Assessment of bacterial endospore viability with fluorescent dyes.

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Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

2004-01-01

To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

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

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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.

Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

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

2012-01-01

Background 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. 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 IC50 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 different

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

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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

Fluoro-luminometric real-time measurement of bacterial viability and killing.

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Lehtinen, Janne; Virta, Marko; Lilius, Esa Matti

2003-10-01

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

Development and validation of a quick easily used biochemical assay for evaluating the viability of small immobile arthropods.

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Phillips, Craig B; Iline, Ilia I; Richards, Nicola K; Novoselov, Max; McNeill, Mark R

2013-10-01

Quickly, accurately, and easily assessing the efficacy of treatments to control sessile arthropods (e.g., scale insects) and stationary immature life stages (e.g., eggs and pupae) is problematic because it is difficult to tell whether treated organisms are alive or dead. Current approaches usually involve either maintaining organisms in the laboratory to observe them for development, gauging their response to physical stimulation, or assessing morphological characters such as turgidity and color. These can be slow, technically difficult, or subjective, and the validity of methods other than laboratory rearing has seldom been tested. Here, we describe development and validation of a quick easily used biochemical colorimetric assay for measuring the viability of arthropods that is sufficiently sensitive to test even very small organisms such as white fly eggs. The assay was adapted from a technique for staining the enzyme hexokinase to signal the presence of adenosine triphosphate in viable specimens by reducing a tetrazolium salt to formazan. Basic laboratory facilities and skills are required for production of the stain, but no specialist equipment, expertise, or facilities are needed for its use.

Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence.

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Lee, Jia-Ying Joey; Miller, James Alastair; Basu, Sreetama; Kee, Ting-Zhen Vanessa; Loo, Lit-Hsin

2018-06-01

Human lungs are susceptible to the toxicity induced by soluble xenobiotics. However, the direct cellular effects of many pulmonotoxic chemicals are not always clear, and thus, a general in vitro assay for testing pulmonotoxicity applicable to a wide variety of chemicals is not currently available. Here, we report a study that uses high-throughput imaging and artificial intelligence to build an in vitro pulmonotoxicity assay by automatically comparing and selecting human lung-cell lines and their associated quantitative phenotypic features most predictive of in vivo pulmonotoxicity. This approach is called "High-throughput In vitro Phenotypic Profiling for Toxicity Prediction" (HIPPTox). We found that the resulting assay based on two phenotypic features of a human bronchial epithelial cell line, BEAS-2B, can accurately classify 33 reference chemicals with human pulmonotoxicity information (88.8% balance accuracy, 84.6% sensitivity, and 93.0% specificity). In comparison, the predictivity of a standard cell-viability assay on the same set of chemicals is much lower (77.1% balanced accuracy, 84.6% sensitivity, and 69.5% specificity). We also used the assay to evaluate 17 additional test chemicals with unknown/unclear human pulmonotoxicity, and experimentally confirmed that many of the pulmonotoxic reference and predicted-positive test chemicals induce DNA strand breaks and/or activation of the DNA-damage response (DDR) pathway. Therefore, HIPPTox helps us to uncover these common modes-of-action of pulmonotoxic chemicals. HIPPTox may also be applied to other cell types or models, and accelerate the development of predictive in vitro assays for other cell-type- or organ-specific toxicities.

The fluorometric microculture cytotoxicity assay.

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Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

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Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

2011-01-01

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation.

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Shapiro-Ilan, David I; Hazir, Selcuk; Lete, Luis

2015-09-01

Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone

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.

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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.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

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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

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

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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.

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

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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

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

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Tsukatani, Tadayuki; Suenaga, Hikaru; Ishiyama, Munetaka; Ezoe, Takatoshi; Matsumoto, Kiyoshi

2011-07-15

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

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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.

Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution

International Nuclear Information System (INIS)

Ali, Muhammad; Pages, Emeline; Ducom, Alexandre; Fontaine, Aurelien; Guillemot, Fabien

2014-01-01

Laser-assisted bioprinting is a versatile, non-contact, nozzle-free printing technique which has demonstrated high potential for cell printing with high resolution. Improving cell viability requires determining printing conditions which minimize shear stress for cells within the jet and cell impact at droplet landing. In this context, this study deals with laser-induced jet dynamics to determine conditions from which jets arise with minimum kinetic energies. The transition from a sub-threshold regime to jetting regime has been associated with a geometrical parameter (vertex angle) which can be harnessed to print mesenchymal stem cells with high viability using slow jet conditions. Finally, hydrodynamic jet stability is also studied for higher laser pulse energies which give rise to supersonic but turbulent jets. (paper)

A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

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Wyatt, C R; Goff, W; Davis, W C

1991-06-24

We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

Energy Technology Data Exchange (ETDEWEB)

Wills, Lauren P. [MitoHealth Inc., Charleston, SC 29403 (United States); Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Beeson, Craig C. [MitoHealth Inc., Charleston, SC 29403 (United States); Peterson, Yuri K. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

2013-10-15

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

International Nuclear Information System (INIS)

Wills, Lauren P.; Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C.; Beeson, Craig C.; Peterson, Yuri K.; Schnellmann, Rick G.

2013-01-01

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants

Aptamer-based viability impedimetric sensor for bacteria.

Science.gov (United States)

Labib, Mahmoud; Zamay, Anna S; Kolovskaya, Olga S; Reshetneva, Irina T; Zamay, Galina S; Kibbee, Richard J; Sattar, Syed A; Zamay, Tatiana N; Berezovski, Maxim V

2012-11-06

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella typhimurium were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable S. typhimurium and a negative selection step against heat killed S. typhimurium and a mixture of related pathogens, including Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. typhimurium down to 600 CFU mL(-1) (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other Salmonella species, including S. enteritidis and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform.

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

Directory of Open Access Journals (Sweden)

Delyan P Ivanov

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

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

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.

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

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.

Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays.

Science.gov (United States)

Fischer, Janine; Prosenc, Marc H; Wolff, Martin; Hort, Norbert; Willumeit, Regine; Feyerabend, Frank

2010-05-01

Magnesium (Mg) alloys are promising materials for the development of biodegradable implants. However, the current in vitro test procedures for cytotoxicity, cell viability and proliferation are not always suitable for this class of materials. In this paper we show that tetrazolium-salt-based assays, which are widely used in practice, are influenced by the corrosion products of Mg-based alloys. Corroded Mg converts tetrazolium salts to formazan, leading to a higher background and falsifying the results of cell viability. Tetrazolium-based assays are therefore not a useful tool for testing the cytotoxicity of Mg in static in vitro assays. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cell viability and PSA secretion assays in LNCaP cells: a tiered in vitro approach to screen chemicals with a prostate-mediated effect on male reproduction within the ReProTect project.

Science.gov (United States)

Lorenzetti, Stefano; Marcoccia, Daniele; Narciso, Laura; Mantovani, Alberto

2010-08-01

Prostate function is critical for male fertility; nevertheless, prostate was so far overlooked in reproductive toxicity assays. Within the EU project ReProTect, the human prostate cell line LNCaP was utilized to identify molecules targeting prostate function by the integrated assessment of cell viability (MTS assay) and prostate-specific antigen (PSA) secretion as specific marker; a training set - five (anti)androgenic chemicals - and a ReProTect feasibility set - ten chemicals - were used. Several compounds reduced PSA only at cytotoxic concentrations. Androgens (DHT, MT) markedly increased PSA as did the herbicide glufosinate ammonium, not known as androgen agonist. Anti-androgens (2OH-flutamide, linuron, vinclozolin, di-n-butyl phthalate) also increased PSA, but the effect of magnitude was much lower than for androgens. The ER-binder bisphenol A reduced PSA, while increasing cell viability. At this stage, the approach can identify chemicals able to interfere with prostate function: further refinements may allow to include prostate effects in reproductive toxicity in vitro testing. Copyright 2010 Elsevier Inc. All rights reserved.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

Science.gov (United States)

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

PBMC and MDAMB-231 cellular viability after telecobalt irradiation

International Nuclear Information System (INIS)

Andrade, Lidia M.; Campos, Tarcisio P.R.

2002-01-01

Radiotherapy by gamma rays are used for cancer treatment. Ionizing radiation effects on cells has been investigated. Studies in vitro were developed using tumor cell lines and ionizing radiation. The aim of this research was to test the cellular viability response of two cell types through MTT assay: human peripheral blood mononuclear cell (PBMC) and human breast carcinoma cell line MDAMB-231. These cells were irradiated with 60 Co source Theratron 80 radiotherapy machine from Atomic Energy Canada Limited with 20 x 20 cm field at 136.4 cGy/min, surface source distance 70 cm. Culture flasks contained 10 4 , 10 5 and 10 6 cells were irradiated with 100 Gy, 25 Gy, and 50 Gy using non fractionated doses. Cellular viability were evaluated 1h, 24h, 48h and 72h after irradiation and samples were measured by optical density at 595nm. Our results show that PBMC cells present lower cellular viability post irradiation. On the other hand, MDAMB-231 cells maintain viability after 50 Gy irradiation at 72h indicating cellular radioresistance. (author)

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

Science.gov (United States)

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

2015-01-01

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

Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

Science.gov (United States)

Kessel, Sarah; Cribbes, Scott; Bonasu, Surekha; Rice, William; Qiu, Jean; Chan, Leo Li-Ying

2017-09-01

The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better

Viability Study for an Unattended UF_6 Cylinder Verification Station: Phase I Final Report

International Nuclear Information System (INIS)

Smith, Leon E.; Miller, Karen A.; Garner, James R.; Branney, Sean; McDonald, Benjamin S.; Webster, Jennifer B.; Zalavadia, Mital A.; Todd, Lindsay C.; Kulisek, Jonathan A.; Nordquist, Heather; Deshmukh, Nikhil S.; Stewart, Scott

2016-01-01

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, "2"3"5U mass, total uranium mass and identification for all declared UF_6 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-measured instrument

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

Science.gov (United States)

Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

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

Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

Science.gov (United States)

Pabst, A M; Walter, C; Grassmann, L; Weyhrauch, M; Brüllmann, D D; Ziebart, T; Scheller, H; Lehmann, K M

2014-05-01

The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each materials at all points of measurement (between -36 % and -71 %; p each ceramic materials could be investigated. This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

OpenAIRE

Lu, Yong; Jiang, Feng; Jiang, Hao; Wu, Kalina; Zheng, Xuguang; Cai, Yizhong; Katakowski, Mark; Chopp, Michael; To, Shing-Shun Tony

2010-01-01

Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose- and time-dependent manner. BrdU and tube formation assays indicated that gallic acid sign...

Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

Directory of Open Access Journals (Sweden)

Emilie L Laurin

Full Text Available Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12 as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

An efficient and economical MTT assay for determining the antioxidant activity of plant natural product extracts and pure compounds.

Science.gov (United States)

Liu, Yunbao; Nair, Muraleedharan G

2010-07-23

Antioxidants scavenge free radicals, singlet oxygen, and electrons in cellular redox reactions. The yellow MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to a purple formazan by mitochondrial enzymes. NADPH is the basis of established in vitro cell viability assays. An antioxidant assay has been developed utilizing the redox reaction between MTT and selected natural product extracts and purified compounds. This simple, fast, and inexpensive MTT antioxidant assay is comparable with the lipid peroxidation inhibitory assay and can be mechanized to achieve high throughput.

Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.

Science.gov (United States)

Xu, Lin; Su, Weiheng; Jin, Jun; Chen, Jiawen; Li, Xiaojun; Zhang, Xuyuan; Sun, Meiyan; Sun, Shiyang; Fan, Peihu; An, Dong; Zhang, Huafei; Zhang, Xiguang; Kong, Wei; Ma, Tonghui; Jiang, Chunlai

2014-07-17

Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

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.

Human periodontal ligament cell viability in milk and milk substitutes.

Science.gov (United States)

Pearson, Robert M; Liewehr, Frederick R; West, Leslie A; Patton, William R; McPherson, James C; Runner, Royce R

2003-03-01

The purpose of this study was to determine the efficacy of several milk substitutes compared to whole milk in maintaining the viability of human periodontal ligament (PDL) cells on avulsed teeth. PDL cells were obtained from freshly extracted, healthy third molars and cultured in Eagle's minimal essential media (EMEM). The cells were plated onto 24-well culture plates and allowed to attach for 24 h. EMEM was replaced with refrigerated whole milk (positive control), reconstituted powdered milk, evaporated milk, or one of two baby formulas (Similac or Enfamil). Tap water served as the negative control. Tissue culture plates were incubated with the experimental media at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined by a cell proliferation assay (CellTiter 96 AQ Assay), with absorbance read at 450 nM. A two-way ANOVA (p effect on PDL cell viability between any of the materials and whole milk. At 2 h, Enfamil and Similac performed significantly better than whole milk, whereas evaporated milk performed worse. At 4 h, Enfamil performed better than whole milk, whereas all other milk substitutes performed worse. At 8 h, all substitutes performed worse than whole milk. These results suggest that Enfamil, which is supplied in powder form that does not require special storage and has a shelf life of 18 months, is a more effective storage medium for avulsed teeth than pasteurized milk for at least 4 h.

A rapid, sensitive and cost-efficient assay to estimate viability of potato cyst nematodes

NARCIS (Netherlands)

Elsen, van den S.J.J.; Ave, M.; Schoenmakers, N.; Landeweert, R.; Bakker, J.; Helder, J.

2012-01-01

Potato cyst nematodes (PCN) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts which can contain 200-500 eggs protected by the hardened cuticle of a dead female, are either time and labor

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

Science.gov (United States)

Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

2013-09-01

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

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

Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

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Xuefei Song

2015-01-01

Full Text Available Purpose. The purpose of this study was to determine the impact of cross-linking (CXL on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham’s F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2 during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA. Results. Following CXL, cell viability and proliferation decreased (P0.06. Five hours after CXL, FGFb secretion increased significantly (P=0.037; however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P>0.12. Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours, normalizing after 24 hours.

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

Low-dose dose-response for reduced cell viability after exposure of human keratinocyte (HEK001 cells to arsenite

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Kenneth T. Bogen

Full Text Available The in vitro arsenite (AsIII cytotoxicity dose-response (DR of human keratinocytes (HEK001 was examined at greater statistical resolution than ever previously reported using the MTT assay to determine cell viability. Fifty-four 96-well plates were treated with AsIII concentrations of 0.25, 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, or 30 μM. Because of unexpected variation in viability response patterns, a two-stage DR analysis was used in which data on plate-specific viability (%, estimated as 100% times the ratio of measured viability in exposed to unexposed cells, were fit initially to a generalized lognormal response function positing that HEK001 cells studied consisted of: a proportion P of relatively highly sensitive (HS cells, a proportion Po of relatively resistant cells, and a remaining (1–P–Po fraction of typical-sensitivity (TS cells exhibiting the intermediate level of AsIII sensitivity characteristic of most cells in each assay. The estimated fractions P and Po were used to adjust data from all 54 plates (and from the 28 plates yielding the best fits to reflect the condition that P = Po = 0 to provide detailed DR analysis specifically for TS cells. Four DR models fit to the combined adjusted data were each very predictive (R2 > 0.97 overall but were inconsistent with at least one of the data set examined (p  0.30 and exceeded 100% significance (p ≤ 10−6. A low-dose hormetic model provided the best fit to the combined adjusted data for TS cells (R2 = 0.995. Marked variability in estimates of P (the proportion of apparent HS cells was unexpected, not readily explained, and warrants further study using additional cell lines and assay methods, and in vivo. Keywords: Arsenic, Arsenate, Cell culture, Cell death, Cytotoxicity, HEK001 cells

The application of flow cytometry and fluorescent probe technology for detection and assessment of viability of plant pathogenic bacteria

NARCIS (Netherlands)

Chitarra, L.G.; Bulk, van den R.W.

2003-01-01

Conventional methods to detect and assess the viability of plant pathogenic bacteria are usually based on plating assays or serological techniques. Plating assays provide information about the number of viable cells, expressed as colony-forming units, but are time-consuming and laborious.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry.

Science.gov (United States)

Davis, W C; Wyatt, C R; Hamilton, M J; Goff, W L

1992-01-01

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethidine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrome, ethidium. Following uptake of the dye, erythrocytes containing viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

Directory of Open Access Journals (Sweden)

W. C. Davis

1992-01-01

Full Text Available Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC on the growth and viability of intraerythrocytic parasites.

Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

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Chiquita Prahasanti

2018-04-01

Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

Efficacy of SYBR 14/propidium iodide viability stain for the amphibian chytrid fungus Batrachochytrium dendrobatidis.

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Stockwell, M P; Clulow, J; Mahony, M J

2010-01-25

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently described pathogen that has been implicated as a causal agent in the global decline in amphibians. Research into its biology and epidemiology has frequently involved in vitro experimentation. However, this research is currently limited by the inability to differentiate between viable and inviable zoospores. Stains are frequently used to determine cell viability, and this study tested a 2-colour fluorescence assay for the detection and quantification of viable B. dendrobatidis zoospores. The results show that the nucleic acid stains SYBR 14 and propidium iodide are effective in distinguishing live from dead zoospores, and a protocol has been optimized for their use. This viability assay provides an efficient and reliable tool that will have applications in B. dendrobatidis challenge and amphibian exposure experiments.

The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

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Wajid, Nadia; Naseem, Rashida; Anwar, Sanam Saiqa; Awan, Sana Javaid; Ali, Muhammad; Javed, Sara; Ali, Fatima

2015-09-01

Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

Study of wettability and cell viability of H implanted stainless steel

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Shafique, Muhammad Ahsan; Ahmad, Riaz; Rehman, Ihtesham Ur

2018-03-01

In the present work, the effect of hydrogen ion implantation on surface wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are implanted in the near-surface of stainless steel to facilitate hydrogen bonding at different doses with constant energy of 500 KeV, which consequently improve the surface wettability. Treated and untreated sample are characterized for surface wettability, incubation of hydroxyapatite and cell viability. Contact angle (CA) study reveals that surface wettability increases with increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite over the surface increase with increasing dose of H-ions. Cell viability study using MTT assay describes improved cell viability in treated samples as compared to the untreated sample. It is found that low dose of H-ions is more effective for cell proliferation and the cell count decreases with increasing ion dose. Our study demonstrates that H ion implantation improves the surface wettability and biocompatibility of stainless steel.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

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Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Viability Theory

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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

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

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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)Urea 2 , Cu(II)Ser 2 and CuCl 2 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.

Release kinetics and cell viability of ibuprofen nanocrystals produced by melt-emulsification.

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Fernandes, A R; Dias-Ferreira, J; Cabral, C; Garcia, M L; Souto, E B

2018-06-01

The clinical use of poorly water-soluble drugs has become a big challenge in pharmaceutical development due to the compromised bioavailability of the drugs in vivo. Nanocrystals have been proposed as a formulation strategy to improve the dissolution properties of these drugs. The benefits of using nanocrystals in drug delivery, when compared to other nanoparticles, are related to their production facilities, simple structure, and suitability for a variety of administration routes. High pressure homogenization (HPH) is the most promising production process, which can be employed at low or high temperatures. Ibuprofen nanocrystals with a mean size below 175 nm, and polydispersity below 0.18, have been produced by melt-emulsification, followed by HPH. Two nanocrystal formulations, differing on the surfactant composition, have been produced, their in vitro ibuprofen release tested in Franz diffusion cells and adjusted to several kinetic models (zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas, Baker-Lonsdale and Weibull model). Cell viability was assessed at 3, 6 and 24 h of incubation on human epithelial colorectal cells (Caco-2) by AlamarBlue ® colorimetric assay. For both formulations, Caco-2 cells viability was dependent on the drug concentration and time of exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid, radiolabeled-microculture method that uses macrophages for in vitro evaluation of Mycobacterium leprae viability and drug susceptibility.

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Mittal, A; Sathish, M; Seshadri, P S; Nath, I

1983-04-01

This paper describes a microculture rapid assay using radiolabeling and mouse macrophages to determine the viability and the drug susceptibility or resistance of Mycobacterium leprae. Comparison of M. leprae resident macrophage cultures maintained in 96-well flat-bottomed plates showed results for viability and susceptibility or resistance to dapsone that were similar to results for concurrent cultures in Leighton tubes with greater numbers of bacilli and macrophages.

Rapid, Radiolabeled-Microculture Method That Uses Macrophages for In Vitro Evaluation of Mycobacterium leprae Viability and Drug Susceptibility

OpenAIRE

Mittal, A.; Sathish, M.; Seshadri, P. S.; Nath, Indira

1983-01-01

This paper describes a microculture rapid assay using radiolabeling and mouse macrophages to determine the viability and the drug susceptibility or resistance of Mycobacterium leprae. Comparison of M. leprae resident macrophage cultures maintained in 96-well flat-bottomed plates showed results for viability and susceptibility or resistance to dapsone that were similar to results for concurrent cultures in Leighton tubes with greater numbers of bacilli and macrophages.

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

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Cristiana Lalli

2015-01-01

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

Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

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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).

FDG uptake in cold and heat treated MCF-7 cells, comparison with cell viability, apoptosis, and tumor marker changes

International Nuclear Information System (INIS)

Zhang, C.; Sun, X.; Huang, G.; Liu, J.

2007-01-01

Full text: Objectives-To investigate the FDG uptake changes in cold and hyperthermia therapy and its correlation with cell viability, apoptosis and tumor marker changes. Methods: An in vitro cultured breast adenocarcinoma cell line, MCF- 7, was divided into 5 groups. Hyperthermia group: cell was treated in 43 degree centigrade 30 min. Hypothermia group: cell was treated in 0 degree centigrade 30 min. Hypo- and hyperthermia group: cell was treated in 0 degree centigrade 30 min and 43 degree centigrade 30 min. chemotherapy group: cell was treated with 21 microgram Cisplatin for 6 hours. And Control group: cell was untreated. The levels 18F-labelled FDG uptake, a 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazoliumbromide viability assay, flow cytometry assay and tumor markers (CA153, CA125) were detected at 24 hour and 48 hour. Results: The change of 18F- FDG uptake (which came out at the 24h) is early than tumor marker (which came out at the 48h) under our study conditions. In treated MCF-7 cells, the levels of 18F-labelled FDG uptake were significantly lower than control group. The levels of 18F-FDG uptake depression were well correlated with cell viability and apoptosis data. Conclusion: FDG uptake is sensitive and well correlated with cell viability and apoptosis assay, and can be used for early response monitoring in hypo- and hyperthermia therapy. (author)

Moringa oleifera's Nutritious Aqueous Leaf Extract Has Anticancerous Effects by Compromising Mitochondrial Viability in an ROS-Dependent Manner.

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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.

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

Science.gov (United States)

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

2015-01-01

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

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

Science.gov (United States)

Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

2018-07-01

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

Evaluation of radical scavenging activity, intestinal cell viability and antifungal activity of Brazilian propolis by-product.

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de Francisco, Lizziane; Pinto, Diana; Rosseto, Hélen; Toledo, Lucas; Santos, Rafaela; Tobaldini-Valério, Flávia; Svidzinski, Terezinha; Bruschi, Marcos; Sarmento, Bruno; Oliveira, M Beatriz P P; Rodrigues, Francisca

2018-03-01

Propolis is a natural adhesive resinous compound produced by honeybees to protect hives from bacteria and fungi, being extremely expensive for food industry. During propolis production, a resinous by-product is formed. This resinous waste is currently undervalued and underexploited. Accordingly, in this study the proximate physical and chemical quality, as well as the antioxidant activity, radical scavenging activity and cell viability of this by-product were evaluated and compared with propolis in order to boost new applications in food and pharmaceutical industries. The results revealed that the by-product meets the physical and chemical quality standards expected and showed that the propolis waste contains similar amounts of total phenolic content (TPC) and total flavonoid content (TFC) to propolis. Also, a good scavenging activity against reactive oxygen and nitrogen species (ROS and RNS, respectively) determined by the assays of superoxide anion radical (O 2 - ), hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl), nitric oxide (NO) and peroxyl radical (ROO) were determined. Linear positive correlations were established between the TPC of both samples and the antioxidant activity evaluated by three different methods (DPPH, ABTS and FRAP assays). The extracts were also screened for cell viability assays in two different intestinal cell lines (HT29-MTX and Caco-2), showing a viability concentration-dependent. Similarly, the Artemia salina assay, used to assess toxicity, demonstrated the concentration influence on results. Finally, the antifungal activity against ATCC species of Candida was demonstrated. These results suggest that propolis by-product can be used as a new rich source of bioactive compounds for different areas, such as food or pharmaceutical. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of drinking desalinated seawater on cell viability and proliferation.

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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.

Effect of radiation dosage changes on the cell viability and the apoptosis induction on normal and tumorigenic cells

International Nuclear Information System (INIS)

Park, In Woo; Choi, Soon Chul; Lee, Sam Sun; Heo, Min Suk

1999-01-01

The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. The study, that was generated for two human normal cells (RHEK, HGF-1) and two human tumor cells (KB, HT-1080), was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5, 1, 2, 4, and 8 Gy were applied to the cells. The two fractions of 1, 2, 4, and 8 Gy were separated with a 4 hour time interval. The irradiation was done with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. 1. In 3-day group, the cell viability of HGF-1 cell was significantly decreased at 2, 4 and 8 Gy irradiation, the cell viability of KB cell was significantly decreased at 8 Gy irradiation and the cell viability of HT-1080 cell was significantly decreased at 4 and 8 Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8 Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8 Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2, 4 and 8 Gy on HGF-1 cell, at 4 and 8 Gy on HT-1080 cell, at 8 Gy on KB cell.4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8 Gy irradiation. However, there was no correlation between cell viability and apoptosis.5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.

Assessment of myocardial viability by MR imaging

International Nuclear Information System (INIS)

Sandstede, Joern J.W.

