Gavin; Zeng, Fu-Yue; Hassig, Christian A; Jackson, Michael R; Cosford, Nicholas D P; Matsuzawa, Shu-Ichi; Reed, John C

    2015-12-01

    Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the "adaptive" signaling cascade mediated by the XBP1 pathway and the "alarm" signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)-Luc cell-based assay in 1536-well format for monitoring IRE1's RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1. © 2015 Society for Laboratory Automation and Screening.

  2. Modelling the comet assay.

    Science.gov (United States)

    McArt, Darragh G; McKerr, George; Howard, C Vyvyan; Saetzler, Kurt; Wasson, Gillian R

    2009-08-01

    The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

  3. Terminology for pregnancy loss prior to viability

    DEFF Research Database (Denmark)

    Kolte, A M; Bernardi, L A; Christiansen, O B

    2015-01-01

    Pregnancy loss prior to viability is common and research in the field is extensive. Unfortunately, terminology in the literature is inconsistent. The lack of consensus regarding nomenclature and classification of pregnancy loss prior to viability makes it difficult to compare study results from...... different centres. In our opinion, terminology and definitions should be based on clinical findings, and when possible, transvaginal ultrasound. With this Early Pregnancy Consensus Statement, it is our goal to provide clear and consistent terminology for pregnancy loss prior to viability....

  4. Morphology and viability of castor bean genotypes pollen grains

    Directory of Open Access Journals (Sweden)

    Maria Selma Alves Silva Diamantino

    2016-01-01

    Full Text Available The objective of this work was to characterize the morphology and viability of the pollen of 15 genotypes of castor bean (Ricinus communis L. and to generate information that can assist in the selection of highly promising male parents for future use in genetic improvement programs aimed at producing seeds for oil extraction. Acetolysis and scanning electron microscopy was used to characterize the morphology of the pollen. The viability of the pollen grains was estimated by in vitro germination and colorimetric analysis (acetocarmine 2% and 2, 3, 5-triphenyltetrazolium chloride 1%. For the in vitro germination, pollen grains were grown in 10 types of solidified culture medium consisting of different concentrations of sucrose, boric acid, calcium nitrate, magnesium sulfate and potassium nitrate. The pollen grains had the following characteristics: medium size, isopolar and subspheroidal shape, radial symmetry, circular ambit, 3-colporate, elongated endoapertures, tectate exine and granulated sexine. The acetocarmine dye overestimated pollen viability. The media M5 and M8 were the most efficient at promoting the germination of pollen grains. The studied genotypes had high levels of viability and can therefore be used as male parents in genetic improvement programs.

  5. 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

  6. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    Science.gov (United States)

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  7. 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.

  8. A highly sensitive carbapenemase assay using laser desorption/ionization mass spectrometry based on a parylene-matrix chip.

    Science.gov (United States)

    Park, Jong-Min; Kim, Jo-Il; Noh, Joo-Yoon; Kim, Mira; Kang, Min-Jung; Pyun, Jae-Chul

    2017-09-01

    A quantitative carbapenemase assay was developed using laser desorption/ionization mass spectrometry (LDI-MS) based on a parylene-matrix chip. As a first step, the reproducibility (spot-to-spot, shot-to-shot, and day-to-day) of LDI-MS based on a parylene-matrix chip and the quantification ranges for four carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) were determined. A carbapenem-susceptibility test was performed using the four carbapenems and 51 bacterial strains that displayed (1) carbapenem resistance with carbapenemase, (2) carbapenem resistance without carbapenemase, or (3) carbapenem susceptibility. The susceptibility test results showed that LDI-MS based on a parylene-matrix chip was more sensitive and selective for detecting the carbapenemase reaction than conventional MALDI-TOF MS based on a 2,5-dihydroxybenzoic acid matrix. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    Directory of Open Access Journals (Sweden)

    Raja Elina Raja Aziddin

    2011-08-01

    Full Text Available The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE on human recombinant cytochrome P450 (CYP enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC50 values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC50 of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC50 of CYP2C19 could not be determined, however, because inhibition was < 50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6, ketoconazole (CYP3A4, tranylcypromine (CYP2C19 and furafylline (CYP1A2 were used as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

  10. High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay.

    Science.gov (United States)

    Jones, P A; King, A V

    2003-01-01

    Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity.

  11. 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.

  12. Detection of aflatoxin B1 in imported food products into Japan by enzyme-linked immunosorbent assay and high performance liquid chromatography.