2003-01-01

Diagnosis of myocardial viability after infarction focuses on the prediction of functional improvement of dysfunctional myocardium after revascularization therapy. Magnetic resonance imaging provides different approaches for the detection of myocardial viability. Measurement of end-diastolic wall thickness is easy to perform and has a high sensitivity, but a low specificity, and can only be used 4 months after myocardial infarction due to infarct healing processes. Low-dose dobutamine stress has a good sensitivity with a high specificity for the prediction of wall motion improvement, but this is only true for patients with a singular dysfunctional area and only slightly depressed cardiac function. Late enhancement allows for direct visualization of necrotic or scarred tissue. By measuring the transmural extent of late enhancement, the probability of mechanical improvement can precisely be given. Imaging of microvascular obstruction by first-pass perfusion or late enhancement gives additional information on viability and patient prognosis. Metabolic imaging techniques, such as 31 P-MR spectroscopy and 23 Na-MR imaging, provide further insights into the mechanisms of myocardial infarction and viability. In conclusion, cardiac MRI offers several clinically usable approaches for the assessment of myocardial viability and will probably become the method of choice in the near future. (orig.)

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Oliveira, R.J. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Mestrado em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mantovani, M.S.; Silva, A.F. da [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Pesarini, J.R. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mauro, M.O. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Doutorado em Biotecnologia e Biodiversidade - Rede Pró Centro-Oeste, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Ribeiro, L.R. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Programa de Pós-Graduação em Patologia, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP (Brazil)

2014-03-28

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

Directory of Open Access Journals (Sweden)

R.J. Oliveira

2014-04-01

Full Text Available The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

International Nuclear Information System (INIS)

Oliveira, R.J.; Mantovani, M.S.; Silva, A.F. da; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

2014-01-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero

Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout.

Science.gov (United States)

Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Watson, Maureen; Gamble, Greg D; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

2011-09-01

Bone erosion is a common manifestation of chronic tophaceous gout. To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus-bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.

Towards a high throughput droplet-based agglutination assay

KAUST Repository

Kodzius, Rimantas; Castro, David; Foulds, Ian G.

2013-01-01

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.

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.

Two-Phase Microfluidic Systems for High Throughput Quantification of Agglutination Assays

KAUST Repository

Castro, David

2018-04-01

Lab-on-Chip, the miniaturization of the chemical and analytical lab, is an endeavor that seems to come out of science fiction yet is slowly becoming a reality. It is a multidisciplinary field that combines different areas of science and engineering. Within these areas, microfluidics is a specialized field that deals with the behavior, control and manipulation of small volumes of fluids. Agglutination assays are rapid, single-step, low-cost immunoassays that use microspheres to detect a wide variety molecules and pathogens by using a specific antigen-antibody interaction. Agglutination assays are particularly suitable for the miniaturization and automation that two-phase microfluidics can offer, a combination that can help tackle the ever pressing need of high-throughput screening for blood banks, epidemiology, food banks diagnosis of infectious diseases. In this thesis, we present a two-phase microfluidic system capable of incubating and quantifying agglutination assays. The microfluidic channel is a simple fabrication solution, using laboratory tubing. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5-10 fold improvement over traditional agglutination assays. It has a user-friendly interface that that does not require droplet generators, in which a pipette is used to continuously insert assays on-demand, with no down-time in between experiments at 360 assays/h. System parameters are explored, using the streptavidin-biotin interaction as a model assay, with a minimum detection limit of 50 ng/mL using optical image analysis. We compare optical image analysis and light scattering as quantification methods, and demonstrate the first light scattering quantification of agglutination assays in a two-phase ow format. The application can be potentially applied to other biomarkers, which we demonstrate using C-reactive protein (CRP) assays. Using our system, we can take a commercially available CRP qualitative slide

Plasma Electrolytic Oxidation of Titanium Implant Surfaces: Microgroove-Structures Improve Cellular Adhesion and Viability.

Science.gov (United States)

Hartjen, Philip; Hoffmann, Alexia; Henningsen, Anders; Barbeck, Mike; Kopp, Alexander; Kluwe, Lan; Precht, Clarissa; Quatela, Olivia; Gaudin, Robert; Heiland, Max; Friedrich, Reinhard E; Knipfer, Christian; Grubeanu, Daniel; Smeets, Ralf; Jung, Ole

2018-01-01

Plasma electrolytic oxidation (PEO) is an established electrochemical treatment technique that can be used for surface modifications of metal implants. In this study we to treated titanium implants with PEO, to examine the resulting microstructure and to characterize adhesion and viability of cells on the treated surfaces. Our aim was to identify an optimal surface-modification for titanium implants in order to improve soft-tissue integration. Three surface-variants were generated on titanium alloy Ti6Al4V by PEO-treatment. The elemental composition and the microstructures of the surfaces were characterized using energy dispersive X-ray spectroscopy, scanning electron microscopy and profilometry. In vitro cytocompatibility of the surfaces was assessed by seeding L929 fibroblasts onto them and measuring the adhesion, viability and cytotoxicity of cells by means of live/dead staining, XTT assay and LDH assay. Electron microscopy and profilometry revealed that the PEO-surface variants differed largely in microstructure/topography, porosity and roughness from the untreated control material as well as from one another. Roughness was generally increased after PEO-treatment. In vitro, PEO-treatment led to improved cellular adhesion and viability of cells accompanied by decreased cytotoxicity. PEO-treatment provides a promising strategy to improve the integration of titanium implants with surrounding tissues. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Science.gov (United States)

Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

2017-12-01

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

High speed measurement of corn seed viability using hyperspectral imaging

Science.gov (United States)

Ambrose, Ashabahebwa; Kandpal, Lalit Mohan; Kim, Moon S.; Lee, Wang-Hee; Cho, Byoung-Kwan

2016-03-01

Corn is one of the most cultivated crops all over world as food for humans as well as animals. Optimized agronomic practices and improved technological interventions during planting, harvesting and post-harvest handling are critical to improving the quantity and quality of corn production. Seed germination and vigor are the primary determinants of high yield notwithstanding any other factors that may play during the growth period. Seed viability may be lost during storage due to unfavorable conditions e.g. moisture content and temperatures, or physical damage during mechanical processing e.g. shelling, or over heating during drying. It is therefore vital for seed companies and farmers to test and ascertain seed viability to avoid losses of any kind. This study aimed at investigating the possibility of using hyperspectral imaging (HSI) technique to discriminate viable and nonviable corn seeds. A group of corn samples were heat treated by using microwave process while a group of seeds were kept as control group (untreated). The hyperspectral images of corn seeds of both groups were captured between 400 and 2500 nm wave range. Partial least squares discriminant analysis (PLS-DA) was built for the classification of aged (heat treated) and normal (untreated) corn seeds. The model showed highest classification accuracy of 97.6% (calibration) and 95.6% (prediction) in the SWIR region of the HSI. Furthermore, the PLS-DA and binary images were capable to provide the visual information of treated and untreated corn seeds. The overall results suggest that HSI technique is accurate for classification of viable and non-viable seeds with non-destructive manner.

A highly sensitive and specific assay for vertebrate collagenase

International Nuclear Information System (INIS)

Sodek, J.; Hurum, S.; Feng, J.

1981-01-01

A highly sensitive and specific assay for vertebrate collagenase has been developed using a [ 14 C]-labeled collagen substrate and a combination of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and fluorography to identify and quantitate the digestion products. The assay was sufficiently sensitive to permit the detection and quantitation of collagenase activity in 0.1 μl of gingival sulcal fluid, and in samples of cell culture medium without prior concentration. The assay has also been used to detect the presence of inhibitors of collagenolytic enzymes in various cell culture fluids. (author)

A COMPARISON OF THREE ASSAY PROCEDURES FOR DETERMINING CHLORINE INACTIVATION OF WATERBORNE PATHOGENIC BACTERIA

Science.gov (United States)

One criterion on which chlorine treatment of water may be based is the concentration (C) in mg/l multiplied by the time (t) in min of exposure or Ct values. We compared different Ct values on waterborne pathogenic bacteria by cultural assay for viability and 2 assays that mea...

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.

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.

ToxCast Profiling in a Human Stem Cell Assay for ...

Science.gov (United States)

Standard practice for assessing disruptions in embryogenesis involves testing pregnant animals of two species, typically rats and rabbits, exposed during major organogenesis and evaluated just prior to term. Under this design the major manifestations of developmental toxicity are observed as one or more apical endpoints including intrauterine death, fetal growth retardation, structural malformations and variations. Alternative approaches to traditional developmental toxicity testing have been proposed in the form of in vitro data (e.g., embryonic stem cells, zebrafish embryos, HTS assays) and in silico models (e.g., computational toxicology). To increase the diversity of assays used to assess developmental toxicity in EPA’s ToxCast program, we tested the chemicals in Stemina’s metabolomics-based platform that utilizes the commecrially available H9 human embryonic stem cell line. The devTOXqP dataset for ToxCast of high-quality based on replicate samples and model performance (82% balanced accuracy, 0.71 sensitivity and 1.00 specificity). To date, 136 ToxCast chemicals (12.8% of 1065 tested) were positive in this platform; 48 triggered the biomarker signal without any change in hESC viability and 88 triggered activity concurrent with effects on cell viability. Work is in progress to complete the STM dataset entry into the TCPL, compare data with results from zFish and mESC platforms, profile bioactivity (ToxCastDB), endpoints (ToxRefDB), chemotypes (DSSTox)

Concentration-dependent effects of fullerenol on cultured hippocampal neuron viability

Directory of Open Access Journals (Sweden)

Zha YY

2012-06-01

Full Text Available Ying-ying Zha,1 Bo Yang,1 Ming-liang Tang,2 Qiu-chen Guo,1 Ju-tao Chen,1 Long-ping Wen,3 Ming Wang11CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science and Technology of China, Hefei, 2Suzhou Institute of NanoTech and NanoBionics, Chinese Academy of Sciences, Suzhou, 3Laboratory of Nano-biology, School of Life Sciences, University of Science and Technology of China, Hefei, People's Republic of ChinaBackground: Recent studies have shown that the biological actions and toxicity of the water-soluble compound, polyhydroxyfullerene (fullerenol, are related to the concentrations present at a particular site of action. This study investigated the effects of different concentrations of fullerenol on cultured rat hippocampal neurons.Methods and results: Fullerenol at low concentrations significantly enhanced hippocampal neuron viability as tested by MTT assay and Hoechst 33342/propidium iodide double stain detection. At high concentrations, fullerenol induced apoptosis confirmed by Comet assay and assessment of caspase proteins.Conclusion: These findings suggest that fullerenol promotes cell death and protects against cell damage, depending on the concentration present. The concentration-dependent effects of fullerenol were mainly due to its influence on the reduction-oxidation pathway.Keywords: fullerenol, nanomaterial, neurotoxicity, neuroprotection, hippocampal neuron

Effect of Lactobacillus reuteri on Cell Viability and PGE2 Production in Human Gingival Fibroblasts.

Science.gov (United States)

A Castiblanco, Gina; Yucel-Lindberg, Tulay; Roos, Stefan; Twetman, Svante

2017-09-01

Emerging evidence suggests that probiotic therapy can play a role in the prevention and management of oral inflammatory diseases through immunomodulation and down-regulation of the inflammatory cascade. The aim of this in vitro study was to investigate the viability of human gingival fibroblasts (HGF) and its production of prostaglandin E 2 (PGE 2 ), when exposed to supernatants of two mixed Lactobacillus reuteri strains (ATCC PTA 5289 and DSM 17938). The experiments were conducted in the presence and absence of the pro-inflammatory cytokine IL-1β. L. reuteri strains were grown and the bacterial supernatant was collected. The cell-free supernatant was diluted to concentrations equivalent to the ones produced by 0.5 to 5.0 × 10 7  CFU/mL bacteria. Cell viability was assessed with the MTT colorimetric assay and the amount of PGE 2 in the cell culture medium was determined using the monoclonal enzyme immune assay kits. Our findings showed that none of the L. reuteri supernatants were cytotoxic or affected the viability of HGF. The most concentrated bacterial supernatant stimulated the production of PGE 2 by the gingival cells in a significant way in the presence of IL-1β (p reuteri might play a role in the resolution of inflammation in HGF. Thus, our findings justify further investigations on the influence of probiotic bacteria on gingival inflammatory reactions.

Oleuropein-Enriched Olive Leaf Extract Affects Calcium Dynamics and Impairs Viability of Malignant Mesothelioma Cells

Directory of Open Access Journals (Sweden)

Carla Marchetti

2015-01-01

Full Text Available Malignant mesothelioma is a poor prognosis cancer in urgent need of alternative therapies. Oleuropein, the major phenolic of olive tree (Olea europaea L., is believed to have therapeutic potentials for various diseases, including tumors. We obtained an oleuropein-enriched fraction, consisting of 60% w/w oleuropein, from olive leaves, and assessed its effects on intracellular Ca2+ and cell viability in mesothelioma cells. Effects of the oleuropein-enriched fraction on Ca2+ dynamics and cell viability were studied in the REN mesothelioma cell line, using fura-2 microspectrofluorimetry and MTT assay, respectively. Fura-2-loaded cells, transiently exposed to the oleuropein-enriched fraction, showed dose-dependent transient elevations of cytosolic Ca2+ concentration (Ca2+i. Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca2+ channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca2+ channels. The oleuropein-enriched fraction and standard oleuropein displayed a significant antiproliferative effect, as measured on REN cells by MTT cell viability assay, with IC50 of 22 μg/mL oleuropein. Data suggest that our oleuropein-enriched fraction from olive leaf extract could have pharmacological application in malignant mesothelioma anticancer therapy, possibly by targeting T-type Ca2+ channels and thereby dysregulating intracellular Ca2+ dynamics.

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....... A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads...

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.

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.

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

Science.gov (United States)

Pérez, L M; Alvarez, B L; Codony, F; Fittipaldi, M; Adrados, B; Peñuela, G; Morató, J

2010-09-01

It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

International Nuclear Information System (INIS)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

Energy Technology Data Exchange (ETDEWEB)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

2015-01-15

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

Science.gov (United States)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

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

Science.gov (United States)

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

2018-05-17

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

Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression

Science.gov (United States)

Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J.; Van der Heide, Emile

2017-01-01

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture. PMID:28761837

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

Science.gov (United States)

Smith, Kate E; Kelly, Amy C; Min, Catherine G; Weber, Craig S; McCarthy, Fiona M; Steyn, Leah V; Badarinarayana, Vasudeo; Stanton, J Brett; Kitzmann, Jennifer P; Strop, Peter; Gruessner, Angelika C; Lynch, Ronald M; Limesand, Sean W; Papas, Klearchos K

2017-11-01

Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic β cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β cell function and leads to increased inflammatory signaling.

The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

Science.gov (United States)

Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Gamble, Gregory D; Dray, Michael; Pitto, Rocco; Bentley, Jarome; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

2013-12-01

Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.

Science.gov (United States)

Radrizzani, Marina; Soncin, Sabrina; Bolis, Sara; Lo Cicero, Viviana; Andriolo, Gabriella; Turchetto, Lucia

2016-01-01

The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.

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

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

Science.gov (United States)

Taghi-Kilani, R; Gyürék, L L; Millard, P J; Finch, G R; Belosevic, M

1996-06-01

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

The reducibility of heLa cell viability by Sargassum polycystum extracts

Science.gov (United States)

Firdaus, M.; Setijawati, D.; Islam, I.; Nursyam, H.; Kartikaningsih, H.; Yufidasari, H. S.; Prihanto, A. A.; Nurdiani, R.; Jaziri, A. A.

2018-04-01

Cervical cancer is the second largest cause of death-related cancer in women. The efficacy of cancer drugs is still low. Bioactive of brown seaweed has been studied by in vitro and in vivo as anticancer. The aim of this study was to evaluate the cytotoxicity of Sargassum polycystum extracts on HeLa cell, to recognize bioactive on extract and estimate the interaction between the bioactive and target protein. S. polycystum was found from Talango Island waters and HeLa cell was obtained from Indonesian Science Institute. Sample was extracted by ethanol, ethyl acetate and hexane, concentrated and finally, extracts were assayed on HeLa cell. The viability of this cell was quantified on ELISA-Reader. The bioactive compounds of the extract were elucidated by GC-MS. The interaction between bioactive and target protein was evaluated by using in silico method. The result showed that the lowest viability of HeLa cell on n-hexane extracts treatment. The n-hexane extract of this seaweed contained benzenepropanoic acid. This compound reduced HeLa cell viability by reducing of thrombin concentration. In conclusion, the benzene propanoic acid of S. polycystum was the cytotoxic agent and it is potential agent for anti-cervical cancer.

Viability of human periodontal ligament fibroblasts in milk, Hank's balanced salt solution and coconut water as storage media.

Science.gov (United States)

Souza, B D M; Lückemeyer, D D; Reyes-Carmona, J F; Felippe, W T; Simões, C M O; Felippe, M C S

2011-02-01

To evaluate the effectiveness of various storage media at 5 °C for maintaining the viability of human periodontal ligament fibroblasts (PDLF). Plates with PDLF were soaked in recently prepared Hank's balanced salt solution (HBSS), skimmed milk, whole milk, Save-A-Tooth(®) system's HBSS (Save), natural coconut water, industrialized coconut water or tap water (negative control) at 5 °C for 3, 6, 24, 48, 72, 96 and 120 h. Minimum essential medium (MEM) at 37 °C served as the positive control. PDL cell viability was determined by MTT assay. Data were statistically analysed by Kruskal-Wallis test complemented by the Scheffé test (α=5%). The greatest number of viable cells was observed for MEM. Skimmed and whole milk, followed by natural coconut water and HBSS, were the most effective media in maintaining cell viability (Pmilk had the greatest capacity to maintain PDLF viability when compared with natural coconut water, HBSS, Save, industrialized coconut water and tap water. © 2010 International Endodontic Journal.

Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe

Directory of Open Access Journals (Sweden)

Guerin Jean F

2011-06-01

Full Text Available Abstract Background The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. Methods Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group and frozen tissue. In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE, DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL in primordial and primary follicles (ApopDETEK Kit system Enzo and morphology in the two groups. 100 Follicles (primordial and primary were counted on both fresh and frozen hemiovary to assess this various tests. Results Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing. After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p Conclusion We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

DEFF Research Database (Denmark)

Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

2011-01-01

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...... relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means...

Radioisotopic 51Cr-leukocyte adherence inhibition (LAI) assay. I

International Nuclear Information System (INIS)

Tsang, P.H.; Tangnavarad, K.; Lesnick, G.; Holland, J.F.; Bekesi, J.G.; Perloff, M.

1980-01-01

A simplified radioisotopic leukocyte adherence inhibition assay ( 51 Cr-LAI assay) was used to determine tumor-directed immune responses in patients with cancer of the breast. Essential steps in development of this assay are the standardization of conditions for optimal 51 Cr uptake by peripheral blood lymphocytes (PBL) and the inclusion of autologous or normal AB serum in the incubation media. A dextrose salt mixture (GNK) was found to enhance intracellular uptake of 51 Cr significantly (8-fold) without affecting viability of the cells or without causing selective loss of lymphocyte subpopulations. The presence of 10% autologous or normal AB serum prevented non-specific LAI responses to unrelated tumor antigens. Experimental results are presented and it is seen that this short term (4 h) 51 Cr-LAI assay provides reproducible and specific results analogous to those using tube-LAI assay. The test has the advantages of being accurate, sensitive and free from technical bias. (Auth.)

Detecting viability transitions of umbilical cord mesenchymal stem cells by Raman micro-spectroscopy

International Nuclear Information System (INIS)

Bai, H; Chen, P; Fang, H; Lin, L; Tang, G Q; Mu, G G; Gong, W; Liu, Z P; Wu, H; Zhao, H; Han, Z C

2011-01-01

Recent research suggests that human umbilical cord derived mesenchymal stem cells (hUC-MSCs) can be promising candidates for cell-based therapy. Since large population and high viability are generally required, detecting viability transitions of these cells is crucial for their population expansion and quality control. Here, as a non-invasive method, Raman micro-spectroscopy is applied to examine hUC-MSCs with different viability. Using peak fitting and statistic t-test, the Raman peaks with obvious differences between the cells with high viability (> 90%) and low viability ( -1 , symmetric stretching of C–C in lipids at 877 cm -1 and CH deformation in proteins at 1342 cm -1 show the most significant changes (p < 0.001). When the cell viability decreases, the intensities of the former two peaks are both about doubled while that of the latter peak reduces by about 30%. Based on these results, we propose that the viability of hUC-MSCs can be characterized by these three peaks. And their intensity changes can be understood from the model of excessive reactive oxygen species interacting with the bio-macromolecules

Study on human chondrocyte culture viability for autologous transplantation in clinical application

Directory of Open Access Journals (Sweden)

Christiane Lombello

2003-06-01

Full Text Available Objective: The limited regenerative capacity of the cartilage tissuemakes the treatment of chondral lesions difficult. The techniquescurrently available to treat cartilage lesions may relieve symptoms,but do not regenerate the injured tissue. Autologous chondrocytetransplantation uses cell biology and cell culture techniques toregenerate the hyaline cartilage. Methods: In this study, we analyzechondrocyte biopsy collection and culture for autologoustransplantation. Ultrastructural analyses of hyaline cartilage biopsieswere performed 0, 6, 24 and 48 hours after collection. The tissue evenafter 48 hours. Eleven cell culture assays were performed to evaluateisolation, viability, morphology, proliferation and absence ofcontaminants. Results: The cell culture techniques used allowedchondrocyte proliferation. Rates on cell viability were maintained abovethe acceptable patterns (above 90. Control of cell culture laboratoryconditions showed absence of contaminants, assuring safety of theprocess. The chondrocytes obtained presented the morphology typicalof cultured cell monolayers. Conclusion: The results indicate viabilityof chondrocyte culture technique for clinical application in autologoustransplantation.

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...

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

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

Science.gov (United States)

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

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.

The type and composition of alginate and hyaluronic-based hydrogels influence the viability of stem cells of the apical papilla.

Science.gov (United States)

Lambricht, Laure; De Berdt, Pauline; Vanacker, Julie; Leprince, Julian; Diogenes, Anibal; Goldansaz, Hadi; Bouzin, Caroline; Préat, Véronique; Dupont-Gillain, Christine; des Rieux, Anne

2014-12-01

The goal of the present work was to evaluate in vitro and in vivo the influence of various types and compositions of natural hydrogels on the viability and metabolic activity of SCAPs. Two alginate, three hyaluronic-based (Corgel™) hydrogel formulations and Matrigel were characterized for their mechanical, surface and microstructure properties using rheology, X-ray photoelectron spectroscopy and scanning electron microscopy, respectively. A characterized SCAP cell line (RP89 cells) was encapsulated in the different experimental hydrogel formulations. Cells were cultured in vitro, or implanted in cyclosporine treated mice. In vitro cell viability was evaluated using a Live/Dead assay and in vitro cellular metabolic activity was evaluated with a MTS assay. In vivo cell apoptosis was evaluated by a TUNEL test and RP89 cells were identified by human mitochondria immunostaining. Hydrogel composition influenced their mechanical and surface properties, and their microstructure. In vitro cell viability was above 80% after 2 days but decreased significantly after 7 days (60-40%). Viability at day 7 was the highest in Matrigel (70%) and then in Corgel 1.5 (60%). Metabolic activity increased over time in all the hydrogels, excepted in alginate SLM. SCAPs survived after 1 week in vivo with low apoptosis (<1%). The highest number of RP89 cells was found in Corgel 5.5 (140cells/mm(2)). Collectively, these data demonstrate that SCAP viability was directly modulated by hydrogel composition and suggest that a commercially available hyaluronic acid-based formulation might be a suitable delivery vehicle for SCAP-based dental pulp regeneration strategies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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.

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

DEFF Research Database (Denmark)

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

2016-01-01

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

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

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

Science.gov (United States)

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

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. 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 Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Human islet viability and function is maintained during high density shipment in silicone rubber membrane vessels

Science.gov (United States)

Kitzmann, Jennifer P; Pepper, Andrew R; Lopez, Boris G; Pawlick, Rena; Kin, Tatsuya; O’Gorman, Doug; Mueller, Kathryn R; Gruessner, Angelika C; Avgoustiniatos, Efstathios S; Karatzas, Theodore; Szot, Greg L; Posselt, Andrew M; Stock, Peter G; Wilson, John R; Shapiro, AM; Papas, Klearchos K

2014-01-01

The shipment of human islets from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). Human islets(IE) were isolated from two manufacturing centers and shipped in 10cm2 surface area SRM vessels in temperature and pressure controlled containers to a distant center following at least two days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a micro centrifuge tube negative control (NC). LD was designed to mimic the standard culture density for human islet preparations (200 IE/cm2), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1–3 vessels. Upon receipt, islets were assessed for viability, measured by oxygen consumption rate normalized to DNA content (OCR/DNA), and quantity, measured by DNA, and, when possible, potency and function with dynamic glucose-stimulated insulin secretion (GSIS) measurements and transplants in immunodeficient B6 rag mice. Post-shipment OCR/DNA was not reduced in HD versus LD, and was substantially reduced in the NC condition. HD islets exhibited normal function post-shipment. Based on the data we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function. PMID:25131090

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

Science.gov (United States)

Ng, Khong Y; Machida, Kazuya

2017-01-01

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

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

Science.gov (United States)

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

2006-01-01

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

Mining Chemical Activity Status from High-Throughput Screening Assays

KAUST Repository

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

2015-01-01

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

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.

STUDY ON POLLEN VIABILITY AS BIOINDICATOR OF AIR QUALITY

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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.

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...

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

International Nuclear Information System (INIS)

Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

2016-01-01

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed a RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive 14 C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of

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.

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

Science.gov (United States)

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

Clonogenic assay: adherent cells.

Science.gov (United States)

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.

Science.gov (United States)

Bircher, Lea; Geirnaert, Annelies; Hammes, Frederik; Lacroix, Christophe; Schwab, Clarissa

2018-04-17

Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (-80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03-2% in protectant-free cultures to 11-37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

Science.gov (United States)

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

Science.gov (United States)

Morato de Souza, Letícia; Guilherme Roque Rinco, Ugo; Aparecida Tavares Aguiar, Daniela; Aparecido de Almeida Junior, Luciano; Cosme-Silva, Leopoldo; Marchini Oliveira, Thais; Teixeira Marques, Nádia Carolina; Thiemy Sakai, Vivien

2018-02-01

This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm-2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P  cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm-2.