    Science.gov (United States)

    Adachi, Y; Hara, M; Kumazawa, N H; Hirano, K; Ueno, I; Egawa, K

    1991-02-01

    In order to detect the presence of aflatoxin B1 (AFB1), the use of the enzyme-linked immunosorbent assay (ELISA) and recovery test was evaluated. The detection limit of ELISA for AFB1 was 1 pg/assay and the recovery from maize spiked with AFB1 exceeded 80%. AFB1 was detected by ELISA in seven out of twelve samples of imported food products including peanut, almond, red pepper, cocoa bean, black pepper, buckwheat, walnut, adlay, soybean, popcorn, and pistachio nut, and by high performance liquid chromatography (HPLC) in four of the samples. However, the content of AFB1 in these samples was less than 10 ng/g of the minimum value authorized by the Japanese sanitation law. These results demonstrate that ELISA is more sensitive than HPLC and imported food products are broadly contaminated with AFB1.

  13. A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).

    Science.gov (United States)

    Shapiro, Adam B; Ross, Philip L; Gao, Ning; Livchak, Stephania; Kern, Gunther; Yang, Wei; Andrews, Beth; Thresher, Jason

    2013-03-01

    LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

  14. Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

    KAUST Repository

    Rodriguez, Jose E.

    2013-05-01

    Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

  15. High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Iwata, Yasuhiro; Klaren, William D; Lebakken, Connie S; Grimm, Fabian A; Rusyn, Ivan

    Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

  16. High-Sensitivity Assays for Plasmodium falciparum Infection by Immuno-Polymerase Chain Reaction Detection of PfIDEh and PfLDH Antigens.

    Science.gov (United States)

    Mu, Jianbing; Andersen, John F; Valenzuela, Jesus G; Wellems, Thomas E

    2017-09-15

    Rapid diagnostic tests based on Plasmodium falciparum histidine-rich protein II (PfHRP-II) and P. falciparum lactate dehydrogenase (PfLDH) antigens are widely deployed for detection of P. falciparum infection; however, these tests often miss cases of low-level parasitemia, and PfHRP-II tests can give false-negative results when P. falciparum strains do not express this antigen. We screened proteomic data for highly expressed P. falciparum proteins and compared their features to those of PfHRP-II and PfLDH biomarkers. Search criteria included high levels of expression, conservation in all parasite strains, and good correlation of antigen levels with parasitemia and its clearance after drug treatment. Different assay methods were compared for sensitive detection of parasitemia in P. falciparum cultures. Among potential new biomarkers, a P. falciparum homolog of insulin-degrading enzyme (PfIDEh) met our search criteria. Comparative enzyme-linked immunosorbent assays with monoclonal antibodies against PfLDH or PfIDEh showed detection limits of 100-200 parasites/µL and 200-400 parasites/µL, respectively. Detection was dramatically improved by use of real-time immuno-polymerase chain reaction (PCR), to parasitemia limits of 0.02 parasite/µL and 0.78 parasite/µL in PfLDH- and PfIDEh-based assays, respectively. The ability of PfLDH- or PfIDEh-based immuno-PCR assays to detect <1 parasite/µL suggests that improvements of bound antibody sensor technology may greatly increase the sensitivity of malaria rapid diagnostic tests.

  17. A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate acceptor

    Directory of Open Access Journals (Sweden)

    Angélique eLEVOYE

    2015-11-01

    Full Text Available Although G protein-coupled receptor (GPCR internalization has long been considered a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z’-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS of compounds that may modulate GPCRs internalization.

  18. Clinical validation of a novel real-time human papillomavirus assay for simultaneous detection of 14 high-risk HPV type and genotyping HPV type 16 and 18 in China.

    Science.gov (United States)

    Yang, Hui; Li, Lie-Jun; Xie, Long-Xu; Luo, Zhao-Yun; Lu, Min; Lin, Min; Zheng, Xiang-Bin; Huang, Yue; Yang, Li-Ye

    2016-02-01

    In the present study, we describe the laboratory workflow and the clinical validation of a novel multiplex real-time PCR-based HPV assay in China. The cross-sectional validation analysis showed that this assay worked well for detection of 14 HR-HPV types and identification of HPV 16 and 18 in a single sensitive assay that is suitable for both clinical usage and high-throughput cervical screening purposes. We predict that this accurate, high-throughput and low-cost HPV assay can greatly reduce the heavy economic burden of HPV detection in China.

  19. Near viability for fully nonlinear differential inclusions

    National Research Council Canada - National Science Library

    Irina Căpraru; Alina Lazu

    2014-01-01

    .... We establish a viability result under Lipschitz hypothesis on F, that consists in proving the existence of solutions of the differential inclusion above, starting from a given set, which remain...