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

Directory of Open Access Journals (Sweden)

Alexandra Ibáñez Escribano

2012-08-01

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

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

International Nuclear Information System (INIS)

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

2014-01-01

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. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

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

Energy Technology Data Exchange (ETDEWEB)

Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

2014-11-15

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. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

Dragon's Blood Sap (Croton Lechleri) As Storage Medium For Avulsed Teeth: In Vitro Study Of Cell Viability.

Science.gov (United States)

Martins, Christine Men; Hamanaka, Elizane Ferreira; Hoshida, Thayse Yumi; Sell, Ana Maria; Hidalgo, Mirian Marubayashi; Silveira, Catarina Soares; Poi, Wilson Roberto

2016-01-01

Tooth replantation success depends on the condition of cementum periodontal ligament after tooth avulsion; which is influenced by storage medium. The dragon's blood (Croton lechleri) sap has been suggested as a promising medium because it supports collagen formation and exhibits healing, anti-inflammatory and antimicrobial properties. Thus, the aim of this study was to evaluate the efficacy of dragon's blood sap as a storage medium for avulsed teeth through evaluation of functional and metabolic cell viability. This in vitro study compared the efficacy of different storage media to maintain the viability of human peripheral blood mononuclear and periodontal ligament cells. A 10% dragon's blood sap was tested while PBS was selected as its control. Ultra pasteurized whole milk was used for comparison as a commonly used storage medium. DMEM and distilled water were the positive and negative controls, respectively. The viability was assessed through trypan blue exclusion test and colorimetric MTT assay after 1, 3, 6, 10 and 24 h of incubation. The dragon's blood sap showed promising results due to its considerable maintenance of cell viability. For trypan blue test, the dragon's blood sap was similar to milk (psap showed better results than all storage media, even better than milk (psap was as effective as milk, the gold standard for storage medium. The experimental sap preserved the membrane of all cells and the functional viability of periodontal ligament cells.

Effects of Multivitamins and Known Teratogens on Chick Cardiomyocytes Micromass Culture Assay

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Samreen Memon

2013-09-01

Full Text Available   Objective(s: This study aimed to find out whether the chick cardiomyocyte micromass (MM system could be employed to predict the teratogenecity of common environmental factors. Different multivitamins and over the counter drugs were used in this study.   Materials and Methods: White Leghorn 5-day-old embryo hearts were dissected and trypsinized to produce a cardiomyocyte cell suspension in Dulbecco's Modified Eagle's Medium. The cultures were incubated at 370C in 5% CO2 in air, and observations were made at 24, 48 and 144 hr, for the detection of cell beating. Cellular viability was assessed using the resazurin assay and cell protein content was assessed by the kenacid blue assay. It was observed that while not affecting total cell number folic acid, vitamin C, sodium fluoride and ginseng did not significantly reduced cell activity and beating. However cadmium chloride significantly reduced the beating, cell viability and cell protein content in micromass cultures. Results: The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter morphology and function, which may result in either teratogenic outcome or cytotoxicity. Conclusion: This could form part of a screen for developmental toxicity related to cardiac function

Relationship between humidity and influenza A viability in droplets and implications for influenza's seasonality.

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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.

Microfractionation revisited: a 1536 well high resolution screening assay

NARCIS (Netherlands)

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

2009-01-01

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

[Knockdown of PRDX6 in microglia reduces neuron viability after OGD/R injury].

Science.gov (United States)

Tan, Li; Zhao, Yong; Jiang, Beibei; Yang, Bo; Zhang, Hui

2016-08-01

Objective To observe the effects of peroxiredoxin 6 (PRDX6) knockdown in the microglia on neuron viability after oxygen-glucose deprivation and reoxygenation (OGD/R). Methods Microglia was treated with lentivirus PRDX6-siRNA and Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33). Twenty-four hours later, it was co-cultured with primary neuron to establish the microglia-neuron co-culture OGD/R model. According to the different treatment of microglia, the cells were divided into normal group, OGD/R group, negative control-siRNA treated OGD/R group, PRDX6-siRNA treated OGD/R group and PRDX6-siRNA combined with MJ33 treated OGD/R group. Western blot analysis and real-time quantitative PCR were respectively performed to detect PRDX6 protein and mRNA levels after knockdown of PRDX6 in microglia. The iPLA2 activity was measured by ELISA. MTS and lactate dehydrogenase (LDH) assay were used to measure neuron viability and cell damage. The oxidative stress level of neuron was determined by measuring superoxide dismutase (SOD) and malonaldehyde (MDA) content. Results In PRDX6-siRNA group, neuron viability was inhibited and oxidative stress damage was aggravated compared with OGD/R group. In PRDX6-siRNA combined with MJ33 group, cell viability was promoted and oxidative stress damage was alleviated compared with PRDX6-siRNA group. Conclusion PRDX6 in microglia protects neuron against OGD/R-induced injury, and iPLA2 activity has an effect on PRDX6.

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

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Jin Yipeng

2017-07-01

Full Text Available Smooth muscle differentiated human adipose derived stem cells (hADSCs provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time

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

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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.

Coconut milk and probiotic milk as storage media to maintain periodontal ligament cell viability: an in vitro study.

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Saini, Divya; Gadicherla, Prahlad; Chandra, Prakash; Anandakrishna, Latha

2017-06-01

The viability of periodontal ligament (PDL) cells is a significant determinant of the long-term prognosis of replanted avulsed teeth. A storage medium is often required to maintain the viability of these cells during the extra-alveolar period. Many studies have been carried out to search for the most suitable storage medium for avulsed teeth, but an ideal solution has not yet been found. The purpose of the study was to compare and analyze the ability of coconut milk and probiotic milk to maintain PDL cell viability. In an in vitro setting, 69 caries free human premolars with normal periodontium that had been extracted for orthodontic purposes were randomly divided into two experimental groups on the basis of storage media used (i.e., coconut milk or probiotic milk) and a Hanks' balanced salt solution (HBSS) control group (23 samples per group). Immediately after extraction, the teeth were stored dry for 20 min and then immersed for 30 min in one of the storage media. The teeth were then subjected to collagenase-dispase assay and labeled with 0.5% trypan blue staining solution for determination of cell viability. The number of viable cells was counted under a light microscope and statistically analyzed using anova and post hoc Tukey test (P ≤ 0.05). Statistical analysis demonstrated there was a significant difference (P coconut milk and probiotic milk as well as HBSS in maintaining cell viability. However, there was no significant difference between probiotic milk and HBSS in ability to maintain PDL cell viability (P > 0.05). Coconut milk may not be suitable as an interim transport media due to poor maintenance of cell viability. However, probiotic milk was able to maintain PDL cell viability as well as HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of Lactobacillus reuteri on Cell Viability and PGE2 Production in Human Gingival Fibroblasts

DEFF Research Database (Denmark)

Castiblanco, Gina A.; Yucel-Lindberg, Tulay; Roos, Stefan

2017-01-01

Emerging evidence suggests that probiotic therapy can play a role in the prevention and management of oral inflammatory diseases through immunomodulation and down-regulation of the inflammatory cascade. The aim of this in vitro study was to investigate the viability of human gingival fibroblasts...... (HGF) and its production of prostaglandin E2 (PGE2), when exposed to supernatants of two mixed Lactobacillus reuteri strains (ATCC PTA 5289 and DSM 17938). The experiments were conducted in the presence and absence of the pro-inflammatory cytokine IL-1β. L. reuteri strains were grown and the bacterial...... immune assay kits. Our findings showed that none of the L. reuteri supernatants were cytotoxic or affected the viability of HGF. The most concentrated bacterial supernatant stimulated the production of PGE2 by the gingival cells in a significant way in the presence of IL-1β (p

Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study

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Devi Gopakumar

2014-01-01

Full Text Available Introduction: Aloe vera (Av, a succulent of Liliaceae family is now a widely used medicinal plant. Its′ application covers a wide spectrum of human diseases, including oral mucosa, gastric mucosa and skin. Aloe vera preparations in the form of gel, mouth washes and cream are applied topically for many oral diseases. The applications include oral lichen planus, candidiasis, oral submucous fibrosis, geographic tongue, etc. Aims and Objectives: To evaluate the cytotoxicity of Av on human fibroblasts. Materials and Methods: Aloe vera preparation (70% was applied on the fibroblast cell lineage and the cell viability was evaluated by microculture tetrazolium technique (MTT colorimetric assay. Results: The cell viability at different concentrations was measured. The cells have maintained their viability at different concentrations used in the study. Conclusion: Our study shows the cell viability at different sample concentrations of Av. This could open up wide clinical applications of Av for reactive, inflammatory and potentially malignant oral and other mucocutaneous diseases.

Influence of nanocrystalline structure and surface properties of TiO2 thin films on the viability of L929 cells

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Osękowska Małgorzata

2015-09-01

Full Text Available In this work the physicochemical and biological properties of nanocrystalline TiO2 thin films were investigated. Thin films were prepared by magnetron sputtering method. Their properties were examined by X-ray diffraction, photoelectron spectroscopy, atomic force microscopy, optical transmission method and optical profiler. Moreover, surface wettability and scratch resistance were determined. It was found that as-deposited coatings were nanocrystalline and had TiO2-anatase structure, built from crystallites in size of 24 nm. The surface of the films was homogenous, composed of closely packed grains and hydrophilic. Due to nanocrystalline structure thin films exhibited good scratch resistance. The results were correlated to the biological activity (in vitro of thin films. Morphological changes of mouse fibroblasts (L929 cell line after contact with the surface of TiO2 films were evaluated with the use of a contrast-phase microscope, while their viability was tested by MTT colorimetric assay. The viability of cell line upon contact with the surface of nanocrystalline TiO2 film was comparable to the control sample. L929 cells had homogenous cytoplasm and were forming a confluent monofilm, while lysis and inhibition of cell growth was not observed. Moreover, the viability in contact with surface of examined films was high. This confirms non-cytotoxic effect of TiO2 film surface on mouse fibroblasts.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

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Leandro Borges Araújo

2018-02-01

Full Text Available Abstract Objective: The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA, calcium hydroxide (CH and BiodentineTM (BD on stem cells from human exfoliated deciduous teeth (SHED in vitro. Material and Methods: SHED were cultured for 1 – 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL, and tested for viability (MTT assay and proliferation (SRB assay. Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1 was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning and culture medium supplemented with 20% FBS were used as controls. Results: MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA (p<0.05. In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH (p<0.05. A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion: Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

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

Science.gov (United States)

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

2018-01-01

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

Automatic titrator for high precision plutonium assay

International Nuclear Information System (INIS)

Jackson, D.D.; Hollen, R.M.

1986-01-01

Highly precise assay of plutonium metal is required for accountability measurements. We have developed an automatic titrator for this determination which eliminates analyst bias and requires much less analyst time. The analyst is only required to enter sample data and start the titration. The automated instrument titrates the sample, locates the end point, and outputs the results as a paper tape printout. Precision of the titration is less than 0.03% relative standard deviation for a single determination at the 250-mg plutonium level. The titration time is less than 5 min

Viability of Pushrod Dilatometry Techniques for High Temperature In-Pile Measurements

Energy Technology Data Exchange (ETDEWEB)

J. E. Daw; J. L. Rempe; D. L. Knudson; K. G. Condie; J. C. Crepeau

2008-03-01

To evaluate the performance of new fuel, cladding, and structural materials for use in advanced and existing nuclear reactors, robust instrumentation is needed. Changes in material deformation are typically evaluated out-of-pile, where properties of materials are measured after samples were irradiated for a specified length of time. To address this problem, a series of tests were performed to examine the viability of using pushrod dilatometer techniques for in-pile instrumentation to measure deformation. The tests were performed in three phases. First, familiarity was gained in the use and accuracy of this system by testing samples with well defined thermal elongation characteristics. Second, high temperature data for steels, specifically SA533 Grade B, Class 1 (SA533B1) Low Alloy Steel and Stainless Steel 304 (SS304), found in Light Water Reactor (LWR) vessels, were aquired. Finally, data were obtained from a short pushrod in a horizontal geometry to data obtained from a longer pushrod in a vertical geometry, the configuration likely to be used for in-situ measurements. Results of testing show that previously accepted data for the structural steels tested, SA533B1 and SS304, are inaccurate at high temperatures (above 500 oC) due to extrpolation of high temperature data. This is especially true for SA533B1, as previous data do not account for the phase transformation of the material between 730 oC and 830 oC. Also, comparison of results for horizontal and vertical configurations show a maximum percent difference of 2.02% for high temperature data.

Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

Science.gov (United States)

Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

2016-07-01

Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

Viability of mesenchymal stem cells during electrospinning

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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.

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

Science.gov (United States)

Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

2011-05-01

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

Multifractal resilience and viability

Science.gov (United States)

Tchiguirinskaia, I.; Schertzer, D. J. M.

2017-12-01

The term resilience has become extremely fashionable and there had been many attempts to provide operational definition and in fact metrics going beyond a set of more or less ad-hoc indicators. The viability theory (Aubin and Saint-Pierre, 2011) have been used to give a rather precise mathematical definition of resilience (Deffuant and Gilbert, 2011). However, it does not grasp the multiscale nature of resilience that is rather fundamental as particularly stressed by Folke et al (2010). In this communication, we first recall a preliminary attempt (Tchiguirinskaia et al., 2014) to define multifractal resilience with the help of the maximal probable singularity. Then we extend this multifractal approach to the capture basin of the viability, therefore the resilient basin. Aubin, J P, A. Bayen, and P Saint-Pierre (2011). Viability Theory. New Directions. Springer, Berlin,. Deffuant, G. and Gilbert, N. (eds) (2011) Viability and Resilience of Complex Systems. Springer Berlin.Folke, C., S R Carpenter, B Walker, M Sheffer, T Chapin, and J Rockstroem (2010). Resilience thinking: integrating re- silience, adaptability and transformability. Ecology and So- ciety, 14(4):20, Tchiguirinskaia,I., D. Schertzer, , A. Giangola-Murzyn and T. C. Hoang (2014). Multiscale resilience metrics to assess flood. Proceedings of ICCSA 2014, Normandie University, Le Havre, France -.

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

Science.gov (United States)

Lushnikova, Iryna; Nikandrova, Yelyzaveta; Skibo, Galyna

2017-10-01

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α + ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α + level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies. © 2017 International Federation for Cell Biology.

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

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.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Classification of sensitizing and irritative potential in a combined in-vitro assay

International Nuclear Information System (INIS)

Wanner, Reinhard; Sonnenburg, Anna; Quatchadze, Maria; Schreiner, Maximilian; Peiser, Matthias; Zuberbier, Torsten; Stahlmann, Ralf

2010-01-01

We have developed a coculture system which in parallel indicates the sensitizing and irritative potential of xenobiotics. The assay is named loose-fit coculture-based sensitization assay (LCSA) and may be performed within 5 days. The system is composed of human monocytes that differentiate to a kind of dendritic cells by 2-day culturing in the presence of allogenic keratinocytes. The culture medium is enriched by a cocktail of recombinant cytokines. On day 3, concentration series of probes are added. On day 5, cells are harvested and analyzed for expression range of CD86 as a marker of sensitizing potential and for uptake of the viability stain 7-AAD as a marker of irritative potential. Estimation of the concentration required to cause a half-maximal increase in CD86 expression allowed quantification of sensitizing potential, and estimation of the concentration required to reduce viability to 50% allowed quantification of irritative potential. Examination of substances with known potential resulted in categorization of test scores. To evaluate our data, we have compared results with those of the validated animal-based sensitization test, the murine local lymph node assay (LLNA, OECD TG 429). To a large extent, results from LCSA and from LLNA achieved analogous grouping of allergens into categories like weak-moderate-strong. However, the new assay showed an improved capacity to distinguish sensitizers from non-sensitizers and irritants. In conclusion, the LCSA contains potential to fulfil the requirements of the EU's programme for the safety of chemicals 'Registration, Evaluation, Authorisation and Restriction of chemical substances' (REACH, 2006) to replace animal models.

New and highly sensitive assay for L-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography-voltammetry.

Science.gov (United States)

Rahman, M K; Nagatsu, T; Kato, T

1980-12-12

This paper describes a new, inexpensive and highly sensitive assay for aromatic L-amino acid decarboxylase (AADC) activity, using L-5-hydroxytryptophan (L-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. L-5-HTP was used as substrate and D-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from L-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using L-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.

An in vitro assay for compounds toxic to rumen protozoa

International Nuclear Information System (INIS)

Campbell, A.J.; Cumming, G.J.; Graham, C.A.; Leng, R.A.

1982-01-01

The viability of protozoa in whole rumen fluid was assessed by measuring the incorporation of Me- 14 C-choline in vitro. The use of the technique as an assay for testing antiprotozoal agents was evaluated with a variety of surfactant detergents which have previously been shown to have antiprotozoal activity in vivo. A good correlation was obtained between the potency of these compounds in vitro and in vivo. (auth)

Cartilage formation measured by a novel PIINP assay suggests that IGF-I does not stimulate but maintains cartilage formation ex vivo

DEFF Research Database (Denmark)

Madsen, S H; Sondergaard, B C; Jensen, Anne-Christine Bay

2009-01-01

explants were cultured in Dulbecco's modified Eagle's medium (DMEM):F12 in the presence of 0, 0.01, 0.1, 1, 10, or 100 ng/mL of IGF-I. The viability of the chondrocytes was measured by the colorimetric Alamar blue assay. Collagen formation was assessed from the conditioned medium by the PIINP assay...

Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine

International Nuclear Information System (INIS)

Shields, P.G.; Povey, A.C.; Wilson, V.L.; Weston, A.; Harris, C.C.

1990-01-01

A highly sensitive and specific assay for the detection of N7-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, 32 P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10(7) unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O6-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/ 32 P-postlabeling assay for O6-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents

A new image-based tool for the high throughput phenotyping of pollen viability: evaluation of inter- and intra-cultivar diversity in grapevine.

Science.gov (United States)

Tello, Javier; Montemayor, María Ignacia; Forneck, Astrid; Ibáñez, Javier

2018-01-01

Low pollen viability may limit grapevine yield under certain conditions, causing relevant economic losses to grape-growers. It is usually evaluated by the quantification of the number of viable and non-viable pollen grains that are present in a sample after an adequate pollen grain staining procedure. Although the manual counting of both types of grains is the simplest and most sensitive approach, it is a laborious and time-demanding process. In this regard, novel image-based approaches can assist in the objective, accurate and cost-effective phenotyping of this trait. Here, we introduce PollenCounter, an open-source macro implemented as a customizable Fiji tool for the high-throughput phenotyping of pollen viability. This tool splits RGB images of stained pollen grains into its primary channels, retaining red and green color fractionated images (which contain information on total and only viable pollen grains, respectively) for the subsequent isolation and counting of the regions of interest (pollen grains). This framework was successfully used for the analysis of pollen viability of a high number of samples collected in a large collection of grapevine cultivars. Results revealed a great genetic variability, from cultivars having very low pollen viability (like Corinto Bianco; viability: 14.1 ± 1.3%) to others with a very low presence of sterile pollen grains (Cuelga; viability: 98.2 ± 0.5%). A wide range of variability was also observed among several clones of cv. Tempranillo Tinto (from 97.9 ± 0.9 to 60.6 ± 5.9%, in the first season). Interestingly, the evaluation of this trait in a second season revealed differential genotype-specific sensitivity to environment. The use of PollenCounter is expected to aid in different areas, including genetics research studies, crop improvement and breeding strategies that need of fast, precise and accurate results. Considering its flexibility, it can be used not only in grapevine, but also in other species showing

Investigation into the dissolution and direct assay of high-fired plutonium dioxide

International Nuclear Information System (INIS)

Patterson, J.K.

1976-01-01

A fusion-melt and dissolution assay method has been developed and tested for the quantitative analysis of high-fired plutonium dioxide. The method employs fusion of the plutonium dioxide at temperatures greater than the melting point of an eutectic mixture of potassium pyrosulfate plus sodium peroxide. The resultant melt is then titrated directly by either controlled potential coulometry or a gravimetric titration, using standardized ceric sulfate as the titrant. It has been concluded from these investigations that by using the techniques described, high-fired plutonium dioxide (stochiometric) can be quantitatively dissolved and assayed to a degree heretofore beyond the state-of-the-art, while showing direct traceability to the Federal standards. After fusion, the dissolution and direct assay is applicable to existing routine analytical procedures. The method was designed so as to minimize physical handling, simplify the chemical operations, and maximize the personal safety of the analyst at an appreciable cost savings per analysis

Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture.

Science.gov (United States)

Guo, Xiaoling; Li, Shanyi; Ji, Qingshan; Lian, Ruiling; Chen, Jiansu

2015-10-01

Human adipose-derived stem cells (hADSCs) are potential adult stem cells source for cell therapy. But hADSCs with multi-passage or cryopreservation often revealed poor growth performance. The aim of our work was to improve the activity of poor post-thaw hADSCs by simple and effective means. We describe here a simple method based on commercially available silicone micro-wells for creating hADSCs spheroids to improve viability and neural differentiation potential on poor post-thaw hADSCs. The isolated hADSCs positively expresse d CD29, CD44, CD105, and negatively expressed CD34, CD45, HLA-DR by flow cytometry. Meanwhile, they had adipogenic and osteogenic differentiation capacity. The post-thaw and post-spheroid hADSCs from poor growth status hADSCs showed a marked increase in cell proliferation by CKK-8 analysis, cell cycle analysis and Ki67/P27 quantitative polymerase chain reaction (qPCR) analysis. They also displayed an increase viability of anti-apoptosis by annexin v and propidium iodide assays and mitochondrial membrane potential assays. After 3 days of neural induction, the neural differentiation potential of post-thaw and post-spheroid hADSCs could be enhanced by qPCR analysis and western blotting analysis. These results suggested that the spheroid formation could improve the viability and neural differentiation potential of bad growth status hADSCs, which is conducive to ADSCs research and cell therapy.

Cadmium affects the mitochondrial viability and the acid soluble thiols concentration in liver, kidney, heart and gills of Ancistrus brevifilis (Eigenmann, 1920

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P. Velasquez-Vottelerd

2015-11-01

Full Text Available The freshwater fish Ancistrus brevifilis, which is found in Venezuelan rivers, is considered a potential sentinel fish in ecotoxicological studies. The cadmium (Cd effect on the mitochondrial viability (MV and acid soluble thiols levels (AST in A. brevifilis tissues (liver, kidney, heart, and gill was evaluated. Forty-two fish with similar sizes and weights were randomly selected, of which 7 fish (with their respective replicate were exposed for 7 and 30 days to a Cd sublethal concentration (0.1 mg.l-1. We determined the MV through a Janus Green B colorimetric assay and we obtained the concentration of AST by Ellman’s method. Mitochondrial viability decreased in fish exposed to Cd for 30 days with the liver being the most affected tissue. We also detected a significant decrease in AST levels was in fishes exposed to Cd for 7 days in liver and kidney tissues; these results suggests that AST levels are elevated in some tissues may act as cytoprotective and adaptive alternative mechanism related to the ROS detoxification, maintenance redox status and mitochondrial viability. Organ-specifics variations were observed in both assays. We conclude that the Cd exposure effect on AST levels and MV, vary across fish tissues and is related to the exposure duration, the molecule dynamics in different tissues, the organism and environmental conditions.

Use of statistical analysis to validate ecogenotoxicology findings arising from various comet assay components.

Science.gov (United States)

Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, Khalid Abdullah; Masoud, Muhammad Shahreef; Mahboob, Shahid

2018-04-01

Cirrhinus mrigala, Labeo rohita, and Catla catla are economically important fish for human consumption in Pakistan, but industrial and sewage pollution has drastically reduced their population in the River Chenab. Statistics are an important tool to analyze and interpret comet assay results. The specific aims of the study were to determine the DNA damage in Cirrhinus mrigala, Labeo rohita, and Catla catla due to chemical pollution and to assess the validity of statistical analyses to determine the viability of the comet assay for a possible use with these freshwater fish species as a good indicator of pollution load and habitat degradation. Comet assay results indicated a significant (P comet head diameter, comet tail length, and % DNA damage. Regression analysis and correlation matrices conducted among the parameters of the comet assay affirmed the precision and the legitimacy of the results. The present study, therefore, strongly recommends that genotoxicological studies conduct appropriate analysis of the various components of comet assays to offer better interpretation of the assay data.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

Science.gov (United States)

Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

2016-01-01

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

Directory of Open Access Journals (Sweden)

Anli Zhang

2015-06-01

Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

High-performance liquid chromatographic radioenzymatic assay for plasma catecyholamines

International Nuclear Information System (INIS)

Klaniecki, T.S.; Corder, C.N.; McDonald, R.H. Jr.; Feldman, J.A.

1977-01-01

A new assay method for plasma catecholamimes (CA) requiring only 50 μl has been developed, which uses high performance liquid chromatography (HPLC). The norepinephrine (NE), dopamine (D), and epinephrine (E) compounds found in plasma are radioactively o-methylated with S-[methyl- 3 H]-adenosyl-L-methionine ( 3 H-SAM) 3 H-SAM by the reaction of catechol-o-methyl transferase (COMT). The reaction is terminated and a standard mixture of nonradioactive o-methylated analogues of NE, D, and E is added to act as a carrier. Following separation by HPLC, the D,L-normetanephrine (NMN), 3-methoxy-4-hydroxyphenylethyl-amine or 3-methoxytyramine (3-MOT), and metanephrine (MN) radioactive peaks are collected which represent NE, D, and E, respectively. Then MNM and MN are oxidized to vanillin, and 3-MOT is acetylated. The products are subsequently separated by solvent extraction. This is necessary in order to avoid high radioactive blanks and to allow quantitation of the radioactivity by liquid scintillation spectrometry. The mean supine levels of NE, D, and E in normal subjects were respectively 182, 33, and 87 pg/ml of plasma. Similar assays on patients with pheochromocytoma revealed 797, 80, and 470 pg/ml

Assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

International Nuclear Information System (INIS)

Mehdi, S.; Wiseman, J.S.

1989-01-01

An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection

The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

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Pornanong Aramwit

2010-05-01

Full Text Available Silk sericin (SS can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

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

Analysis of hepatitis B surface antigen (HBsAg) using high-sensitivity HBsAg assays in hepatitis B virus carriers in whom HBsAg seroclearance was confirmed by conventional assays.

Science.gov (United States)

Ozeki, Itaru; Nakajima, Tomoaki; Suii, Hirokazu; Tatsumi, Ryoji; Yamaguchi, Masakatsu; Kimura, Mutsuumi; Arakawa, Tomohiro; Kuwata, Yasuaki; Ohmura, Takumi; Hige, Shuhei; Karino, Yoshiyasu; Toyota, Joji

2018-02-01

We investigated the utility of high-sensitivity hepatitis B surface antigen (HBsAg) assays compared with conventional HBsAg assays. Using serum samples from 114 hepatitis B virus (HBV) carriers in whom HBsAg seroclearance was confirmed by conventional HBsAg assays (cut-off value, 0.05 IU/mL), the amount of HBsAg was re-examined by high-sensitivity HBsAg assays (cut-off value, 0.005 IU/mL). Cases negative for HBsAg in both assays were defined as consistent cases, and cases positive for HBsAg in the high-sensitivity HBsAg assay only were defined as discrepant cases. There were 55 (48.2%) discrepant cases, and the range of HBsAg titers determined by high-sensitivity HBsAg assays was 0.005-0.056 IU/mL. Multivariate analysis showed that the presence of nucleos(t)ide analog therapy, liver cirrhosis, and negative anti-HBs contributed to the discrepancies between the two assays. Cumulative anti-HBs positivity rates among discrepant cases were 12.7%, 17.2%, 38.8%, and 43.9% at baseline, 1 year, 3 years, and 5 years, respectively, whereas the corresponding rates among consistent cases were 50.8%, 56.0%, 61.7%, and 68.0%, respectively. Hepatitis B virus DNA negativity rates were 56.4% and 81.4% at baseline, 51.3% and 83.3% at 1 year, and 36.8% and 95.7% at 3 years, among discrepant and consistent cases, respectively. Hepatitis B surface antigen reversion was observed only in discrepant cases. Re-examination by high-sensitivity HBsAg assays revealed that HBsAg was positive in approximately 50% of cases. Cumulative anti-HBs seroconversion rates and HBV-DNA seroclearance rates were lower in these cases, suggesting a population at risk for HBsAg reversion. © 2017 The Japan Society of Hepatology.

A Functional High-Throughput Assay of Myelination in Vitro

Science.gov (United States)

2014-07-01

Human induced pluripotent stem cells, hydrogels, 3D culture, electrophysiology, high-throughput assay 16. SECURITY CLASSIFICATION OF: 17...image the 3D rat dorsal root ganglion ( DRG ) cultures with sufficiently low background as to detect electrically-evoked depolarization events, as...of voltage-sensitive dyes. 8    We have made substantial progress in Task 4.1. We have fabricated neural fiber tracts from DRG explants and

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Science.gov (United States)

Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

2015-12-01

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

A study of the 51Cr release assay system in micro-method

International Nuclear Information System (INIS)

Kiya, Katsuzo; Harada, Kiyoshi; Uozumi, Tohru; Toge, Tetsuya; Hattori, Takao.

1981-01-01

Conditions of 51 Cr release assay in microculture were investigated to measure the natural cytotoxic (NC) activity of mouse spleen cells. Malignant glioma (MG) cells of C57BL/6 mouse, induced by 20-methylcholanthrene, were labeled with Na 2 51 CrO 4 . Spleen cells collected from the same mouse strain were suspended in Eagle's MEM. Labeled MG cells and spleen cells were incubated for several hours in a CO 2 incubator. Then the activity of the supernatant was measured by an automatic gamma counter. The optimum conditions of 51 Cr release assay in micro-culture were, (1) number of the target cells: 5 x 10 3 / well (2) FCS concentration: 10% (3) E/T ratio: less than 100 : 1, 50 : 1 was possible (4) incubation time: 15 hours. The number of the target cells at labeling incubation was set to 2 x 10 6 /ml. Though the natural release of 51 Cr was not effected by the viability of the target cells, it was suggested that the NC activity was dependent on the viability of both cells, target and effector cells. (Tsunoda, M.)

Study of the /sup 51/Cr release assay system in micro-method

Energy Technology Data Exchange (ETDEWEB)

Kiya, K.; Harada, K.; Uozumi, T. (Hiroshima Univ. (Japan). School of Medicine); Toge, T.; Hattori, T.

1981-05-01

Conditions of /sup 51/Cr release assay in microculture were investigated to measure the natural cytotoxic (NC) activity of mouse spleen cells. Malignant glioma (MG) cells of C57BL/6 mouse, induced by 20-methylcholanthrene, were labeled with Na/sub 2//sup 51/CrO/sub 4/. Spleen cells collected from the same mouse strain were suspended in Eagle's MEM. Labeled MG cells and spleen cells were incubated for several hours in a CO/sub 2/ incubator. Then the activity of the supernatant was measured by an automatic gamma counter. The optimum conditions of /sup 51/Cr release assay in micro-culture were, (1) number of the target cells: 5 x 10/sup 3// well (2) FCS concentration: 10% (3) E/T ratio: less than 100 : 1, 50 : 1 was possible (4) incubation time: 15 hours. The number of the target cells at labeling incubation was set to 2 x 10/sup 6//ml. Though the natural release of /sup 51/Cr was not effected by the viability of the target cells, it was suggested that the NC activity was dependent on the viability of both cells, target and effector cells.

Ebselen alters mitochondrial physiology and reduces viability of rat hippocampal astrocytes.

Science.gov (United States)

Santofimia-Castaño, Patricia; Salido, Ginés M; González, Antonio

2013-04-01

The seleno-organic compound and radical scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) have been extensively employed as an anti-inflammatory and neuroprotective compound. However, its glutathione peroxidase activity at the expense of cellular thiols groups could underlie certain deleterious actions of the compound on cell physiology. In this study, we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular viability, the intracellular free-Ca(2+) concentration ([Ca(2+)]c), the mitochondrial free-Ca(2+) concentration ([Ca(2+)]m), and mitochondrial membrane potential (ψm) were analyzed. The caspase-3 activity was also assayed. Our results show that cell viability was reduced by treatment of cells with ebselen, depending on the concentration employed. In the presence of ebselen, we observed an initial transient increase in [Ca(2+)]c that was then followed by a progressive increase to an elevated plateau. We also observed a transient increase in [Ca(2+)]m in the presence of ebselen that returned toward a value over the prestimulation level. The compound induced depolarization of ψm and altered the permeability of the mitochondrial membrane. Additionally, a disruption of the mitochondrial network was observed. Finally, we did not detect changes in caspase-3 activation in response to ebselen treatment. Collectively, these data support the likelihood of ebselen, depending on the concentration employed, reduces viability of rat hippocampal astrocytes via its action on the mitochondrial activity. These may be early effects that do not involve caspase-3 activation. We conclude that, depending on the concentration used, ebselen might exert deleterious actions on astrocyte physiology that could compromise cell function.

Effects of corticosteroid on the expressions of neuropeptide and cytokine mRNA and on tenocyte viability in lateral epicondylitis

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Han Soo

2012-10-01

Full Text Available Abstract Background The purpose of this study was to determine the reaction mechanism of corticosteroid by analyzing the expression patterns of neuropeptides (substance P (SP, calcitonin gene related peptide (CGRP and of cytokines (interleukin (IL-1α, tumor growth factor (TGF-β after corticosteroid treatment in lateral epicondylitis. In addition, we also investigated whether corticosteroid influenced tenocyte viability. Methods The corticosteroid triamcinolone acetonide (TAA was applied to cultured tenocytes of lateral epicondylitis, and the changes in the mRNA expressions of neuropeptides and cytokines and tenocyte viabilities were analyzed at seven time points. Quantitative real-time polymerase chain reaction and an MTT assay were used. Results The expression of SP mRNA was maximally inhibited by TAA at 24 hours but recovered at 72 hours, and the expressions of CGRP mRNA and IL-1α mRNA were inhibited at 24 and 3 hours, respectively. The expression of TGF-β mRNA was not significant. Tenocyte viability was significantly reduced by TAA at 24 hours. Conclusions We postulate that the reaction mechanism predominantly responsible for symptomatic relief after a corticosteroid injection involves the inhibitions of neuropeptides and cytokines, such as, CGRP and IL-1α. However the tenocyte viability was compromised by a corticosteroid.

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

Science.gov (United States)

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

2017-12-01

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

Impact of age and diagnosis on viability during centrifugation and cryopreservation of peripheral blood stem cell products.

Science.gov (United States)

Civriz Bozdag, S; Bay, M; Ayyıldız, E; Topcuoglu, P; Ilhan, O

2012-08-01

The viability of the hematopoietic stem cells infused to the patient is important for transplant outcome. We evaluated 31 peripheral blood stem cell product collected from 15 patients. We aimed to check the viabilities of the cells from patients with different age and diagnosis, in different stages of the cryopreservation procedure. We showed a markedly decreased viability rate after centrifugation and addition of DMSO. Percentages of viabilities were similar between young and old patients in each step. Type of hematological malignancy did not make a significant influence on the viability. High speed centrifugation has a negative impact on the viability. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation.

Science.gov (United States)

Zhu, Hong; Jia, Zhenquan; Trush, Michael A; Li, Y Robert

2016-05-01

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H 2 O 2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H 2 O 2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H 2 O 2 formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H 2 O 2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H 2 O 2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H 2 O 2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

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...

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

DEFF Research Database (Denmark)

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

2009-01-01

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

A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability.

Science.gov (United States)

Larsson, Marie C; Lerm, Maria; Ängeby, Kristian; Nordvall, Michaela; Juréen, Pontus; Schön, Thomas

2014-11-01

The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and γ-irradiation on viability of neuroblastoma cells

International Nuclear Information System (INIS)

Roessler, Jochen; Zambrzycka, Izabella; Lagodny, Jeanette; Kontny, Udo; Niemeyer, Charlotte Marie

2006-01-01

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-α and -β-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and γ-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 μM STI-571 for 72 h, two cell lines responding already to 10 μM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-α and -β. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and γ-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed γ-irradiation therapy

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.

Nuclear Power Options Viability Study. Volume 4. Bibliography

Energy Technology Data Exchange (ETDEWEB)

Trauger, D B; White, J D; Sims, J W [eds.

1986-09-01

Documents in the Nuclear Power Options Viability Study (NPOVS) bibliography are classified under one of four headings or categories as follows: nuclear options; light water reactors; liquid metal reactors; and high temperature reactors. The collection and selection of these documents, beginning early in 1984 and continuing through March of 1986, was carried out in support of the study's objective: to explore the viabilities of several nuclear electric power generation options for commercial deployment in the United States between 2000 and 2010. There are approximately 550 articles, papers, reports, and books in the bibliography that have been selected from some 2000 surveyed. The citations have been made computer accessible to facilitate rapid on-line retrieval by keyword, author, corporate author, title, journal name, or document number.

Nuclear Power Options Viability Study. Volume 4. Bibliography

International Nuclear Information System (INIS)

Trauger, D.B.; White, J.D.; Sims, J.W.

1986-09-01

Documents in the Nuclear Power Options Viability Study (NPOVS) bibliography are classified under one of four headings or categories as follows: nuclear options; light water reactors; liquid metal reactors; and high temperature reactors. The collection and selection of these documents, beginning early in 1984 and continuing through March of 1986, was carried out in support of the study's objective: to explore the viabilities of several nuclear electric power generation options for commercial deployment in the United States between 2000 and 2010. There are approximately 550 articles, papers, reports, and books in the bibliography that have been selected from some 2000 surveyed. The citations have been made computer accessible to facilitate rapid on-line retrieval by keyword, author, corporate author, title, journal name, or document number

A high-throughput assay of NK cell activity in whole blood and its clinical application

International Nuclear Information System (INIS)

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

2014-01-01

Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as 51 Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer

A high-throughput assay of NK cell activity in whole blood and its clinical application

Energy Technology Data Exchange (ETDEWEB)

Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: kylee117@yuhs.ac [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: jkim63@yuhs.ac [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)

2014-03-14

Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2',7'-dichlorofluorescin diacetate (DCFDA) assay.

Science.gov (United States)

Figueroa, Daniela; Asaduzzaman, Mohammad; Young, Fiona

2018-04-07

The detection of reactive oxygen species (ROS) using 2',7'-dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 μM) for 45 min, and then exposed to TBHP (0-50 μM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 μM DCFDA with 25 μM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology. Published by Elsevier Inc.

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

Non-destructive technique for determining the viability of soybean (Glycine max) seeds using FT-NIR spectroscopy.

Science.gov (United States)

Kusumaningrum, Dewi; Lee, Hoonsoo; Lohumi, Santosh; Mo, Changyeun; Kim, Moon S; Cho, Byoung-Kwan

2018-03-01

The viability of seeds is important for determining their quality. A high-quality seed is one that has a high capability of germination that is necessary to ensure high productivity. Hence, developing technology for the detection of seed viability is a high priority in agriculture. Fourier transform near-infrared (FT-NIR) spectroscopy is one of the most popular devices among other vibrational spectroscopies. This study aims to use FT-NIR spectroscopy to determine the viability of soybean seeds. Viable and artificial ageing seeds as non-viable soybeans were used in this research. The FT-NIR spectra of soybean seeds were collected and analysed using a partial least-squares discriminant analysis (PLS-DA) to classify viable and non-viable soybean seeds. Moreover, the variable importance in projection (VIP) method for variable selection combined with the PLS-DA was employed. The most effective wavelengths were selected by the VIP method, which selected 146 optimal variables from the full set of 1557 variables. The results demonstrated that the FT-NIR spectral analysis with the PLS-DA method that uses all variables or the selected variables showed good performance based on the high value of prediction accuracy for soybean viability with an accuracy close to 100%. Hence, FT-NIR techniques with a chemometric analysis have the potential for rapidly measuring soybean seed viability. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Toxicological Effects of Organophosphate Pesticide on Ceolomocytes Viability of Earthworm E. Foetida Using NRRA

Directory of Open Access Journals (Sweden)

Sameena Farrukh

2015-03-01

Full Text Available Background: In recent years, there has been a growing interest in the development of sub-lethal earthworm biomarkers as they are relevant indicators of environmental change and they are among the five key indicators for ecotoxicological testing of industrial chemicals determined by the OECD. In the present study, the effects of an organophosphate pesticide dichlorovos on lysosomes of coelomocytes of earthworm E. foetida are studied using Neutral Red Retention Assay (NRRA. Methods: Earthworms were exposed to three sub-lethal concentrations of the pesticide for 7, 14, 21, and 28 days and neutral red retention assay was done following the method employed by Weeks and Sevendsen and Booth et al. Results: It was observed that the pesticide significantly affected the coelomocyte viability within 28 days of exposure. The neutral red retention time of lysosomal membrane significantly decreased at all concentrations when compared with well-matched controls. Conclusion: After the analysis of results, it was concluded that the neutral red retention time assay in earthworms can be used to link changes in the permeability of lysosomal membranes to ecologically relevant life cycle effects caused by such toxic substances.

Effect of salt hyperosmotic stress on yeast cell viability

Directory of Open Access Journals (Sweden)

Logothetis Stelios

2007-01-01

Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

Microculture virus titration--a simple colourimetric assay for influenza virus titration.

Science.gov (United States)

Levi, R; Beeor-Tzahar, T; Arnon, R

1995-03-01

Influenza antigens can be detected by several well established methods. However, when it is important to determine the titre of infective virions, a bioassay should be employed. The standard and the most widely used tests for influenza infectivity are titration carried out in embryonated hen eggs, or the plaque assay employing tissue culture techniques. A simple colourimetric assay for influenza virus detection and titration is described. Samples of allantoic fluid or mice lung homogenates were used to infect MDCK cultures in microplate wells. After an incubation period, the tetrazolium (MTT) colourimetric assay was used to determine cell viability, and when compared to untreated culture control enabled the detection and titration of several influenza strains. When samples were assayed simultaneously in embryonated eggs and by the MCVT method, good correlation in determined titres was obtained. The availability of an additional method for influenza titration allows more flexibility in the choice of titration method according to the specific needs of the study. Furthermore, this method lends itself to full automatization. Similar procedures should also be applicable to titration of other cytopathic viruses.

SmATG7 is required for viability in the homothallic ascomycete Sordaria macrospora.

Science.gov (United States)

Nolting, Nicole; Bernhards, Yasmine; Pöggeler, Stefanie

2009-08-01

In filamentous ascomycetes, autophagy is involved in several developmental processes. Nevertheless, until now little is known about its role in fruiting-body development. We therefore isolated a gene of the homothallic ascomycete Sordaria macrospora with high sequence similarity to the Saccharomyces cerevisiae autophagy-related gene ATG7, encoding a core autophagy regulator. This is the first characterization of an ATG7 homolog in filamentous ascomycetes. A S. cerevisiae complementation assay demonstrated that the S. macrospora Smatg7 gene functionally replaces the yeast homolog. We were not able to generate a homokaryotic knock-out mutant in S. macrospora, suggesting that Smatg7 is required for viability. However, a heterokaryotic DeltaSmatg7/Smatg7 strain and transformants generated by RNA interference showed considerable morphological phenotypes during fruiting-body development. Using real-time PCR, we demonstrated that in the wild type, the transcriptional expression of Smatg7 is markedly up-regulated under amino acid starvation conditions and at late stages during sexual development. Moreover, we showed that transcriptionally down-regulation of Smatg7 disturbs autophagy in S. macrospora.

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%.

Algae viability over time in a ballast water sample

Science.gov (United States)

Gollasch, Stephan; David, Matej

2018-03-01

The biology of vessels' ballast water needs to be analysed for several reasons, one of these being performance tests of ballast water management systems. This analysis includes a viability assessment of phytoplankton. To overcome logistical problems to get algae sample processing gear on board of a vessel to document algae viability, samples may be transported to land-based laboratories. Concerns were raised how the storage conditions of the sample may impact algae viability over time and what the most appropriate storage conditions were. Here we answer these questions with a long-term algae viability study with daily sample analysis using Pulse-Amplitude Modulated (PAM) fluorometry. The sample was analysed over 79 days. We tested different storage conditions: fridge and room temperature with and without light. It seems that during the first two weeks of the experiment the viability remains almost unchanged with a slight downwards trend. In the continuing period, before the sample was split, a slightly stronger downwards viability trend was observed, which occurred at a similar rate towards the end of the experiment. After the sample was split, the strongest viability reduction was measured for the sample stored without light at room temperature. We concluded that the storage conditions, especially regarding temperature and light exposure, have a stronger impact on algae viability compared to the storage duration and that inappropriate storage conditions reduce algal viability. A sample storage time of up to two weeks in a dark and cool environment has little influence on the organism viability. This indicates that a two week time duration between sample taking on board a vessel and the viability measurement in a land-based laboratory may not be very critical.

Determination of Nitrate Reductase Assay Depending on the Microbial Growth

International Nuclear Information System (INIS)

El-Kabbany, H.M.

2012-01-01

A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

Stereospecific high-performance liquid chromatographic assay of sotalol in plasma.

Science.gov (United States)

Carr, R A; Foster, R T; Bhanji, N H

1991-09-01

A convenient high-performance liquid chromatographic (HPLC) assay was developed for determination of sotalol (STL) enantiomers in plasma. Following addition of the internal standard (IS; racemic atenolol), enantiomers of STL and IS were extracted using ethyl acetate. After evaporation of the organic layer, samples were derivatized with a solution of S-(+)-1-(1-naphthyl)ethyl isocyanate (NEIC). The resulting diastereomers were chromatographed with normal-phase HPLC with chloroform:hexane:methanol [65:33:2 (v/v)] as the mobile phase at a flow rate of 2 ml/min. The fluorescence detection wavelength was set at 220 nm for excitation with no emission filter. The suitability of the assay for pharmacokinetic studies was determined by measuring STL enantiomers in the plasma of a healthy subject after administration of a single 160-mg oral, racemic dose of STL.

Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.

Science.gov (United States)

Létant, Sonia E; Murphy, Gloria A; Alfaro, Teneile M; Avila, Julie R; Kane, Staci R; Raber, Ellen; Bunt, Thomas M; Shah, Sanjiv R

2011-09-01

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

Effect of Sodium Dodecyl Sulfate (SDS) and Tween 80 on Cell Viability in an Air-Cathode Microbial Fuel Cell

KAUST Repository

Fregoso, Luisa

2011-07-01

Microbial fuel cells (MFCs) generate current via electrochemical reactions produced by bacteria attached to the anode that oxidize organic matter. Due to their high volume use in household products, some concentration of surfactant will reach wastewater treatment plants. The average surfactant concentration in wastewater ranges from 10 to 20 mg L-1, and up to 300 mg L-1, for domestic and industrial wastewaters, respectively. This study aimed to demonstrate the feasibility of enhancing power production by adding Tween 80 and SDS surfactants to air-cathode MFCs, and their effect in cell viability at the anodic biofilm. In order to analyze the effect of anionic and nonionic surfactants in MFCs performance, eight MFCs were spiked with two types of surfactants, the anionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant Tween® 80 at two different concentrations 10 and 100 mg L-1. Cell viability at the anodic biofilms was examined using the LIVE/DEAD BacLight viability assay and images were visualized with a confocal laser scanning microscope. The electrochemical results demonstrate that, for an air-cathode MFC operating on 1 g L-1 acetate in a fed-batch mode, reactors where SDS was added show a lower overall performance, maximum PD of 544 mW m-2, CE of 12.3%, Rint of 322 Ω (10 mg L-1) and maximum PD of 265 mW m-2, CE of 9.4%, Rint of 758 Ω (100 mg L-1). Reactors where Tween 80 was added show quite stable performance, maximum PD of 623 mW m-2, CE of 15.4%, Rint of 216 Ω (10 mg L-1) and maximum PD of 591 mW m-2, CE of 10.8%, Rint of 279 Ω (100 mg L-1), compared with reactors operating at only acetate as a substrate, maximum PD of 574 mW m-2. Confocal microscopy images confirm this observation and biofilm viability appeared severely compromised in SDS reactors, especially at high concentrations. This study has opened up a whole new research area in determining which types of surfactants are toxic to the anodic biofilm and to further investigate the

Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.

Science.gov (United States)

Radrizzani, Marina; Soncin, Sabrina; Lo Cicero, Viviana; Andriolo, Gabriella; Bolis, Sara; Turchetto, Lucia

2016-01-01

Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 .

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

Measurement and Analysis Plan for Investigation of Spent-Fuel Assay Using Lead Slowing-Down Spectroscopy

International Nuclear Information System (INIS)

Smith, Leon E.; Haas, Derek A.; Gavron, Victor A.; Imel, G.R.; Ressler, Jennifer J.; Bowyer, Sonya M.; Danon, Y.; Beller, D.

2009-01-01

Under funding from the Department of Energy Office of Nuclear Energy's Materials, Protection, Accounting, and Control for Transmutation (MPACT) program (formerly the Advanced Fuel Cycle Initiative Safeguards Campaign), Pacific Northwest National Laboratory (PNNL) and Los Alamos National Laboratory (LANL) are collaborating to study the viability of lead slowing-down spectroscopy (LSDS) for spent-fuel assay. Based on the results of previous simulation studies conducted by PNNL and LANL to estimate potential LSDS performance, a more comprehensive study of LSDS viability has been defined. That study includes benchmarking measurements, development and testing of key enabling instrumentation, and continued study of time-spectra analysis methods. This report satisfies the requirements for a PNNL/LANL deliverable that describes the objectives, plans and contributing organizations for a comprehensive three-year study of LSDS for spent-fuel assay. This deliverable was generated largely during the LSDS workshop held on August 25-26, 2009 at Rensselaer Polytechnic Institute (RPI). The workshop itself was a prominent milestone in the FY09 MPACT project and is also described within this report.

A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

Directory of Open Access Journals (Sweden)

Gentry-Nielsen Martha J

2006-07-01

Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

Correlation of bacterial viability with uptake of (14C) acetate into phenolic glycolipid-1 of Mycobacterium leprae within Schwannoma cells

International Nuclear Information System (INIS)

Mistry, Y.; Antia, N.H.; Mukherjee, R.

1989-01-01

The viability of Mycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of ( 14 C) acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays, viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellular Mycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellular Mycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, as Mycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host's protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a unique Mycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets. (author). 19 refs., 5 tabs

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices.

Science.gov (United States)

Pearson, Brooke; Mills, Alexander; Tucker, Madeline; Gao, Siyue; McLandsborough, Lynne; He, Lili

2018-06-01

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay

DEFF Research Database (Denmark)

Harndahl, Mikkel; Rasmussen, Michael; Røder, Gustav Andreas

2011-01-01

and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.......Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely...... timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal...

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

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...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

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....

LIVE/DEAD YEAST VIABILITY STAINING AS A TOOL FOR IMPROVING ARTISANAL PILSNER BEER PRODUCTION

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Benedetta Bottari

2014-10-01

Full Text Available The production of an artisanal beer, made by brewers using traditional practices on a small scale, is founded on the empirical adjustment of parameters, including yeasts handling and serial repitching. The aim of this study was to monitor yeast viability during different stages of artisanal beer productions through the Live/Dead Yeast viability staining and to correlate it with fermentation dynamics in order to increase process standardization and to maintain the quality of final products. Yeast viability and fermentation activities were evaluated during seven fermentation cycles of an artisanal pilsner beer. Yeast inoculated with higher viability performed generally better in fermentation, resulting in faster sugar consumption, faster ethanol production and stability. Handling yeast and serial repitching based on Live/Dead viability measurements, could be the key way to ensure reliable manufacture of high quality beer and to improve process standardization particularly for microbreweries, where variability of production can be a challenging point.

Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

International Nuclear Information System (INIS)

Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

1982-01-01

We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.

Science.gov (United States)

Raghavan, Shreya; Ward, Maria R; Rowley, Katelyn R; Wold, Rachel M; Takayama, Shuichi; Buckanovich, Ronald J; Mehta, Geeta

2015-07-01

Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. Copyright © 2015 Elsevier Inc. All rights reserved.

A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II

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Kanchanmala Deshpande

2010-06-01

Full Text Available 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% CVin inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II was measurable as low as 1 pg.mL−1 within15 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%.

Heterozygous effects of irradiated chromosomes on viability in Drosophila melanogaster

International Nuclear Information System (INIS)

Simmons, M.J.

1976-01-01

Two large experiments were conducted in order to evaluate the heterozygous effects of irradiated chromosomes on viability. Mutations were accumulated on several hundred second chromosomes by delivering doses of 2,500R over either two or four generations for total x-ray exposures of 5,000R or 10,000R. Chromosomes treated with 5,000R were screened for lethals after the first treatment, and surviving nonlethals were used to generate families of fully treated chromosomes. The members of these families shared the effects of the first irradiation, but differed with respect to those of the second. The chromosomes treated with 10,000R were not grouped into families since mutations were accumulated independently on each chromosome in that experiment. Heterozygous effects on viability of the irradiated chromosomes were tested in both isogenic (homozygous) and nonisogenic (heterozygous) genetic backgrounds. In conjunction with these tests, homozygous viabilities were determined by the marked-inversion technique. This permitted a separation of the irradiated chromosomes into those which were drastic when made homozygous and those which were not. The results indicate that drastic chromosomes have deleterious effects in heterozygous condition, since viability was reduced by 2 to 4 percent in tests performed with the 10,000R chromosomes, and by 1 percent in those involving the 5,000R material. Within a series of tests, the effects were more pronounced when the genetic background was homozygous. These results suggest that the mutants induced by high doses of x-rays are principally drastic ones which show deleterious effects on viability in heterozygous condition

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

Science.gov (United States)

Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

2009-05-15

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, pautomated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

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...

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...

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.

Membrane damage and viability loss of thermally treated and high hydrostatic pressurized E. coli 0157:H7 and Salmonella spp. in apple juice

Science.gov (United States)

Differences in membrane damage including leakage of intracellular UV-materials and loss of viability of Salmonella spp. and Escherichia coli O157:H7 bacteria in apple juice following thermal death time disk (TDT) and high hydrostatic pressure treatments were investigated. Salmonella and E. coli O157...

Seed viability of five wild Saudi Arabian species by germination and X-ray tests.

Science.gov (United States)

Al-Hammad, B A; Al-Ammari, B S

2017-09-01

Our objective was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of five wild species ( Moringa peregrina , Abrus precatorius , Arthrocnemum macrostachyum , Acacia ehrenbergiana and Acacia tortilis ) from Saudi Arabia. Usually several days were required to determine the viability of all five species via germination tests. However, X-ray test will give immediate results on filled/viable seeds. Seeds of all species, except Acacia ehrenbergiana and Acacia tortilis showed high viability in both germination (96-72% at 25/15 °C, 94-70% at 35/25 °C) and X-ray (100-80%) test. Furthermore, there was a general agreement between the germination (19%, 14% at 25/15 °C and 17% and 12% at 35/25 °C) and X-ray (8%, 4%) tests in which seed viability of Acacia ehrenbergiana and Acacia tortilis was very low due to insect damaged embryo as shown in X-ray analysis. Seeds of Abruspreca torius have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (83% at 25/15 °C and 81% at 35/25 °C) and X-ray (96%) tests. Most of the nongerminated seeds of the five species except those of Acacia ehrenbergiana and Acacia tortilis , were alive as judged by the tetrazolium test (TZ). Thus, for the five species examined, the X-ray test was proved to be a good and rapid predictor of seed viability.

Effects of Antiseptic Solutions Commonly Used in Dentistry on Bone Viability, Bone Morphology, and Release of Growth Factors.

Science.gov (United States)

Sawada, Kosaku; Fujioka-Kobayashi, Masako; Kobayashi, Eizaburo; Schaller, Benoit; Miron, Richard J

2016-02-01

Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-β1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-β1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1β protein release, although no change was observed for BMP2. The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of

Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells

DEFF Research Database (Denmark)

Zor, Kinga; Mark, Christina; Heiskanen, Arto

2017-01-01

at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same...

Edaravone enhances the viability of ischemia/reperfusion flaps.

Science.gov (United States)

Zhang, Dong-Yi; Kang, Shen-Song; Zhang, Zheng-Wen; Wu, Rui

2017-02-01

The purpose of the experiment was to study the efficacy of edaravone in enhancing flap viability after ischemia/reperfusion (IR) and its mechanism. Forty-eight adult male SD rats were randomly divided into 3 groups: control group (n=16), IR group (n=16), and edaravone-treated IR group (n=16). An island flap at left lower abdomen (6.0 cm×3.0 cm in size), fed by the superficial epigastric artery and vein, was created in each rat of all the three groups. The arterial blood flow of flaps in IR group and edaravone-treated IR group was blocked for 10 h, and then the blood perfusion was restored. From 15 min before reperfusion, rats in the edaravone-treated IR group were intraperitoneally injected with edaravone (10 mg/kg), once every 12 h, for 3 days. Rats in the IR group and control group were intraperitoneally injected with saline, with the same method and frequency as the rats in the edaravone-treated IR group. In IR group and edaravone-treated IR group, samples of flaps were harvested after reperfusion of the flaps for 24 h. In the control group, samples of flaps were harvested 34 h after creation of the flaps. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were determined, and changes in organizational structure and infiltration of inflammatory cells were observed by hematoxylin-eosin (HE) staining, apoptotic cells of vascular wall were marked by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the apoptotic rate of cells in vascular wall was calculated. The ultrastructural changes of vascular endothelial cells were observed by transmission electron microscopy (TEM). Seven days after the operation, we calculated the flap viability of each group, and marked vessels of flaps by immunohistochemical staining for calculating the average number of subcutaneous vessels. The results showed that the content of MDA, the number of multicore inflammatory cells and apoptotic rate of cells in vascular wall

Pollen viability and its effect on fruit set of oil palm (Elaeis guineensis Jacq.

Directory of Open Access Journals (Sweden)

ALFIN WIDIASTUTI

2008-01-01

Full Text Available The research was aimed at studying (1 the decline of pollen viability during storage, and (2 the effect of pollen amount on fruit set of oil palm (Elaeis guineensis Jacq.. The experiment was conducted at PT. Dami Mas Sejahtera and PT. Sinar Mas Agro Resource and Technology (SMART Tbk, Riau from February to August 2004. The first experiment was investigated up to six months storage period in the refrigerator, whereas in the second experiment a randomized complete block design with two factors was used: length of storage, i.e. 0, 1 and 2 months and amount of pollen, i.e. 0.022, 0.044, 0.066, 0.088, and 0.11 g mixed with powder to 10g to pollinate an inflorescence. The result showed that the viability of pollen started to decline three months after storage from about 92% to 83%, and declined to about 75% after six months of storage. Result of the second experiment showed that storage of pollen up to two months did not affect percentage of normal fruit, although the percentage of parthenocarpic fruits was decreased. This could be due to the high viability of pollen as the viability was remained high (about 90% after being stored for two months in the refrigerator. Pollen with high viability could be used in a smaller amount to pollinate a female inflorescence without affecting fruit set of about 70-76%.SD037 had a higher reproductive success than SD038 and SD39.

Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

Directory of Open Access Journals (Sweden)

Ana Amélia Rodrigues

2013-02-01

Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

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.

Sewage sludge pasteurization by gamma radiation: financial viability case studies

International Nuclear Information System (INIS)

Swinwood, J.F.; Kotler, J.

1990-01-01

This paper examines the financial viability of sewage sludge pasteurization by gamma radiation, by examining the following three North American scenarios: 1. Small volume sewage treatment plant experiencing high sludge disposal costs; 2. Large volume sewage treatment plant experiencing low sludge disposal costs; 3. Large volume sewage treatment plant experiencing high sludge disposal costs. (author)

Sewage sludge pasteurization by gamma radiation: financial viability case studies

Energy Technology Data Exchange (ETDEWEB)

Swinwood, J.F.; Kotler, J. (Nordion International Inc., Kanata, Ontario (Canada))

1990-01-01

This paper examines the financial viability of sewage sludge pasteurization by gamma radiation, by examining the following three North American scenarios: 1. Small volume sewage treatment plant experiencing high sludge disposal costs; 2. Large volume sewage treatment plant experiencing low sludge disposal costs; 3. Large volume sewage treatment plant experiencing high sludge disposal costs. (author).

Sewage sludge pasteurization by gamma radiation: Financial viability case studies

Science.gov (United States)

Swinwood, Jean F.; Kotler, Jiri

This paper examines the financial viability of sewage sludge pasteurization by gamma radiation, by examining the following three North American scenarios: 1) Small volume sewage treatment plant experiencing high sludge disposal costs. 2) Large volume sewage treatment plant experiencing low sludge disposal costs. 3) Large volume sewage treatment plant experiencing high sludge disposal costs.

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

International Nuclear Information System (INIS)

Arteaga-Cardona, Fernando; Gutiérrez-García, Eric; Hidalgo-Tobón, Silvia; López-Vasquez, Ciro; Brito-Barrera, Yazmín A.; Flores-Tochihuitl, Julia; Angulo-Molina, Aracely; Reyes-Leyva, Julio R.; González-Rodríguez, Roberto; Coffer, Jeffery L.; Pal, Umapada

2016-01-01

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_2, were measured on a 1.5 T clinical MRI instrument. Values in the range from 205 to 257 mM"−"1 s"−"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.

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.

A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

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Jennifer R. McCall

2014-09-01

Full Text Available Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/ veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

Science.gov (United States)

Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

2015-04-01

The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

High throughput comet assay to study genotoxicity of nanomaterials

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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

Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system

Science.gov (United States)

Nie, Jianhui; Wu, Xiaohong; Ma, Jian; Cao, Shouchun; Huang, Weijin; Liu, Qiang; Li, Xuguang; Li, Yuhua; Wang, Youchun

2017-01-01

Pseudoviruses are useful virological tools because of their safety and versatility; however the low titer of these viruses substantially limits their wider applications. We developed a highly efficient pseudovirus production system capable of yielding 100 times more rabies pseudovirus than the traditional method. Employing the high-titer pseudoviruses, we have developed robust in vitro and in vivo neutralization assays for the evaluation of rabies vaccine, which traditionally relies on live-virus based assays. Compared with current rapid fluorescent focus inhibition test (RFFIT), our in vitro pseudovirus-based neutralization assay (PBNA) is much less labor-intensive while demonstrating better reproducibility. Moreover, the in vivo PBNA assay was also found to be superior to the live virus based assay. Following intravenous administration, the pseudovirus effectively infected the mice, with dynamic viral distributions being sequentially observed in spleen, liver and brain. Furthermore, data from in vivo PBNA showed great agreement with those generated from the live virus model but with the experimental time significantly reduced from 2 weeks to 3 days. Taken together, the effective pseudovirus production system facilitated the development of novel PBNA assays which could replace live virus-based traditional assays due to its safety, rapidity, reproducibility and high throughput capacity. PMID:28218278

Viability and growth characteristics of Lactobacillus in soymilk supplemented with B-vitamins.

Science.gov (United States)

Ewe, Joo-Ann; Wan-Abdullah, Wan-Nadiah; Liong, Min-Tze

2010-02-01

Ten strains of Lactobacillus were evaluated for their viability in soymilk. Lactobacillus acidophilus ATCC 314, L. acidophilus FTDC 8833, L. acidophilus FTDC 8633 and L. gasseri FTDC 8131 displayed higher viability in soymilk and were thus selected to be evaluated for viability and growth characteristics in soymilk supplemented with B-vitamins. Pour plate analyses showed that the supplementation of all B-vitamins studied promoted the growth of lactobacilli to a viable count exceeding 7 log CFU/ml. alpha-Galactosidase specific activity of lactobacilli as determined spectrophotometrically showed an increase upon supplementation of B-vitamins. High-performance liquid chromatography analyses revealed that this led to increased hydrolysis of soy oligosaccharides and subsequently higher utilization of simple sugars. Production of organic acids as determined via high-performance liquid chromatography also showed an increase, accompanied by a decrease in pH of soymilk. Additionally, the supplementation of B-vitamins also promoted the synthesis of riboflavin and folic acid by lactobacilli in soymilk. Our results indicated that B-vitamin-supplemented soymilk is a good proliferation medium for strains of lactobacilli.

An easy 'one tube' method to estimate viability of Cryptosporidium oocysts using real-time qPCR

NARCIS (Netherlands)

Paziewska-Harris, A.; Schoone, G.; Schallig, H. D. F. H.

2016-01-01

Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes

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.

Effects of voluntary exercise on the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups born from morphine- dependent mothers during pregnancy.

Science.gov (United States)

Haydari, Sakineh; Safari, Manouchehr; Zarbakhsh, Sam; Bandegi, Ahmad Reza; Miladi-Gorji, Hossein

2016-11-10

This study was designed to investigate whether free access to a running wheel during pregnancy in morphine-dependent mothers would influence the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups. Pregnant rats were made dependent by chronic administration of morphine in drinking water simultaneously with free access to a running wheel. Male pups are weaned at 21days of birth and their bones marrows were aspirated from the femurs and tibias and also the bone marrow stromal cells (BMSCs) cultured. MTT assay was used to determine cell viability and proliferation rate. The level of BDNF was measured in the supernant of BMSCs culture by ELISA. The sedentary morphine-dependent mothers' pups showed a significant increase in the percentage cell viability and proliferation rate and also a significant decrease in the BDNF protein levels in BMSCs. The rat pups borne from exercising the control and morphine-dependent mothers exhibited an increase in the percentage viability, proliferation rate and BDNF levels of the BMSCs. This study showed that maternal exercise during pregnancy in morphine-dependent and non-dependent mothers, with increasing of BDNF levels increased the proliferation and viability of BMSCs in the rat pups. Also, chronic administration of morphine during pregnancy was able to increase the proliferation and viability of BMSCs in the rat pups. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Seed viability of five wild Saudi Arabian species by germination and X-ray tests

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B.A. Al-Hammad

2017-09-01

Full Text Available Our objective was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of five wild species (Moringa peregrina, Abrus precatorius, Arthrocnemum macrostachyum, Acacia ehrenbergiana and Acacia tortilis from Saudi Arabia. Usually several days were required to determine the viability of all five species via germination tests. However, X-ray test will give immediate results on filled/viable seeds. Seeds of all species, except Acacia ehrenbergiana and Acacia tortilis showed high viability in both germination (96–72% at 25/15 °C, 94–70% at 35/25 °C and X-ray (100–80% test. Furthermore, there was a general agreement between the germination (19%, 14% at 25/15 °C and 17% and 12% at 35/25 °C and X-ray (8%, 4% tests in which seed viability of Acacia ehrenbergiana and Acacia tortilis was very low due to insect damaged embryo as shown in X-ray analysis. Seeds of Abruspreca torius have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min, and they exhibited high viability in both the germination (83% at 25/15 °C and 81% at 35/25 °C and X-ray (96% tests. Most of the nongerminated seeds of the five species except those of Acacia ehrenbergiana and Acacia tortilis, were alive as judged by the tetrazolium test (TZ. Thus, for the five species examined, the X-ray test was proved to be a good and rapid predictor of seed viability.

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.

Effect of Zebularine loaded MePEG-PCL nanoparticles on viability, attachment of in vitro cultured lens epithelial cells

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Si-Wei Liu

2015-01-01

Full Text Available AIM: To investigate the effect of zebularine(Zebloaded Poly(ethylene glycol-block-poly(ε-caprolactonemethyl ether(MePEG-PCLnanoparticles(NPson the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells(LECs. METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L(ZebF1 group, 100μmol/L(ZebF2 group, Zeb -loaded MePEG-PCL NPs 50μmol/L(ZebNP1 group, Zeb -loaded MePEG-PCL NPs 100μmol/L(ZebNP2 group, MePEG-PCL empty NPs(NPs groupor blank medium(group Crespectively. A tetrazolium dye assay(MTTtest and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis. RESULTS: Determined by MTT colorimetric method: Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner(PPP ZebNP1>ZebF2(PCONCLUSION: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups, as well as enhancing the apoptosis.

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

Science.gov (United States)

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

2013-06-15

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

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

Science.gov (United States)

Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

2015-02-01

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals. © 2014 SETAC.

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

International Nuclear Information System (INIS)

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

2012-01-01

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

A high-throughput colorimetric assay for glucose detection based on glucose oxidase-catalyzed enlargement of gold nanoparticles

Science.gov (United States)

Xiong, Yanmei; Zhang, Yuyan; Rong, Pengfei; Yang, Jie; Wang, Wei; Liu, Dingbin

2015-09-01

We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose.We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose. Electronic supplementary information (ESI) available: Experimental section and additional figures. See DOI: 10.1039/c5nr03758a

A high throughput biochemical fluorometric method for measuring lipid peroxidation in HDL.

Directory of Open Access Journals (Sweden)

Theodoros Kelesidis

Full Text Available Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001 and cell-free assays (r = 0.46, p<0.001. HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01 and subendocardial viability ratio (r = -0.21, p = 0.05 and physiological parameters such as metabolic and anthropometric parameters (p<0.05. In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.

Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

Science.gov (United States)

Vahabi, Surena; Vaziri, Shahram; Torshabi, Maryam

2015-01-01

Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF) and platelet-rich fibrin (PRF) on proliferation and viability of human gingival fibroblasts (HGFs). Materials and Methods: Anitua’s PRGF and Choukran’s PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-valuesPRGF treatment induced statistically significant (PPRGF than in PRF group (PPRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF. PMID:26877740

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

Science.gov (United States)

Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

2015-01-01

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay.

Science.gov (United States)

Li, Qunyi; Rottländer, Mario; Xu, Mingkai; Christoffersen, Claus Tornby; Frederiksen, Kristen; Wang, Ming-Wei; Jensen, Henrik Sindal

2011-11-01

To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC(50) values of the positive control compound (retigabine) determined by the thallium and (86)rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC(50) values of less than 20 μmol/L and 2 demonstrated EC(50) values of less than 1 μmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels. Copyright © 2011 Elsevier Inc. All rights reserved.

Doctors' perspectives on the viability of rural practice.

Science.gov (United States)

Jones, J A; Humphreys, J S; Adena, M A

2004-01-01

Private practitioners play a vital role in meeting the health needs of rural communities. However, the prospect of operating a private practice business in rural Australia seems to be increasingly unattractive, because many communities are forced to recruit salaried or overseas-trained doctors. This study focuses on rural practices as businesses whose viability influences their attractiveness for the recruitment and retention of practitioners. The specific objectives are to ascertain which factors contribute to or threaten practice viability in rural areas, and whether they vary according to the degree of rurality or geographical remoteness. This study is based on data collected from a national study into the viability of rural general practice undertaken jointly by the Rural Doctors Association of Australia and Monash University School of Rural Health Bendigo. The Rural Remote and Metropolitan Area (RRMA) classification was used as the indicator of rurality. The study surveyed all general practitioners practising in rural or remote regions of Australia (RRMAs 3 to 7). Only practitioners with some financial interest in the practice were selected for this analysis. Free-text responses to the two questions 'What are the key factors contributing to the viability of your practice?' and 'What factors would put the viability of your practice at risk?' were analysed using qualitative content analysis. Factors were derived iteratively through higher-level aggregation of responses. Chi-square tests were used to make comparisons across the RRMA categories. The national survey achieved a response rate of 35% of the entire population of GPs practising in RRMA 3 to 7 regions. Of these, 1050 respondents were relevant to this analysis. Seven major factors were identified by practitioners as the main contributors to practice viability. 'Practice characteristics' was nominated by 59% of respondents, followed by 'Income' (31%), 'Personal circumstances', 'Workforce' and 'Community

Long-term vascular contractility assay using genipin-modified muscular thin films

International Nuclear Information System (INIS)

Hald, Eric S; Steucke, Kerianne E; Reeves, Jack A; Win, Zaw; Alford, Patrick W

2014-01-01

Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases. (paper)

Link overlap, viability, and mutual percolation in multiplex networks

International Nuclear Information System (INIS)

Min, Byungjoon; Lee, Sangchul; Lee, Kyu-Min; Goh, K.-I.

2015-01-01

Many real-world complex systems are best modeled by multiplex networks. The multiplexity has proved to have broad impact on the system’s structure and function. Most theoretical studies on multiplex networks to date, however, have largely ignored the effect of the link overlap across layers despite strong empirical evidences for its significance. In this article, we investigate the effect of the link overlap in the viability of multiplex networks, both analytically and numerically. After a short recap of the original multiplex viability study, the distinctive role of overlapping links in viability and mutual connectivity is emphasized and exploited for setting up a proper analytic framework. A rich phase diagram for viability is obtained and greatly diversified patterns of hysteretic behavior in viability are observed in the presence of link overlap. Mutual percolation with link overlap is revisited as a limit of multiplex viability problem, and the controversy between existing results is clarified. The distinctive role of overlapping links is further demonstrated by the different responses of networks under random removals of overlapping and non-overlapping links, respectively, as well as under several link-removal strategies. Our results show that the link overlap facilitates the viability and mutual percolation; at the same time, the presence of link overlap poses a challenge in analytical approaches to the problem

A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis

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Lyon, Elaine; Laver, Thomas; Yu, Ping; Jama, Mohamed; Young, Keith; Zoccoli, Michael; Marlowe, Natalia

2010-01-01

Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide “ladder” extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening. PMID:20431035

Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

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Anna Lavut

Full Text Available Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical

Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.

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Ranganathan, Raj; Lenti, Gena; Tassone, Nicholas M; Scannell, Brian J; Southern, Cathrine A; Karver, Caitlin E

2018-02-15

Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (K M  = 15 ± 2 μM), WEHD-MCA (K M  = 93 ± 19 μM), WEHDG-MCA (K M  = 21 ± 6 μM) and WEHDA-MCA (K M  = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases. Copyright © 2017 Elsevier Inc. All rights reserved.

Viability of lactobacillus acidophilus in various vaginal tablet formulations

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Fazeli M.R.

2006-07-01

Full Text Available The lactobacilli which are present in vaginal fluids play an important role in prevention of vaginosis and there are considerable interests in formulation of these friendly bacteria into suitable pharmaceutical dosage forms. Formulating these microorganisms for vaginal application is a critical issue as the products should retain viability of lactobacilli during formulation and also storage. The aim of this study was to examine the viability and release of Lactobacillus acidophilus from slow-release vaginal tablets prepared by using six different retarding polymers and from two effervescent tablets prepared by using citric or adipic acid. The Carbomer–based formulations showed high initial viablility compared to those based on HPMC-LV, HPMC-HV, Polycarbophil and SCMC polymers which showed one log decrease in viable cells. All retarding polymers in slow release formulations presented a strong bacterial release at about 2 h except Carbomer polymers which showed to be poor bacterial releasers. Although effervescent formulations produced a quick bacterial release in comparison with polymer based slow-release tablets, they were less stable in cold storage. Due to the strong chelating characteristic of citric acid, the viability was quickly lost for aqueous medium of citric acid in comparison with adipic acid based effervescent tablets.

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

Viability and vigour of ageing winter wheat grains

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Stanisław Grzesiuk

2014-01-01

Full Text Available The viability and vigour of ageing winter wheat caryopses of the cvs. Grana and Jana were tested. Viability was determined on the basis of germination capacity and rate, and vigour on the basis of the over-all activity of hydrogenases in the sprouts, exudate conductometry, analysis of sprout growth, oxygen uptake and mitochondrial protein content in the sprouts. What is called energy (or rate of germination and over-all dehydrogenase activity in embryos and sprouts and the electroconductivity of exudates were found to be very good measures of the vigour of ageing caryopses. The latter two indices of vigour should be determined at a strictly defined moment of swelling and germination. Good measures of caryopse vigour are also respiration during swelling and at the beginning of germination and mitochondrial protein content in the sprouts or seedlings. There is a high correlation between the vigour of ageing grain and its bioenergetic indices.

A viability analysis for a stock/price model

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Jerry, Chakib; Raissi, Nadia

2012-09-01

We examine the conditions for the sustainability of a stock/price system based on the use of a marine renewable resource. Instead of studying the environmental and economic interactions in terms of optimal control, we focus on the viability of the system. These viability/crisis situations are defined by a set of economic state constraints. This constraints combine a guaranteed consumption and a minimum income for fishermen. Using the mathematical concept of viability kernel, we reveal that with only economics constraints we guarantee a perennial stock/price system.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

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Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Assessment of cellular viability on calcium sulphate/hydroxyapatite injectable scaffolds

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Randa Alfotawi

2013-10-01

Full Text Available Cements for maxillofacial reconstruction of jaw defects through calcification of rotated muscle have been tested. The objective of this study was to investigate the visibility of loading of two types of commercially available cements, Cerament™ Spine Support and Cerament Bone Void Filler with mesenchymal cells and cytokines (bone morphogenetic protein to act as a biomimetic scaffolding for future clinical application. Determination of basic biocompatibility (cell viability using methyl thiazolyl tetrazolium and live/dead assay was carried out using MG-63 cells at various time points. Next, in order to inform potential subsequent in vivo experiments, a collagen tissue mimic was used for characterization of rabbit mesenchymal stromal cells using immunofluorescent cytoskeleton staining, and simultaneous and then sequential injection of Cerament Spine Support cement and cells into collagen gels. Results indicated that Cerament Spine Support was more biocompatible and that sequential injection of cement and then rabbit mesenchymal stromal cells into the tissue mimics is an optimal approach for clinical applications.

LncRNA-LET inhibits cell viability, migration and EMT while induces apoptosis by up-regulation of TIMP2 in human granulosa-like tumor cell line KGN.

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Han, Qingfang; Zhang, Wenke; Meng, Jinlai; Ma, Li; Li, Aihua

2018-04-01

Polycystic ovary syndrome (PCOS) is a common endocrine disease characterized by hyperandrogenism, irregular menses, and polycystic ovaries. Several long non-coding RNAs (lncRNAs) are aberrantly expressed in PCOS patients; however, little is known about the effects of the lncRNA-low expression in tumor (lncRNA-LET) on PCOS. We aimed to explore the effects of lncRNA-LET on human granulosa-like tumor cell line, KGN. Expression of lncRNA-LET in normal IOSE80 cells and granulosa cells was determined by qRT-PCR. KGN cell viability, apoptosis and migration were measured by trypan blue exclusion method, flow cytometry assay and wound healing assay, respectively. TGF-β1 was used to induce epithelial-mesenchymal transition (EMT) process. LncRNA-LET expression and mRNA expressions of TIMP2 and EMT-related proteins were measured by qRT-PCR. Western blot analysis was used to measure the protein expression of apoptosis-related proteins, EMT-related proteins, TIMP2, and the proteins in the Wnt/β-catenin and Notch signaling pathways. lncRNA-LET was down-regulated in KGN cells, and its overexpression inhibited cell viability and migration, and promoted apoptosis in KGN cells. Overexpression of lncRNA-LET increased the expression of E-cadherin and decreased the expressions of N-cadherin and vimentin in KGN cells. These effects of lncRNA-LET on KGN cells were reversed by TIMP2 suppression. Overexpression of TIMP2 inhibited cell viability, migration and EMT process, and increased apoptosis by activating the Wnt/β-catenin and Notch pathways. Overexpression of lncRNA-LET inhibits cell viability, migration and EMT process, and increases apoptosis in KGN cells by up-regulating the expression of TIMP2 and activating the Wnt/β-catenin and notch signaling pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

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Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

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

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Ikechukwu Okoli

2009-09-01

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.

In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes.

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Gajski, Goran; Dinter, Domagoj; Garaj-Vrhovac, Vera

2010-11-01

This study aimed to evaluate the effect of proguanil, a chemical substance used for treatment and prevention of malaria on viability and DNA integrity in human lymphocytes in vitro. Two different concentrations of proguanil obtained from the plasma concentrations were used: 130ng/ml used for prophylactic treatment and 520ng/ml used in treatment of malaria. Testing was done with and without metabolic activation. Viability of lymphocytes decreased in time and dose dependent manner. Comet assay parameters showed similar effects, indicating that some damage to DNA molecule can occur. Frequency of sister chromatid exchanges did not show significant deviation from the control samples. As for the proliferation kinetics no significant changes were noticed. Since majority of DNA damaging effect is induced after metabolic activation it is to be concluded that activity of proguanil is dependent upon the active metabolite cycloguanil and that monitoring should be conducted especially among frequent travellers. Copyright © 2010 Elsevier B.V. All rights reserved.

Automation of the dicentric chromosome assay and related assays

International Nuclear Information System (INIS)

Balajee, Adayabalam S.; Dainiak, Nicholas

2016-01-01

Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

Determination of seed viability of eight wild Saudi Arabian species by germination and X-ray tests.

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Al-Turki, Turki A; Baskin, Carol C

2017-05-01

Our purpose was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of eight wild species ( Salvia spinosa , Salvia aegyptiaca , Ochradenus baccatus , Ochradenus arabicus , Suaeda aegyptiaca , Suaeda vermiculata , Prosopisfarcta and Panicumturgidum ) from Saudi Arabia. Several days were required to determine viability of all eight species via germination tests, while immediate results on filled/viable seeds were obtained with the X-ray test. Seeds of all the species, except Sa.aegyptiaca , showed high viability in both the germination (98-70% at 25/15 °C, 93-66% at 35/25 °C) and X-ray (100-75%) test. Furthermore, there was general agreement between the germination (10% at 25/15 °C and 8% at 35/25 °C) and X-ray (5%) tests that seed viability of Sa.aegyptiaca was very low, and X-ray analysis revealed that this was due to poor embryo development. Seeds of P.farcta have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (98% at 25/15 °C and 93% at 35/25 °C) and X-ray (98%) test. Most of the nongerminated seeds of the eight species except those of Sa.aegyptiaca were alive as judged by the tetrazolium test (TZ). Thus, for the eight species examined, the X-ray test was a good and rapid predictor of seed viability.

Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

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ZHAO, BING; HU, MENGCAI

2013-01-01

Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer. PMID:24843386

Bioactive and biocompatible copper containing glass-ceramics with remarkable antibacterial properties and high cell viability designed for future in vivo trials.

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Popescu, R A; Magyari, K; Vulpoi, A; Trandafir, D L; Licarete, E; Todea, M; Ştefan, R; Voica, C; Vodnar, D C; Simon, S; Papuc, I; Baia, L

2016-07-19

In the present study our interest is focused on finding the efficiency of 60SiO2·(32 - x)CaO·8P2O5·xCuO (mol%) glass-ceramics, with 0 ≤ x ≤ 4 mol%, in terms of bioactivity, biocompatibility, antibacterial properties and cell viability in order to determine the most appropriate composition for their further use in in vivo trials. The sol-gel synthesized samples show a preponderantly amorphous structure with a few crystallization centers associated with the formation of an apatite and calcium carbonate crystalline phases. The Fourier Transform Infrared (FT-IR) spectra revealed slightly modified absorption bands due to the addition of copper oxide, while the information derived from the measurements performed by transmission electron microscopy, UV-vis and electron paramagnetic resonance spectroscopy showed the presence of ions and metallic copper species. X-Ray photoelectron spectroscopic analysis indicated the presence of copper metallic species, in a reduced amount, only on the sample surface with the highest Cu content. Regarding in vitro assessment of bioactivity, the results obtained by X-ray diffraction, FT-IR spectroscopy and scanning electron microscopy, demonstrated the formation of a calcium phosphate layer on all investigated sample surfaces. The inhibitory effect of the investigated samples was more significant on the Pseudomonas aeruginosa than the Staphylococcus aureus strain, the sample with the lowest concentration of copper oxide (0.5 mol%) being also the most efficient in both bacterial cultures. This sample also exhibits a very good bactericidal activity, for the other samples it was necessary to use a higher quantity to inhibit and kill the bacterial species. The secondary structure of adsorbed albumin presents few minor changes, indicating the biocompatibility of the glass-ceramics. The cell viability assay shows a good proliferation rate on samples with 0.5 and 1.5 mol% CuO, although all glass-ceramic samples exhibited a good in vivo

A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.

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Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

2017-03-01

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

Bacterial Viability within Dental Calculus: An Untrodden, Inquisitive Clinico-Patho- Microbiological Research.

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Gupta, Swati; Jain, P K; Kumra, Madhumani; Rehani, Shweta; Mathias, Yulia; Gupta, Ramakant; Mehendiratta, Monica; Chander, Anil

2016-07-01

Chronic inflammatory periodontal diseases i.e. gingivitis and periodontitis are one of the most common afflictions faced by human beings. Dental plaque, which is a pool of pathogenic microorganisms, remains to be current mainstay in etiopathogenesis. Dental calculus, which is a mineralized product of this plaque remains ignored and is considered merely as an ash heap of minor significance. However, the intriguing array in disease etiopathogenesis bulldozed researchers to suspect the role of calculus in disease chrysalis but still the viability of bacteria inside calculus and thus its pathogenicity remains an intricacy; the answer to which lies in the Pandora's Box. The present study was undertaken to investigate the viability of bacteria within dental calculus along with their identification. Also, to classify dental calculus on the basis of mineralization and to observe the variation of viable microflora found in dental calculus with the extent of mineralization and disease severity. A total of 60 samples were obtained, by harvesting two samples of supragingival calculus from each patient having chronic inflammatory periodontal disease. These samples were divided into two groups (Group A and Group B). Samples of Group A were kept non-irradiated and samples of Group B were exposed to UV radiation. The samples were categorized into less, moderately and highly mineralized according to the force required for crushing them. All the crushed calculus samples were then divided into three parts. These were used for dark-field microscopy, gram staining and bacterial cultures. Bacterial identification of the cultures obtained was also carried out by performing various biochemical assays. The present study revealed the presence of motile spirochaetes within the samples under dark-field microscope. Gram staining revealed presence of numerous gram positive cocci and gram negative bacilli. Bacterial cultures showed growth of variety of aerobic and capnophilic microorganisms. The

Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation

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Elena Grossini

2016-09-01

Full Text Available We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article “Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions” (Grossini et al., in press [1].

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

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Mindy I Davis

Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Endogenous Locus Reporter Assays.

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Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

2018-01-01

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Pollen Viability and Autogamy Fitness in Bauhinia forficata Link (Fabaceae

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Luana Camila Capitani

2018-05-01

Full Text Available ABSTRACT Bauhinia forficata (Fabaceae occurs in many phytophysiognomies of southern Brazil, however its ecological relevance is not well understood. The present study was developed in the Central Depression of Rio Grande do Sul and aimed to determine variations in pollen viability along flowering, ability to perform autogamy and dye efficiency for the viability test. Pollen viability was determined by colorimetry as well as the ability to perform autogamy by isolating floral buds, being evaluated in eleven matrices. Average pollen viability was 81.43%, with the highest average value obtained with the dye 2,3,5- Triphenyltetrazolium Chloride (TTC (84.11%. Safranin was not a good indicator at the tested concentration. No correlation was found between pollen viability and flowering time. The species demonstrated an inability to perform autogamy.

Radiobiological and PK assays at advance Centre for Training Research and Education in Cancer (ACTREC)

International Nuclear Information System (INIS)

Sastri, Goda Jayant; Gota, Vikram

2014-01-01

Radiobiological, pharmacokinetic and biodistribution studies are of paramount importance for drug development and more so in the development of newer radiation modulators. Radiobiological studies have now graduated from simple cell survival and viability assays to more complex molecular and imaging studies to study radiation modulation both in in-vitro and in-vivo models. Tata Memorial Centre and its research centre (ACTREC) is a premiere cancer centre in India dedicated to cancer research. The Department of Radiation Oncology treats approximately 7000 new patients in a year and is uniquely placed to do both translational radiation and clinical research in the field of drug development. The Clinical Biology Lab of the Department of Radiation Oncology at ACTREC in collaboration with other labs at ACTREC has standardized cell survival assays, DNA damage assays such as Gamma H2AX assay (by flow as well as confocal microscopy), Micronuclei assay and COMET assays using CASP software for quantification. We have also done apoptotic assays. These assays have been conducted for development newer drug formulations (for e.g liposomal radiosensitizers). We also have a strong imaging division having sophisticated microscopes (confocal and single molecule super resolution microscopes) for in-vitro optical imaging and a dedicated preclinical PET/CT/SPECT for in-vivo imaging. The clinical 3T MRI and PET/CT is being used to study the effect of hypoxia in various cancers

A method for assay of special nuclear material in high level liquid waste streams

International Nuclear Information System (INIS)

Venkata Subramani, C.R.; Swaminathan, K.; Asuvathraman, R.; Kutty, K.V.G.

2003-01-01

The assay of special nuclear material in the high level liquid waste streams assumes importance as this is the first stage in the extraction cycle and considerable losses of plutonium could occur here. This stream contains all the fission products as also the minor actinides and hence normal nuclear techniques cannot be used without prior separation of the special nuclear material. This paper presents the preliminary results carried out using wavelength dispersive x-ray fluorescence as part of the developmental efforts to assay SNM in these streams by instrumental techniques. (author)

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

Science.gov (United States)

Greer, Michael S; Zhou, Ting; Weselake, Randall J

2014-08-01

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-(14)C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Enhancement of viability of radiosensitive (PBMC and resistant (MDA-MB-231 clones in low-dose-rate cobalt-60 radiation therapy

Directory of Open Access Journals (Sweden)

Patrícia Lima Falcão

2015-06-01

Full Text Available Abstract Objective: In the present study, the authors investigated the in vitro behavior of radio-resistant breast adenocarcinoma (MDA-MB-231 cells line and radiosensitive peripheral blood mononuclear cells (PBMC, as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy. Materials and Methods: The cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed. Results: Radiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation. Conclusion: Low dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer.

Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

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Elena Serena

Full Text Available INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin and functional properties. The spontaneous contraction frequency was (0.83±0.2 Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2O(2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2O(2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

In vitro germination and viability of pollen grain of coconut accessions1

Directory of Open Access Journals (Sweden)

Catrine Regina Feitosa Moura

Full Text Available Storage as a means of maintaining the pollen viability is important for the preservation of the genetic variability, facilitates the exchange of germplasm and greatly contributes to the generation of variability obtained from artificial crosses, increasing the efficiency of breeding programs. The objective of this study was to select different culture media for the in vitro germination of pollen grain of dwarf and tall coconut accessions, as well as to determine the viability of pollen grain at room temperature conditions. For this purpose, Brazil Green Dwarf (BGD and Brazilian Tall (BRA pollen grains derived from the Coconut Active Germplasm Bank of Embrapa Coastal Tablelands Sergipe were used. To evaluate the effect of different culture media on the in vitro germination of pollen grains of anão verde do Brasil de Jiqui (AVeBrJ and gigante do Brasil Praia do Forte (GBrPF accessions, they were inoculated on to Petri dishes containing 2 ml of culture media. The pollen viability was assessed by staining with 1% acetic carmine and in vitro germination at 0, 24, 48 and 72 hours. The culture medium of Lora is suitable to assess the in vitrogermination of pollen grain of the AVeBrJ and GBrPF accessions. The pollen grain of the AVeBrJ accession showed intermediate viability (66.87% at room temperature up to 23.14 hours by in vitro germination. The pollen grain of the GBrPF accession showed high viability, above 70%, at room temperature up to 120 hours by in vitro germination.

High-pressure liquid chromatographic assay of Bay n 7133 in human serum.

OpenAIRE

Fasching, C E; Hughes, C E; Hector, R F; Peterson, L R

1984-01-01

A high-pressure liquid chromatographic method that includes a Sep-Pak (Waters Associates, Inc., Milford , Mass.) preparation of human serum was employed for the quantitative assay of Bay n 7133. Drug levels of 0.1 to 20 micrograms/ml could be detected. No interference from amphotericin B was found in the chromatographic analysis of Bay n 7133.

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.

Myocardial viability assessment using nuclear imaging

International Nuclear Information System (INIS)

Matsunari, Ichiro; Hisada, Kinichi; Taki, Junichi; Nakajima, Kenichi; Tonami, Norihisa

2003-01-01

Myocardial assessment continues to be an issue in patients with coronary artery disease and left ventricular dysfunction. Nuclear imaging has long played an important role in this field. In particular, PET imaging using 18 F-fluorodeoxyglucose is regarded as the metabolic gold standard of tissue viability, which has been supported by a wide clinical experience. Viability assessment using SPECT techniques has gained more wide-spread clinical acceptance than PET, because it is more widely available at lower cost. Moreover, technical advances in SPECT technology such as gated-SPECT further improve the diagnostic accuracy of the test. However, other imaging techniques such as dobutamine echocardiography have recently emerged as competitors to nuclear imaging. It is also important to note that they sometimes may work in a complementary fashion to nuclear imaging, indicating that an appropriate use of these techniques may significantly improve their overall accuracy. In keeping these circumstances in mind, further efforts are necessary to further improve the diagnostic performance of nuclear imaging as a reliable viability test. (author) 107 refs

Viability Assessment Volume 1

International Nuclear Information System (INIS)

1998-01-01

Since May 1996, under its draft Civilian Radioactive Waste Management Program Plan (DOE 1996), DOE has been carrying out a 5-year program of work to support the decision in 2001 by the Secretary of Energy on whether or not to recommend the site to the President. Part of this program was to address major unresolved technical issues and to complete an assessment of the viability of the Yucca Mountain site by 1998. Affirming the DOE plans, Congress directed DOE in the 1997 Energy and Water Development Appropriations Act to provide a viability assessment of the Yucca Mountain site to Congress and the President. This Viability Assessment (VA) document is the DOE report to Congress and the President. They are expected to use the VA to make an informed decision about program direction and funding. Drawing on 15 years of scientific investigation and design work at Yucca Mountain, the VA summarizes a large technical basis of field investigations, laboratory tests, models, analyses, and engineering, described in cited references. The VA identifies the major uncertainties relevant to the technical defensibility of DOE analyses and designs, the DOE approach to managing these uncertainties, and the status of work toward the site recommendation and LA. The VA also identifies DOE plans for the remaining work, and the estimated costs of completing an LA and constructing and operating a repository. The attention to uncertainties is important because DOE must evaluate how the repository will perform during the next 10,000 years or longer. Uncertainties exist because of variability in the natural (geologic and hydrologic) systems at Yucca Mountain and because of imperfect scientific understanding of the natural processes that might affect the repository system. This is Volume 1 and it covers, Introduction and Site Characteristics, includes a high-level summary of the results of the VA and some additional background information. (The overview is bound separately.) Section 1 of Volume

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...

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

KAUST Repository

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

2016-01-01

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

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...

Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.

Science.gov (United States)

Pape, B E; Cary, P L; Clay, L C; Godolphin, W

1983-01-01

Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.

Analysis of the Interactions of Botanical Extract Combinations Against the Viability of Prostate Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Lynn S. Adams

2006-01-01

Full Text Available Herbal medicines are often combinations of botanical extracts that are assumed to have additive or synergistic effects. The purpose of this investigation was to compare the effect of individual botanical extracts with combinations of extracts on prostate cell viability. We then modeled the interactions between botanical extracts in combination isobolographically. Scutellaria baicalensis, Rabdosia rubescens, Panax-pseudo ginseng, Dendranthema morifolium, Glycyrrhiza uralensis and Serenoa repens were collected, taxonomically identified and extracts prepared. Effects of the extracts on cell viability were quantitated in prostate cell lines using a luminescent ATP cell viability assay. Combinations of two botanical extracts of the four most active extracts were tested in the 22Rv1 cell line and their interactions assessed using isobolographic analysis. Each extract significantly inhibited the proliferation of prostate cell lines in a time- and dose-dependent manner except repens. The most active extracts, baicalensis, D. morifolium, G. uralensis and R. rubescens were tested as two-extract combinations. baicalensis and D. morifolium when combined were additive with a trend toward synergy, whereas D. morifolium and R. rubescens together were additive. The remaining two-extract combinations showed antagonism. The four extracts together were significantly more effective than the two-by-two combinations and the individual extracts alone. Combining the four herbal extracts significantly enhanced their activity in the cell lines tested compared with extracts alone. The less predictable nature of the two-way combinations suggests a need for careful characterization of the effects of each individual herb based on their intended use.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Science.gov (United States)

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

2015-08-01

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

Present assessment of myocardial viability by nuclear imaging.

Science.gov (United States)

Saha, G B; MacIntyre, W J; Brunken, R C; Go, R T; Raja, S; Wong, C O; Chen, E Q

1996-10-01

Prospective delineation of viable from nonviable myocardium in patients with coronary artery disease in an important factor in deciding whether a patient should be revascularized or treated medically. Two common techniques--single-photon emission computed tomography (SPECT) and positron-emission computed tomography (PET)--are used in nuclear medicine using various radiopharmaceuticals for the detection of myocardial viability in patients. Thallium-201 (201Tl) and technetium-99m (99mTc)-sestamibi are the common radiopharmaceuticals used in different protocols using SPECT, whereas fluoride-18 (18F)-fluorodeoxyglucose (FDG) and rubidium-82 (82Rb) are most widely used in PET. The SPECT protocols involve stress/redistribution, stress/redistribution/reinjection, and rest/redistribution imaging techniques. Many studies have compared the results of 201Tl and (99mTc)-sestamibi SPECT with those of FDG PET; in some studies, concordant results have been found between delayed thallium and FDG results, indicating that 201Tl, although considered a perfusion agent, shows myocardial viability. Discordant results in a number of studies have been found between sestamibi and FDG, suggesting that the efficacy of sestamibi as a viability marker has yet to be established. Radiolabeled fatty acids such as iodine-123 (123I)-para-iodophenylpentadecanoic acid and carbon-11 (11C)-palmitic acid have been used for the assessment of myocardial viability with limited success. 11C-labeled acetate is a good marker of oxidative metabolism in the heart and has been used to predict the reversibility of wall motion abnormalities. (18F)-FDG is considered the marker of choice for myocardial viability, although variable results are obtained under different physiological conditions. Detection of myocardial viability can be greatly improved by developing new equipment and radiopharmaceuticals of better quality.

Photothermal effects of multi-walled carbon nanotubes on the viability of BT-474 cancer cells

International Nuclear Information System (INIS)

Chou, Hung-Tao; Wang, Tsung-Pao; Lee, Chi-Young; Tai, Nyan-Hwa; Chang, Hwan-You

2013-01-01

Functionalized multi-walled carbon nanotubes (f-MWCNTs) were conjugated to an antibody of BT-474 cancer cells (f-MWCNTs-ab), and the photothermal effect of the f-MWCNTs-ab for BT-474 cancer cell destruction was demonstrated. After near-infrared irradiation, the f-MWCNTs-ab were more capable of killing cancer cells and possessed higher cell specificity than f-MWCNTs. Quantitative results showed that the viability of the cancer cells was affected by the concentration of the f-MWCNTs-ab solution, irradiation time, and settling time after irradiation. The membrane impermeable fluorescence dye ethidium bromide was used to detect cell viability after near-infrared irradiation, and the results agreed with those obtained from the Alamar Blue cell viability assay. The EtBr fluorescence results suggest that the cell membrane, attached to f-MWCNTs-ab, was damaged after irradiation, which led to cell death and necrosis. Using confocal microscopy, a few f-MWCNTs-ab were detected in the cell, indicating the endocytosis effect. The results not only explain the improved efficiency of thermotherapy but also indicate that necrosis may result from protein denaturation attributing to the heated f-MWCNTs-ab in the cell. Highlights: ► f-MWCNTs conjugated with anti-HER2 antibody by chemical method. ► Kill breast cancer cells by using low dose f-MWCNTs-ab due to photothermal effect. ► Use EtBr fluorescent to prove that the cell membrane was broken by heated f-MWCNTs. ► Few f-MWCNTs-ab were detected in the cell indicating the endocytosis effect. ► Necrosis may result from protein denaturation due to contact with the heated CNTs.

The effects of storage conditions on the viability of ...

African Journals Online (AJOL)

SARAKA DANIEL

2015-01-07

Jan 7, 2015 ... E-mail: ndsaraka@yahoo.fr, ndsaraka@gmail.com. Tel: 225 ..... sample viability and control of contaminants that may mask the ... viability and composition of the Escherichia coli flora in faecal ... Microbiologie alimentaire, 8e.

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.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

Science.gov (United States)

Anumula, Kalyan Rao

2012-07-01

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

The modified high-density survival assay is the useful tool to predict the effectiveness of fractionated radiation exposure

International Nuclear Information System (INIS)

Kuwahara, Yoshikazu; Mori, Miyuki; Oikawa, Toshiyuki; Shimura, Tsutomu; Fukumoto, Manabu; Ohtake, Yosuke; Ohkubo, Yasuhito; Mori, Shiro

2010-01-01

The high-density survival (HDS) assay was originally elaborated to assess cancer cell responses to therapeutic agents under the influence of intercellular communication. Here, we simplified the original HDS assay and studied its applicability for the detection of cellular radioresistance. We have recently defined clinically relevant radioresistant (CRR) cells, which continue to proliferate with daily exposure to 2 gray (Gy) of X-rays for more than 30 days in vitro. We established human CRR cell lines, HepG2-8960-R from HepG2, and SAS-R1 and -R2 from SAS, respectively. In an attempt to apply the HDS assay to detect radioresistance with clinical relevance, we simplified the original HDS assay by scoring the total number of surviving cells after exposure to X-rays. The modified HDS assay successfully detected radioresistance with clinical relevance. The modified HDS assay detected CRR phenotype, which is not always detectable by clonogenic assay. Therefore, we believe that the modified HDS assay presented in this study is a powerful tool to predict the effectiveness of fractionated radiotherapy against malignant tumors. (author)

A business model design framework for viability : a business ecosystem approach

NARCIS (Netherlands)

D'Souza, Austin; Velthuijsen, Hugo; Wortmann, J.C.; Huitema, George

2015-01-01

Purpose: To facilitate the design of viable business models by proposing a novel business model design framework for viability. Design: A design science research method is adopted to develop a business model design framework for viability. The business model design framework for viability is

Cytotoxic Effect of Iron Oxide Nanoparticles on Mouse Embryonic Stem Cells by MTT Assay

Directory of Open Access Journals (Sweden)

Homa Mohseni Kouchesfehani

2016-07-01

Full Text Available Background: Despite the wide range of applications, there is a serious lack of information on the impact of the nanoparticles on human health and the environment. The present study was done to determine the range of dangerous concentrations of iron oxide nanoparticle and their effects on mouse embryonic stem cells. Methods: Iron oxide nanoparticles with less than 20 nanometers diameter were encapsulated by a PEG-phospholipid. The suspension of iron oxide nanoparticles was prepared using the culture media and cell viability was determined by MTT assay. Results: MTT assay was used to examine the cytotoxicity of iron oxide nanoparticle s. Royan B1 cells were treated with medium containing different concentrations (10, 20, 30, 40, 50, and 60µg/ml of the iron oxide nanoparticle. Cell viability was determined at 12 and 24 hours after treatment which showed significant decreases when concentration and time period increased. Conclusion: The main mechanism of nanoparticles action is still unknown, but in vivo and in vitro studies in different environments suggest that they are capable of producing reactive oxygen species (ROS. Therefore, they may have an effect on the concentration of intracellular calcium, activation of transcription factors, and changes in cytokine. The results of this study show that the higher concentration and duration of treatment of cells with iron oxide nanoparticles increase the rate of cell death.

Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation.

Science.gov (United States)

Liu, Di; Zhang, Hong; Gu, Wenjuan; Zhang, Mengren

2014-06-01

Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFH-DA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucose-treated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation.

Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

Directory of Open Access Journals (Sweden)

Surena Vahabi

2016-01-01

Full Text Available Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF and platelet-rich fibrin (PRF on proliferation and viability of human gingival fibroblasts (HGFs.Materials and Methods: Anitua's PRGF and Choukran's PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-values<0.05 were considered statistically significant.Results: PRGF treatment induced statistically significant (P<0.001 proliferation of HGF cells compared to the negative control (100% viability at 24, 48 and 72 hours in values of 123%±2.25%, 102%±2.8% and 101%±3.92%, respectively. The PRF membrane treatment of HGF cells had a statistically significant effect on cell proliferation (21%±1.73%, P<0.001 at 24 hours compared to the negative control. However, at 48 and 72 hours after treatment, PRF had a negative effect on HGF cell proliferation and caused 38% and 60% decrease in viability and proliferation compared to the negative control, respectively. The HGF cell proliferation was significantly higher in PRGF than in PRF group (P< 0.001.Conclusion: This study demonstrated that PRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF.

Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)

2015-10-27

We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

Prolonging the viability of Lactobacillus plantarum through the addition of prebiotics into the medium.

Science.gov (United States)

Altieri, Clelia; Bevilacqua, Antonio; Sinigaglia, Milena

2011-08-01

This article reports on the effects of prebiotics on the growth/death kinetics, metabolism, and biomass production by 2 strains of Lactobacillus plantarum (c19 and DSMZ 2601, isolated from table olives and purchased from a Public Collection, respectively). The research was divided into 3 different steps, in order to highlight the optimal combination for cell viability and experiments were performed under the conditions of an accelerated shelf life test; thus, 3 combinations were pointed out (fructooligosaccharides [FOS], 5 g/L; glucose + inulin, 2.5 + 2.5 g/L; glucose + FOS, 2.5 + 2.5 g/L). A sample containing only glucose was used as control. The results highlighted that the 3 combinations aforementioned prolonged cell viability over the time both under low and high inoculum conditions (3 and 9 log CFU/mL, respectively); however, FOS alone caused a reduction of biomass production, even if cell number was not affected by this compound. Therefore, as a final result of this research, the combination glucose + FOS could be proposed as a suitable mean to achieve an optimal production of biomass and prolong cell viability over the time. Food producers require a prolonged viability of probiotic bacteria in functional foods; this goal is usually achieved by refrigeration. In this article, the prolongation of cell viability through the addition of prebiotics was proposed. © 2011 Institute of Food Technologists®

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

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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.

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

Effect of different cryo-protectants on the viability of frozen/thawed semen from boars of the Piau breed.

Science.gov (United States)

Pinho, R O; Lima, D M A; Shiomi, H H; Siqueira, J B; Silva, H T; Lopes, P S; Guimarães, S E F; Guimarães, J D

2014-05-01

The objective of this study was to evaluate the effect of different cryo-protectants (glycerol, dimethylacetamide and dimethylformamide alone or combined and added to lactose-egg yolk extender) on the viability of frozen/thawed semen from the Piau breed as assessed by in vitro testing. Frozen semen samples (n=20) were used from five male swine. Five different freezing extenders, including 2% glycerol (Group 1 - G), 2% glycerol and 3% dimethylacetamide (Group 2 - GA), 2% glycerol and 3% dimethylformamide (Group 3 - GF), 5% dimethylacetamide (Group 4 - A) and 5% dimethylformamide (group 5 - F), were evaluated. To assess post-thawing sperm quality, sperm motility and morphology were evaluated. Sperm viability was determined using the hypoosmotic swelling test, supravital staining, and a fluorescent assay (carboxyfluorescein diacetate and propidium iodide). The mean total sperm motility of semen immediately after thawing was 46.2±1.3, 57.7±1.5, 53.2±2.1, 51.7±1.2, and 46.5±1.6% for groups 1-5, respectively. Groups 2 (GA) and 3 (GF) had greater motility values (P0.05). The other complementary tests (hypoosmotic swelling test and supra-vital staining) demonstrated that sperm in Groups 2 (GA), 3 (GF) and 4 (A) had the greatest viability and there were no significant differences among these three groups (P>0.05). The most effective cryo-protectant combinations likely minimized and controlled the deleterious processes that occur in the sperm cell during freezing/thawing, thus improving post-thawing sperm viability. In conclusion, the combination of amides (3%) and glycerol (2%) or dimethylacetamide (5%) alone were more efficient at cryo-protection than glycerol alone for semen freezing in the Piau swine breed. Copyright © 2014 Elsevier B.V. All rights reserved.

High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format.

Science.gov (United States)

Mercurio, Meagan D; Dambergs, Robert G; Herderich, Markus J; Smith, Paul A

2007-06-13

The methyl cellulose precipitable (MCP) tannin assay and a modified version of the Somers and Evans color assay were adapted to high-throughput (HTP) analysis. To improve efficiency of the MCP tannin assay, a miniaturized 1 mL format and a HTP format using 96 well plates were developed. The Somers color assay was modified to allow the standardization of pH and ethanol concentrations of wine samples in a simple one-step dilution with a buffer solution, thus removing inconsistencies between wine matrices prior to analysis and allowing for its adaptation to a HTP format. Validation studies showed that all new formats were efficient, and results were reproducible and analogous to the original formats.

Ovarian fluid mediates the temporal decline in sperm viability in a fish with sperm storage.

Directory of Open Access Journals (Sweden)

Clelia Gasparini

Full Text Available A loss of sperm viability and functionality during sperm transfer and storage within the female reproductive tract can have important fitness implications by disrupting fertilization and impairing offspring development and survival. Consequently, mechanisms that mitigate the temporal decline in sperm function are likely to be important targets of selection. In many species, ovarian fluid is known to regulate and maintain sperm quality. In this paper, we use the guppy Poecilia reticulata, a highly polyandrous freshwater fish exhibiting internal fertilization and sperm storage, to determine whether ovarian fluid (OF influences the decline in sperm viability (the proportion of live sperm in the ejaculate over time and whether any observed effects depend on male sexual ornamentation. To address these questions we used a paired experimental design in which ejaculates from individual males were tested in vitro both in presence and absence of OF. Our results revealed that the temporal decline in sperm viability was significantly reduced in the presence of OF compared to a saline control. This finding raises the intriguing possibility that OF may play a role in mediating the decline in sperm quality due to the deleterious effects of sperm ageing, although other possible explanations for this observation are discussed. Interestingly, we also show that the age-related decline in sperm viability was contingent on male sexual ornamentation; males with relatively high levels of iridescence (indicating higher sexual attractiveness exhibited a more pronounced decline in sperm viability over time than their less ornamented counterparts. This latter finding offers possible insights into the functional basis for the previously observed trade-off between these key components of pre- and postcopulatory sexual selection.

Viability of dielectrophoretically trapped neuronal cortical cells in culture

NARCIS (Netherlands)

Heida, Tjitske; Vulto, P; Rutten, Wim; Marani, Enrico

2001-01-01

Negative dielectrophoretic trapping of neural cells is an efficient way to position neural cells on the electrode sites of planar micro-electrode arrays. The preservation of viability of the neural cells is essential for this approach. This study investigates the viability of postnatal cortical rat

Flow cytometric assessment of viability of lactic acid bacteria

NARCIS (Netherlands)

Bunthof, C.J.; Bloemen, K.; Breeuwer, P.; Rombouts, F.M.; Abee, T.

2001-01-01

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA

Sperm viability staining in ecology and evolution: potential pitfalls

DEFF Research Database (Denmark)

Holman, Luke

2009-01-01

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 a nu...

n Vitro Immunomodulatory Effect of R10 Fraction of Garlic on Viability and Production of TNF-? in CD8+ T Cells

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T. Ghazanfari

2014-01-01

Full Text Available Introduction & Objective: -cells, especially CD8+ T lymphocytes are the most important cells in anti-tumor response. Previously R10 fraction of garlic extract was reported as an immuno-modulator which induced an effective cellular immunity and Th1 responses. In this study the in vitro immunomodulatory effect of R10 on CD8+ T cells viability and production of TNF-? were evaluated. Materials & Methods: In this experimental study, using monoclonal antibodies attached to magnetic beads with isolating columns by magnetic bead method, CD8+ T cells from spleen cells of Balb/C mice were isolated. R10 fraction based on molecular weight was prepared using Ultra filtration. MTT assay was used to evaluate cell viability. TNF-? level was meas-ured in the supernatant of culture of CD8+ T cells by ELISA. Obtained data was compared and analyzed using Nonparametric Test and Keraskel & Wanny's Test tests.. Results: The findings indicate that all dilutions of R10 fraction increased cell viability of CD8+ T cells in comparison with the negative control group and in the presence of ConA with dilution of 1:50 of R10 fraction significantly increased cell viability of CD8+ T Cells com-pared to ConA alone. Secretion of TNF-? significantly increased by all dilutions of R10 frac-tion. Conclusion: These findings suggest that R10 fraction of garlic can be used as an Immuno-modulator drug candidate for induction of cellular Immunity in tumor therapy. Sci J Hamadan Univ Med Sci 2014; 20 (4:273-279

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

Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

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I.C. Oliveira

2012-09-01

Full Text Available A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8 pfu/mL without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL, after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

Science.gov (United States)

Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

2013-01-01

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

Random assay in radioimmunoassay: Feasibility and application compared with batch assay

Energy Technology Data Exchange (ETDEWEB)

Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

2016-12-15

The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay.

Science.gov (United States)

Londoño-Velasco, Elizabeth; Martínez-Perafán, Fabián; Carvajal-Varona, Silvio; García-Vallejo, Felipe; Hoyos-Giraldo, Luz Stella

2016-05-01

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p  0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.

Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay.

Science.gov (United States)

Mann, Sarah K; Czuba, Ewa; Selby, Laura I; Such, Georgina K; Johnston, Angus P R

2016-10-01

The internalization of nanoparticles into cells is critical for effective nanoparticle mediated drug delivery. To investigate the kinetics and mechanism of internalization of nanoparticles into cells we have developed a DNA molecular sensor, termed the Specific Hybridization Internalization Probe - SHIP. Self-assembling polymeric 'pHlexi' nanoparticles were functionalized with a Fluorescent Internalization Probe (FIP) and the interactions with two different cell lines (3T3 and CEM cells) were studied. The kinetics of internalization were quantified and chemical inhibitors that inhibited energy dependent endocytosis (sodium azide), dynamin dependent endocytosis (Dyngo-4a) and macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) were used to study the mechanism of internalization. Nanoparticle internalization kinetics were significantly faster in 3T3 cells than CEM cells. We have shown that ~90% of the nanoparticles associated with 3T3 cells were internalized, compared to only 20% of the nanoparticles associated with CEM cells. Nanoparticle uptake was via a dynamin-dependent pathway, and the nanoparticles were trafficked to lysosomal compartments once internalized. SHIP is able to distinguish between nanoparticles that are associated on the outer cell membrane from nanoparticles that are internalized. This study demonstrates the assay can be used to probe the kinetics of nanoparticle internalization and the mechanisms by which the nanoparticles are taken up by cells. This information is fundamental for engineering more effective nanoparticle delivery systems. The SHIP assay is a simple and a high-throughput technique that could have wide application in therapeutic delivery research.

Pollen viability and germination in Jatropha ribifolia and Jatropha ...

African Journals Online (AJOL)

The aim of this work is to assess pollen viability using the staining technique and in vitro germination with different concentrations of sucrose in Jatropha ribifolia and Jatropha mollissima, contributing to the knowledge of the reproductive biology and subsidizing their conservation, management and utilization. Pollen viability ...

Influence of boundary-layer dynamics on pollen dispersion and viability

Science.gov (United States)

Arritt, Raymond W.; Viner, Brian J.; Westgate, Mark E.

2013-04-01

Adoption of genetically modified (GM) crops has raised concerns that GM traits can accidentally cross into conventional crops or wild relatives through the transport of wind-borne pollen. In order to evaluate this risk it is necessary to account both for dispersion of the pollen grains and environmental influences on pollen viability. The Lagrangian approach is suited to this problem because it allows tracking the environmental temperature and moisture that pollen grains experience as they travel. Taking advantage of this capability we have combined a high-resolution version of the WRF meteorological model with a Lagrangian particle dispersion model to predict maize pollen dispersion and viability. WRF is used to obtain fields of wind, turbulence kinetic energy, temperature, and humidity which are then used as input to the Lagrangian dispersion model. The dispersion model in turn predicts transport of a statistical sample of a pollen cloud from source plants to receptors. We also use the three-dimensional temperature and moisture fields from WRF to diagnose changes in moisture content of the pollen grains and consequent loss of viability. Results show that turbulent motions in the convective boundary layer counteract the large terminal velocity of maize pollen grains and lift them to heights of several hundred meters, so that they can be transported long distances before settling to the ground. We also found that pollen lifted into the upper part of the boundary layer remains more viable than has been inferred using surface observations of temperature and humidity. This is attributed to the thermal and moisture structure that typifies the daytime atmospheric boundary layer, producing an environment of low vapor pressure deficit in the upper boundary layer which helps maintain pollen viability.

Stationary and protable instruments for assay of HEU [highly enriched uranium] solids holdup

International Nuclear Information System (INIS)

Russo, P.A.; Sprinkle, J.K. Jr.; Stephens, M.M.; Brumfield, T.L.; Gunn, C.S.; Watson, D.R.

1987-01-01

Two NaI(Tl)-based instruments, one stationary and one portable, designed for automated assay of highly enriched uranium (HEU) solids holdup, are being evaluated at the scrap recovery facility of the Oak Ridge Y-12 Plant. The stationary instrument, a continuous monitor of HEU within the filters of the chip burner exhaust system, measures the HEU deposits that accumulate erratically and rapidly during chip burner operation. The portable system was built to assay HEU in over 100 m of elevated piping used to transfer UO 3 , UO 2 , and UF 4 powder to, from, and between the fluid bed conversion furnances and the powder storage hoods. Both instruments use two detector heads. Both provide immediate automatic readout of accumulated HEU mass. The 186-keV 235 U gamma ray is the assay signature, and the 60-keV gamma ray from an 241 Am source attached to each detector is used to normalize the 186-keV rate. The measurement geometries were selected for compatibility with simple calibration models. The assay calibrations were calculated from these models and were verified and normalized with measurements of HEU standards built to match geometries of uniform accumulations on the surfaces of the process equipment. This instrumentation effort demonstrates that simple calibration models can often be applied to unique measurement geometries, minimizing the otherwise unreasonable requirements for calibration standards and allowing extension of the measurements to other process locations

Punicalagin and (-)-Epigallocatechin-3-Gallate Rescue Cell Viability and Attenuate Inflammatory Responses of Human Epidermal Keratinocytes Exposed to Airborne Particulate Matter PM10.

Science.gov (United States)

Seok, Jin Kyung; Lee, Jeong-Won; Kim, Young Mi; Boo, Yong Chool

2018-01-01

Airborne particulate matter with a diameter of < 10 µm (PM10) causes oxidative damage, inflammation, and premature skin aging. In this study, we evaluated whether polyphenolic antioxidants attenuate the inflammatory responses of PM10-exposed keratinocytes. Primary human epidermal keratinocytes were exposed in vitro to PM10 in the absence or presence of punicalagin and (-)-epigallocatechin-3-gallate (EGCG), which are the major polyphenolic antioxidants found in pomegranate and green tea, respectively. Assays were performed to determine cell viability, production of reactive oxygen species (ROS), and expression of NADPH oxidases (NOX), proinflammatory cytokines, and matrix metalloproteinase (MMP)-1. PM10 decreased cell viability and increased ROS production in a dose-dependent manner. It also increased the expression levels of NOX-1, NOX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and MMP-1. Punicalagin was not cytotoxic up to 300 μM, and (-)-EGCG was cytotoxic above 30 μM, respectively. Further, punicalagin (3-30 μM) and EGCG (3-10 μM) rescued the viability of PM10-exposed cells. They also attenuated ROS production and the expression of NOX-1, NOX-2, TNF-α, IL-1β, IL-6, IL-8, and MMP-1 stimulated by PM10. This study demonstrates that polyphenolic antioxidants, such as punicalagin and (-)-EGCG, rescue keratinocyte viability and attenuate the inflammatory responses of these cells due to airborne particles. © 2018 S. Karger AG, Basel.

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.

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.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine

International Nuclear Information System (INIS)

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2016-01-01

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

Science.gov (United States)

Urusov, Alexandr E; Petrakova, Alina V; Gubaydullina, Milyausha K; Zherdev, Anatoly V; Eremin, Sergei A; Kong, Dezhao; Liu, Liqiang; Xu, Chuanlai; Dzantiev, Boris B

2017-05-01

To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration. The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Changes in viability of two Antarctic marine bacteria exposed to solar radiation in the water column: influence of vertical mixing

International Nuclear Information System (INIS)

Hernandez, E.A.

2007-01-01

The effect of UV radiation on two Antarctic marine bacterial strains (UVps and UVvi) was studied in the water column of Potter Cove (South Shetland, Antarctica). Quartz flasks were filled with the bacterial suspensions and exposed to solar radiation at 0 m, 1 m and 3 m depth. Assays using flasks exposed to direct solar radiation and others using flasks covered with/by interferential filters which discriminate between UVA and UVB, were performed. In other assays, a vertical mixing of 4 m/h was simulated. Both strains showed a significant decrease in viability (expressed as colony - forming units) when exposed to a surface UVB dose of 8.4 kJ m -2 . Studies with interferential filters showed a significant decrease at 0 and 1 m depth under both UV treatments. The UVps strain appeared to be more sensitive to UVB than to UVA. Damage produced by UVB was attenuated by the vertical mixing when the surface UVB dose was 4.8 kJ m -2 . This effect was not observed when surface UVB dose was 7.7 kJ m -2 . These results show that the negative effect caused by UVB radiation on the bacterio plankton would be significant only in the first meter of water column of the Antarctic coastal waters with high levels of suspended particulate material. (author) [es

Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Science.gov (United States)

Tweedie, David; Brossi, Arnold; Chen, DeMoa; Ge, Yuan-Wen; Bailey, Jason; Yu, Qian-Sheng; Kamal, Mohammad A; Sambamurti, Kumar; Lahiri, Debomoy K; Greig, Nigel H

2006-09-01

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

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

Science.gov (United States)

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

2014-05-01

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

Photothermal effects of multi-walled carbon nanotubes on the viability of BT-474 cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chou, Hung-Tao [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Wang, Tsung-Pao [Department of Medical Science, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Lee, Chi-Young [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Tai, Nyan-Hwa, E-mail: nhtai@mx.nthu.edu.tw [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Chang, Hwan-You, E-mail: hychang@mx.nthu.edu.tw [Department of Medical Science, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China)

2013-03-01

Functionalized multi-walled carbon nanotubes (f-MWCNTs) were conjugated to an antibody of BT-474 cancer cells (f-MWCNTs-ab), and the photothermal effect of the f-MWCNTs-ab for BT-474 cancer cell destruction was demonstrated. After near-infrared irradiation, the f-MWCNTs-ab were more capable of killing cancer cells and possessed higher cell specificity than f-MWCNTs. Quantitative results showed that the viability of the cancer cells was affected by the concentration of the f-MWCNTs-ab solution, irradiation time, and settling time after irradiation. The membrane impermeable fluorescence dye ethidium bromide was used to detect cell viability after near-infrared irradiation, and the results agreed with those obtained from the Alamar Blue cell viability assay. The EtBr fluorescence results suggest that the cell membrane, attached to f-MWCNTs-ab, was damaged after irradiation, which led to cell death and necrosis. Using confocal microscopy, a few f-MWCNTs-ab were detected in the cell, indicating the endocytosis effect. The results not only explain the improved efficiency of thermotherapy but also indicate that necrosis may result from protein denaturation attributing to the heated f-MWCNTs-ab in the cell. Highlights: Black-Right-Pointing-Pointer f-MWCNTs conjugated with anti-HER2 antibody by chemical method. Black-Right-Pointing-Pointer Kill breast cancer cells by using low dose f-MWCNTs-ab due to photothermal effect. Black-Right-Pointing-Pointer Use EtBr fluorescent to prove that the cell membrane was broken by heated f-MWCNTs. Black-Right-Pointing-Pointer Few f-MWCNTs-ab were detected in the cell indicating the endocytosis effect. Black-Right-Pointing-Pointer Necrosis may result from protein denaturation due to contact with the heated CNTs.

Effect of surface organic coatings of cellulose nanocrystals on the viability of mammalian cell lines

Directory of Open Access Journals (Sweden)

Jimenez AS

2017-09-01

Full Text Available Ambar S Jimenez,1 Francesca Jaramillo,1 Usha D Hemraz,2 Yaman Boluk,3 Karina Ckless,1 Rajesh Sunasee1 1Department of Chemistry, State University of New York at Plattsburgh, Plattsburgh, NY, USA; 2National Research Council, Montreal, QC, Canada, 3Department of Civil & Environmental Engineering, University of Alberta and National Institute for Nanotechnology, National Research Council, Edmonton, AB, Canada Abstract: Cellulose nanocrystals (CNCs have emerged as promising candidates for a number of bio-applications. Surface modification of CNCs continues to gain significant research interest as it imparts new properties to the surface of the nanocrystals for the design of multifunctional CNCs-based materials. A small chemical surface modification can potentially lead to drastic behavioral changes of cell-material interactions thereby affecting the intended bio-application. In this work, unmodified CNCs were covalently decorated with four different organic moieties such as a diaminobutane fragment, a cyclic oligosaccharide (β-cyclodextrin, a thermoresponsive polymer (poly[N-isopropylacrylamide], and a cationic aminomethacrylamide-based polymer using different synthetic covalent methods. The effect of surface coatings of CNCs and the respective dose-response of the above organic moieties on the cell viability were evaluated on mammalian cell cultures (J774A.1 and MFC-7, using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. Overall, the results indicated that cells exposed to surface-coated CNCs for 24 h did not display major changes in cell viability, membrane permeability as well as cell morphology. However, with longer exposure, all these parameters were somewhat affected, which appears not to be correlated with either anionic or cationic surface coatings of CNCs used in this study. Keywords: cellulose nanocrystals, surface coating, cell viability, MTT, LDH

The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

Science.gov (United States)

Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

2016-05-01

Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

Science.gov (United States)

Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

2002-01-01

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Pollen diversity, viability and floral structure of some Musa genotypes

African Journals Online (AJOL)

Pollen diversity, viability and floral structure of some Musa genotypes. ... This experiment was designed to study the floral structure, pollen morphology and the potential pollen viability of five Musa genotypes obtained ... HOW TO USE AJOL.

Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples

Directory of Open Access Journals (Sweden)

Dorien M. Rotteveel-de Groot

2016-08-01

Full Text Available Objectives: Thyroglobulin (Tg measurements are used to monitor for residual thyroid tissue in patients with differentiated thyroid cancer (DTC after thyroidectomy and radioiodine ablative therapy. In recent years highly sensitive Tg assays have been developed. In this study the analytical performance of the new Roche Elecsys Tg II assay was evaluated and compared with the well documented Access2 Tg assay (Beckman–Coulter. Design and methods: Analytical performance was examined using various Clinical and Laboratory Standards Institute (CLSI evaluation protocols. Tg negative patient sera were used to establish an upper reference limit (URL for the Elecsys Tg II assay. Results: Non-linearity, drift and carry-over according to CLSI EP10 and EP6 in a measuring range of 0.04–500 ng/mL were non-significant. Total precision according to CLSI EP5 was 10% at a Tg concentration of 0.08 ng/mL. A patient serum comparison performed according to a modified CLSI EP9 protocol showed a significant difference of a factor of approximately 1.4, despite using an identical CRM calibrator. The Elecsys Tg II assay measured Tg with a two-fold higher sensitivity than the Access2 assay. Finally, using human sera without Tg, an URL of 0.05 ng/mL was determined. Conclusions: In our hands the highly sensitive Elecsys Tg II assay shows a good analytical performance and a higher sensitivity compared to the Access2 Tg assay. An URL of 0.05 ng/mL for the Elecsys Tg II assay was determined which may improve the clinical utility of the assay for the detection of residual DTC or disease recurrence. Keywords: Thyroglobulin, Roche Elecsys Tg II assay, validation, reporting limit

Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples.

Science.gov (United States)

Rotteveel-de Groot, Dorien M; Ross, H Alec; Janssen, Marcel J R; Netea-Maier, Romana T; Oosting, Janine D; Sweep, Fred C G J; van Herwaarden, Antonius E

2016-08-01

Thyroglobulin (Tg) measurements are used to monitor for residual thyroid tissue in patients with differentiated thyroid cancer (DTC) after thyroidectomy and radioiodine ablative therapy. In recent years highly sensitive Tg assays have been developed. In this study the analytical performance of the new Roche Elecsys Tg II assay was evaluated and compared with the well documented Access2 Tg assay (Beckman-Coulter). Analytical performance was examined using various Clinical and Laboratory Standards Institute (CLSI) evaluation protocols. Tg negative patient sera were used to establish an upper reference limit (URL) for the Elecsys Tg II assay. Non-linearity, drift and carry-over according to CLSI EP10 and EP6 in a measuring range of 0.04-500 ng/mL were non-significant. Total precision according to CLSI EP5 was 10% at a Tg concentration of 0.08 ng/mL. A patient serum comparison performed according to a modified CLSI EP9 protocol showed a significant difference of a factor of approximately 1.4, despite using an identical CRM calibrator. The Elecsys Tg II assay measured Tg with a two-fold higher sensitivity than the Access2 assay. Finally, using human sera without Tg, an URL of 0.05 ng/mL was determined. In our hands the highly sensitive Elecsys Tg II assay shows a good analytical performance and a higher sensitivity compared to the Access2 Tg assay. An URL of 0.05 ng/mL for the Elecsys Tg II assay was determined which may improve the clinical utility of the assay for the detection of residual DTC or disease recurrence.

Increased viability of fibroblasts when pretreated with ceria nanoparticles during serum deprivation.

Science.gov (United States)

Genier, Francielli S; Bizanek, Maximilian; Webster, Thomas J; Roy, Amit K

2018-01-01

Conditions of cellular stress are often the cause of cell death or dysfunction. Sustained cell stress can lead to several health complications, such as extensive inflammatory responses, tumor growth, and necrosis. To prevent disease and protect human tissue during these conditions and to avoid medication side effects, nanomaterials with unique characteristics have been applied to biological systems. This paper introduces the pretreatment in human dermal fibroblasts with cerium oxide nanoparticles during nutritional stress. For this purpose, human dermal fibroblast cells received cell culture media with concentrations of 250 µg/mL and 500 µg/mL of nano-cerium oxide before being exposed to 24, 48, and 72 hours of serum starvation. Contrast images demonstrated higher cell confluence and cell integrity in cells pretreated with ceria nanoparticles compared to untreated cells. It was confirmed by MTS assay after 72 hours of serum starvation that higher cell viability was achieved with ceria nanoparticles. The results demonstrate the potential of cerium oxide nanoparticles as protective agents during cellular starvation.

Survey of the Definition of Fetal Viability and the Availability, Indications, and Decision Making Processes for Post-Viability Termination of Pregnancy for Fetal Abnormalities and Health Conditions in Canada.

Science.gov (United States)

Hull, Danna; Davies, Gregory; Armour, Christine M

2016-06-01

The purpose of this study was to explore the definition of fetal viability and the availability, indications, and decision making processes for post-viability termination of pregnancy for fetal abnormalities and health conditions in Canada. An online survey of members of the Canadian Association of Genetic Counsellors, the Canadian College of Medical Geneticists, and the Canadian Society for Maternal-Fetal Medicine who provide direct counselling to, or management of, prenatal patients in Canada (total sample size 815). Results of this study showed that the majority of respondents indicated that their centre will offer post-viability termination of pregnancy (98/123; 80 %). Sixty-seven percent (68/101) of respondents reported the definition of fetal viability to be 24 weeks' gestation. Most respondents reported that a collaborative decision making process was used to determine if post-viability termination of pregnancy would be offered (136/170; 80 %). For conditions presumed to be lethal/likely lethal, the majority of respondents would "sometimes" or "always" offer post-viability termination of pregnancy, whereas for conditions presumed to have a mild effect, the majority of respondents would "rarely" or "never" offer post-viability termination of pregnancy. Ninety percent (77/86) of respondents reported that perinatal hospice is offered as an alternative to termination of pregnancy. In conclusion, this study suggests that although post-viability termination is available in many provinces in Canada, variation in the definition of fetal viability and indications appear to exist. While these variations may lead to unequal access to post-viability termination of pregnancy across Canada, they might also represent the complexity of the decision making process and the importance of examining individual factors to ensure that the most appropriate decision is made in each case.

Cell-mediated cytotoxicity for melanome tumor cells: detection by a (3H)proline release assay

International Nuclear Information System (INIS)

Saal, J.G.; Rieber, E.P.; Riethmueller, G.

1976-01-01

An in vitro lymphocyte-mediated cytotoxicity assay using [ 3 H]proline-labelled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [ 3 H]proline by filtering the total culture fluid containing both trypsinised tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (p > 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [ 3 ]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [ 3 H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells. (Auth.)

The effect of extremely high glucose concentrations on 21 routine chemistry and thyroid Abbott assays: interference study

OpenAIRE

?uhadar, Serap; K?seo?lu, Mehmet; ?inpolat, Yasemin; Bu?dayc?, G?ler; Usta, Murat; Semerci, Tuna

2016-01-01

Abstract Introduction: Extremely high glucose concentrations have been shown to interfere with creatinine assays especially with Jaffe method in peritoneal dialysate. Because diabetes is the fastest growing chronic disease in the world, laboratories study with varying glucose concentrations. We investigated whether different levels of glucose spiked in serum interfere with 21 routine chemistry and thyroid assays at glucose concentrations between 17-51 mmol/L. Materials and methods: Base...

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.

An updated methodology to review developing-country vaccine manufacturer viability.

Science.gov (United States)

Luter, Nicholas; Kumar, Ritu; Hozumi, Dai; Lorenson, Tina; Larsen, Shannon; Gowda, Bhavya; Batson, Amie

2017-07-05

In 1997, Milstien, Batson, and Meaney published "A Systematic Method for Evaluating the Potential Viability of Local Vaccine Producers." The paper identified characteristics of successful vaccine manufacturers and developed a viability framework to evaluate their performance. This paper revisits the original study after two decades to determine the ability of the framework to predict manufacturer success. By reconstructing much of the original dataset and conducting in-depth interviews, the authors developed informed views on the continued viability of manufacturers in low- and middle-income country markets. Considering the marked changes in the market and technology landscape since 1997, the authors find the viability framework to be predictive and a useful lens through which to evaluate manufacturer success or failure. Of particular interest is how incumbent and potentially new developing-country vaccine manufacturers enter and sustain production in competitive international markets and how they integrate (or fail to integrate) new technology into the production process. Ultimately, most manufacturers will need to meet global quality standards to be viable. As governments and donors consider investments in vaccine producers, the updated viability factors will be a useful tool in evaluating the prospects of manufacturers over the mid to long term. The paper emphasizes that while up-front investments are important, other critical factors-including investments in a national regulatory authority, manufacturer independence, and ability to adapt and adopt new technology-are necessary to ensure viability. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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.

Improved assay for thymine base damage in E. coli using high performance liquid chromatography

International Nuclear Information System (INIS)

Claycamp, H.G.

1985-01-01

A simple high performance liquid chromatography (HPLC) technique has been established for the simultaneous assay of thymine and thymidine radiation damage products. The HPLC procedure uses an isocratic mobile phase of 4% acetonitrile in 0.2 M ammonium dihydrogen phosphate (pH 5.0), a reversed-phase octadecylsilicate (5 micro-spherical packing) 0.45 x 25 cm column, and a variable wavelength UV detector. This procedure affords much better resolution than other published procedures that use 10 micron columns or separate assays for bases and nucleosides. For example, irradiation of 5 x 10 -3 M thymidine solutions have been performed to calibrate the system for base damage assays in E. coli. This yields up to 15 resolvable residues within 20 minutes. Sensitivity of the system (at 2210 nm) for 5,6- dihydrothymine (DHT) is about 10 -10 moles. Preliminary results show that this translates to about 0.4 DHT residues per 10 6 daltons of E. coli DNA. This is comparable to the sensitivities of monoclonal assays to thymine damage products that have recently been reported by others. Since it is feasible that the sensitivity of this system can be improved by 2-3 times, this HPLC technique should provide a simple and rapid means of detecting E. coli base damage release and base damage in nucleoside hydrolysates of DNA

Viability of seeds of two representatives from Apocynaceae family

Directory of Open Access Journals (Sweden)

Y.A. Aviekin

2015-05-01

Full Text Available The viability of some Apocynaceae seeds depending on the duration of storage under conditions of low temperature was studied. Extracted embryos from the seeds of Pachypodium lamerei Drake and Adenium obesum (Forssk. Roem. et Schult with different storage history were analyzed. Embryos were stained by acidic fuchsine what allows identification of viable and destructed cells. Destructed cells were stained much more intensively, while viable cells remained unstained. Observed results showed dependence of viability of P. lamerei and A. obesum seeds from term of storage. It was obtained that the seeds of investigated succulent species, just like in many other tropical plants, rapidly lost their viability and should be described as microbiotic.

Cell viability score as an integrated indicator for cytotoxicity of benzalkonium chloride-containing antiglaucoma eyedrops.

Science.gov (United States)

Ayaki, Masahiko; Iwasawa, Atsuo; Niwano, Yoshimi

2012-01-01

We evaluated the in vitro cytotoxicity of benzalkonium chloride (BAK)-containing antiglaucoma eyedrops. We prepared cell cultures of SIRC, BCE C/D-1b, RC-1, and Chang conjunctiva. The viability of cell cultures was determined using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of test solutions. %CVS50 and %CVS40/80 of each eyedrop solution were 71 and 26 for Lumigan(®) (0.002% bimatoprost with 0.005% BAK), 100 and 99 for Tapros(®) (0.0015% tafluprost, a new formula from 2010 with 0.001% BAK), 39 and -29 for 2% Trusopt(®) (2% dorzolamide with 0.0075% BAK), 28 and -43 for Xalacom(®) (latanoprost/0.5% timolol with 0.02% BAK), 88 and 66 for DuoTrav(®) (travoprost/0.5% timolol with no BAK), 36 and -35 for Cosopt(®) (2% dorzolamide/0.5% timolol with 0.0075% BAK) and 53 and -1 for Combigan(®) (0.15% brimonidin/0.5% timolol with 0.005% BAK). Only Xalacom(®) and Tapros(®) did not show an apparent decrease in %CVS as compared to the corresponding concentration of BAK. In conclusion, the cytotoxicity of tested eyedrops was dependent on BAK. Only the eyedrops containing latanoprost or tafluprost showed a reduction in the cytotoxicity of BAK.

Cytokinesis-block micronucleus assay evolves into a 'cytome' assay of chromosomal instability, mitotic dysfunction and cell death

International Nuclear Information System (INIS)

Fenech, Michael

2006-01-01

The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r = 0.75-0.77, P < 0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a 'cytome' method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations

RAS - Screens & Assays - Drug Discovery

Science.gov (United States)

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

Low-level laser therapy with helium-neon laser improved viability of osteoporotic bone marrow-derived mesenchymal stem cells from ovariectomy-induced osteoporotic rats

Science.gov (United States)

Fallahnezhad, Somaye; Piryaei, Abbas; Tabeie, Faraj; Nazarian, Hamid; Darbandi, Hasan; Amini, Abdoldllah; Mostafavinia, Ataroalsadat; Ghorishi, Seyed Kamran; Jalalifirouzkouhi, Ali; Bayat, Mohammad

2016-09-01

The purpose of this study was to evaluate the influences of helium-neon (He-Ne) and infrared (IR) lasers on the viability and proliferation rate of healthy and ovariectomy-induced osteoporotic (OVX) bone marrow mesenchymal stem cells (BMMSCs) in vitro. MSCs harvested from the BM of healthy and OVX rats were culture expanded. He-Ne and IR lasers were applied three times at energy densities of 0.6, 1.2, and 2.4 J/cm2 for BMMSCs. BMMSCs viability and proliferation rate were evaluated by MTT assay on days 2, 4, 6, 14, and 21. The results showed that healthy BMMSCs responded optimally to 0.6 J/cm2 using an IR laser after three times of laser radiation. Moreover, it was found that OVX-BMMSCs responded optimally to 0.6 J/cm2 with He-Ne laser and one-time laser radiation. It is concluded that the low-level laser therapy (LLLT) effect depends on the physiological state of the BMMSCs, type of the laser, wavelength, and number of laser sessions. The biostimulation efficiency of LLLT also depends on the delivered energy density. LLLT can enhance the viability and proliferation rate of healthy and especially osteoporotic autologous BMMSCs, which could be very useful in regenerative medicine.

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

Science.gov (United States)

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

2018-02-01

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

Two-Phase Microfluidic Systems for High Throughput Quantification of Agglutination Assays

KAUST Repository

Castro, David

2018-01-01

assay, with a minimum detection limit of 50 ng/mL using optical image analysis. We compare optical image analysis and light scattering as quantification methods, and demonstrate the first light scattering quantification of agglutination assays in a two

Viability Test Device for anisakid nematodes

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Michael Kroeger

2018-03-01

Full Text Available Up to now the visual inspection of mobility of isolated anisakid larvae serves as a measure of viability and possible risk of infection. This paper presents a new method to rule out unreliability – caused by the temporary immobility of the larvae and by the human uncertainty factor of visual observation. By means of a Near infrared (NIR imaging method, elastic curvature energies and geometric shape parameters were determined from contours, and used as a measure of viability. It was based on the modelling of larvae as a cylindrical membrane system. The interaction between curvatures, contraction of the longitudinal muscles, and inner pressure enabled the derivation of viability from stationary form data. From series of spectrally signed images within a narrow wavelength range, curvature data of the larvae were determined. Possible mobility of larvae was taken into account in statistical error variables. Experiments on individual living larvae, long-term observations of Anisakis larvae, and comparative studies of the staining method and the VTD measurements of larvae from the tissue of products confirmed the effectiveness of this method. The VTD differentiated clearly between live and dead nematode larvae isolated from marinated, deep-frozen and salted products. The VTD has been proven as excellent method to detect living anisakid nematode larvae in fishery products and is seen as useful tool for fish processing industry and control authorities. Keywords: Biophysics

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

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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.

Cardiac troponins--Translational biomarkers in cardiology: Theory and practice of cardiac troponin high-sensitivity assays.

Science.gov (United States)

Adamcova, Michaela; Popelova-Lencova, Olga; Jirkovsky, Eduard; Simko, Fedor; Gersl, Vladimir; Sterba, Martin

2016-01-01

Tn is a unique translational biomarker in cardiology whose potential has not been diminished in the new era of high sensitive assays. cTns can be valuable markers in cardiac diseases as well as in infectious diseases and respiratory diseases. Furthermore, the role of cTns is growing in the routine evaluation of cardioxicity and in determining the efficacy/safety ratio of novel cardioprotective strategies in clinical settings. cTns can detect myocardial injury not only in a wide spectrum of laboratory animals in experimental studies in vivo, but also in isolated heart models or cardiomyocytes in vitro. The crucial issue regarding the cross-species usage of cardiac troponin investigation remains the choice of cardiac troponin testing. This review summarizes the recent proteomic data on aminoacid sequences of cTnT and cTnI in various species, as well as selected analytical characteristics of human cardiac troponin high-sensitivity assays. Due to the highly phylogenetically conserved structure of troponins, the same bioindicator can be investigated using the same method in both clinical and experimental cardiology, thus contributing to a better understanding of the pathogenesis of cardiac diseases as well as to increased effectiveness of troponin use in clinical practice. Measuring cardiac troponins using commercially available human high-sensitivity cardiac troponin tests with convenient antibodies selected on the basis of adequate proteomic knowledge can solve many issues which would otherwise be difficult to address in clinical settings for various ethical and practical reasons. Our survey could help elaborate the practical guidelines for optimizing the choice of cTns assay in cardiology. © 2016 International Union of Biochemistry and Molecular Biology.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

Science.gov (United States)

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2016-06-01

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

Science.gov (United States)

Lorenz, Daniel A; Song, James M; Garner, Amanda L

2015-01-21

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway.

Science.gov (United States)

Lan, Tian; Wu, Teng; Gou, Hongju; Zhang, Qianqian; Li, Jiangchao; Qi, Cuiling; He, Xiaodong; Wu, Pingxiang; Wang, Lijing

2013-11-01

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway. © 2013 Wiley Periodicals, Inc.

Viability of Lactobacillus acidophilus in synbiotic guava mousses and its survival under in vitro simulated gastrointestinal conditions.

Science.gov (United States)

Buriti, Flávia C A; Castro, Inar A; Saad, Susana M I

2010-02-28

The effects of refrigeration, freezing and substitution of milk fat by inulin and whey protein concentrate (WPC) on Lactobacillus acidophilus La-5 viability and resistance to gastric and enteric simulated conditions in synbiotic guava mousses effects were investigated. Refrigerated mousses supplemented with WPC presented the best probiotic viability, ranging from 7.77 to 6.24 log cfu/g during 28 days of storage. The highest probiotic populations, above 7.45 log cfu/g, were observed for all frozen mousses during 112 days of storage. Decreased L. acidophilus survival during the in vitro gastrointestinal simulation was observed both for refrigerated and frozen mousses. Nonetheless, for the refrigerated mousses, the addition of inulin enhanced the probiotic survival during the in vitro assays in the first week of storage. L. acidophilus survival in simulated gastrointestinal fluids was also improved through freezing. The frozen storage may be used to provide increased shelf-life for synbiotic guava mousses. Even though the protective effect of inulin and WPC on the probiotic microorganism tested was shown to be more specific for the refrigerated products, the partial replacement of milk fat by these ingredients may also help, as it improves the nutritional value of mousses in both storage conditions. (c) 2009 Elsevier B.V. All rights reserved.

In vitro evidence of glucose-induced toxicity in GnRH secreting neurons: high glucose concentrations influence GnRH secretion, impair cell viability, and induce apoptosis in the GT1-1 neuronal cell line.

Science.gov (United States)

Pal, Lubna; Chu, Hsiao-Pai; Shu, Jun; Topalli, Ilir; Santoro, Nanette; Karkanias, George

2007-10-01

To evaluate for direct toxic effects of high glucose concentrations on cellular physiology in GnRH secreting immortalized GT1-1 neurons. Prospective experimental design. In vitro experimental model using a cell culture system. GT1-1 cells were cultured in replicates in media with two different glucose concentrations (450 mg/dL and 100 mg/dL, respectively) for varying time intervals (24, 48, and 72 hours). Effects of glucose concentrations on GnRH secretion by the GT1-1 neurons were evaluated using a static culture model. Cell viability, cellular apoptosis, and cell cycle events in GT1-1 neurons maintained in two different glucose concentrations were assessed by flow cytometry (fluorescence-activated cell sorter) using Annexin V-PI staining. Adverse influences of high glucose concentrations on GnRH secretion and cell viability were noted in cultures maintained in high glucose concentration (450 mg/dL) culture medium for varying time intervals. A significantly higher percentage of cells maintained in high glucose concentration medium demonstrated evidence of apoptosis by a fluorescence-activated cell sorter. We provide in vitro evidence of glucose-induced cellular toxicity in GnRH secreting GT1-1 neurons. Significant alterations in GnRH secretion, reduced cell viability, and a higher percentage of apoptotic cells were observed in GT1-1 cells maintained in high (450 mg/dL) compared with low (100 mg/dL) glucose concentration culture medium.

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.

High frequency lateral flow affinity assay using superparamagnetic nanoparticles

International Nuclear Information System (INIS)

Lago-Cachón, D.; Rivas, M.; Martínez-García, J.C.; Oliveira-Rodríguez, M.; Blanco-López, M.C.; García, J.A.

2017-01-01

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.

A comparative in vitro study of the viability of human keratinocytes grown on irradiated human amnion membrane and fibrin glue scaffolds

International Nuclear Information System (INIS)

Dorai, A.A.; Lim, C.K.; Azman, W.S.; Halim, A.S.

2008-01-01

Full text: The dried irradiated human amnion membrane has been used as a biological dressing for various clinical conditions. Being another biological membrane its potential as a scaffold to grow human keratinocytes is not known yet. To compare the growth patterns and cell viability of keratinocytes using fibrin glue and air dried amnion membrane as a scaffold. Keratinocytes were obtained from skin samples of six patients undergoing elective surgery. Fibrin glue (Tisseel, Baxter ) was diluted and used to coat the wells. Human dried amnion membrane was obtained and placed into the 24 well plates. Keratinocytes were seeded into the fibrin and amnion scaffold. Cell viability assay (MTT) was performed after 24, 48 and 72 hours. Finally the measurements were done by the Enzyme-Linked Immunosorbent Assay (ELISA) reader at 570 nm. Six patients consented for the study. The cells growing on the amnion scaffold showed a decreasing trend (20.67%, 17.94% and 16.78% respectively for 24, 48 and 72 hours). The cells growing on the fibrin scaffold showed a steady increase in number at 24, 48 and 72 hours (73.03%, 74.12% and 79.66%). The percentage of growth of normal human keratinocytes were significantly greater in the fibrin scaffold group (Mann - Whitney p = 0.002) for 24, 48 and 72 hours. The air dried irradiated human amnion membrane can be used as a scaffold to grow keratinocytes but however the growth pattern does not sustain with time. Fibrin glue supports the growth of human keratinocytes and shows an increasing pattern of growth with time. (Author)

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

Science.gov (United States)

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

2012-03-01

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

Preservation of seed viability during 25 years of storage under standard genebank conditions

NARCIS (Netherlands)

Treuren, van R.; Groot, de E.C.; Hintum, van T.J.L.

2013-01-01

Maintaining sufficient viability is critical to the sustainability of ex situ conserved seed collections. For this reason, accessions are regenerated when viability falls below a predefined threshold. Viability is monitored by determining the germination ability of accessions at predefined time

Development and viability of a translocated beaver Castor fiber population in the Netherlands

NARCIS (Netherlands)

Nolet, B.A.; Baveco, J.M.

1996-01-01

We monitored survival, reproduction and emigration of a translocated beaver Castor fiber population in the Netherlands for five years and used a stochastic model to assess its viability. Between 1988 and 1991, 42 beavers were released in the Biesbosch National Park. The mortality was initially high

A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide.

Science.gov (United States)

Taylor, Steven W; Clarke, Nigel J; Chen, Zhaohui; McPhaul, Michael J

2016-04-01

Measurements of fasting levels of insulin and C-peptide are useful in documenting insulin resistance and may help predict development of diabetes mellitus. However, the specific insulin and C-peptide levels associated with specific degrees of insulin resistance have not been defined, owing to marked variability among immunoassays and lack of standardization. Herein, we describe a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for intact insulin and C-peptide. Insulin and C-peptide were enriched from patient sera using monoclonal antibodies immobilized on magnetic beads and processed on a robotic liquid handler. Eluted peptides were analyzed by LC-MS/MS. Bovine insulin and a stable isotopically-labeled (13C/15N) C-peptide were utilized as internal standards. The assay had an analytical measurement range of 3 to 320 μIU/ml (18 to 1920 pmol/l) for insulin and 0.11 to 27.2 ng/ml (36 to 9006 pmol/l) for C-peptide. Intra- and inter-day assay variation was less than 11% for both peptides. Of the 5 insulin analogs commonly prescribed to treat diabetes, only the recombinant drug insulin lispro caused significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide. We developed and validated a high-throughput, quantitative, multiplexed LC-MS/MS assay for intact insulin and C-peptide. Copyright © 2016 Elsevier B.V. All rights reserved.

The Effect of Histone Hyperacetylation on Viability of Basal-Like Breast Cancer Cells MDA-MB-231

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Aliasghar Rahimian

2017-06-01

Full Text Available Background The Basal-Like breast cancer, is always known for lack of expression of estrogen receptor (ER, progesterone receptor (PR and as well, absence of epidermal growth factor receptor 2 (HER2 gene amplification. Improper expression pattern of ER, PR, and Her2, makes Basal-Like breast tumors resistant to the current hormonal and anti HER2 treatments. In recent decades, several studies have been conducted to investigate the regulatory role of chemical modifications of core histones in gene expression. Their results have shown that histone acetylation is involved in regulation of cell survival. Acetylation of core histones is regulated by the epigenetic-modifying enzymes named Histone Deacetylases (HDACs. As a new approach to control the viability of breast tumor cells resistant to the hormonal and anti-HER2 treatments, we have targeted the HDACs. Using Trichostatin A (TSA as a known HDACs inhibitor, we have tried to hyperacetylate the core histones of MDA-MB-231 cells as an in vitro model of Basal-Like breast tumors. Then we have investigated the effect of histone hyperacetylation on viability of MDA-MB-231 cells. Methods MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS and were incubated at 37°C, in a humidified incubator with 5% CO2 atmosphere. Then cells were treated with different concentrations of TSA including: 50, 100, 200, 400, 800 and 1000 nM or control (1% DMSO. After 24 and 48 hours, viability of cells was evaluated by MTT assay. Results After 24 and 48h exposure to different concentrations of TSA, MDA-MB-231 cells showed a maximum tolerable dose. At higher concentrations, TSA decreased the percentage of cell viability through a time-dose dependent manner. IC50 value for 48h treatment was 600 nM. Conclusions Our results indicate that HDACs inhibition and subsequently hyperacetylation of histones, leads to cytotoxic effects on breast tumor cells resistant to the current treatments. Following

Have you stress tested your assay?

Directory of Open Access Journals (Sweden)

Zheng Cao

2016-08-01

Full Text Available Objectives: When a clinical assay is stressed with extraordinarily high volume of specimens over a short period of time, extra caution may be needed to avoid systematic errors and biases. Here we report our experience with a HgbA1c assay used for high volume wellness screening purpose, to illustrate the importance of stress testing during assay validation. Design and Methods: Over 15,000 whole blood specimens were tested for HgbA1c in a period of 2 months. HgbA1c was tested by an immunoturbidimetric method on a high through-put automation line. The HgbA1c population distribution in our study was compared to that from the NHANES database. Daily distributions of HgbA1c values ≥6%, means and medians were plotted. Correlation studies were performed between the high through-put immunoturbidimetric assay and a medium through-put HPLC method. Results: We observed a shift of HgbA1c distribution to the higher values compared to the NHANES. A bias of 15–20% was noted from further stress testing where large number of samples were batched and tested using the immunoturbidimetric assay. A 5–7% higher bias remained after implementing a cuvette washing program after each HgbA1c sample. We hypothesized this bias was caused by build-up of blood cell fragments in the cuvettes when continuous whole blood samples are run through the system. Our experience suggests stress testing needs to be incorporated early in the test validation process for high volume batched screening applications. This seemingly extra validation step may save significant troubleshooting and retesting efforts down the road. Keywords: Hemoglobin A1c, Immunoturbidimetric assay, HPLC, Quality assurance, Systematic bias, High volume, Automation

Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

Science.gov (United States)

Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

2005-02-01

The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

Effect of Chlorine on Giardia lamblia Cyst Viability

OpenAIRE

Jarroll, Edward L.; Bingham, Alan K.; Meyer, Ernest A.

1981-01-01

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5°C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water...

Assessment of microbial viability in municipal sludge following ultrasound and microwave pretreatments and resulting impacts on the efficiency of anaerobic sludge digestion.

Science.gov (United States)

Cella, Monica Angela; Akgul, Deniz; Eskicioglu, Cigdem

2016-03-01

A range of ultrasonication (US) and microwave irradiation (MW) sludge pretreatments were compared to determine the extent of cellular destruction in micro-organisms within secondary sludge and how this cellular destruction translated to anaerobic digestion (AD). Cellular lysis/inactivation was measured using two microbial viability assays, (1) Syto 16® Green and Sytox® Orange counter-assay to discern the integrity of cellular membranes and (2) a fluorescein diacetate assay to understand relative enzymatic activity. A range of MW intensities (2.17-6.48 kJ/g total solids or TS, coinciding temperatures of 60-160 °C) were selected for comparison via viability assays; a range of corresponding US intensities (2.37-27.71 kJ/g TS, coinciding sonication times of 10-60 min at different amplitudes) were also compared to this MW range. The MW pretreatment of thickened waste activated sludge (tWAS) caused fourfold to fivefold greater cell death than non-pretreated and US-pretreated tWAS. The greatest microbial destruction occurred at MW intensities greater than 2.62 kJ/g TS of sludge, after which increased energy input via MW did not appear to cause greater microbial death. In addition, the optimal MW pretreatment (80 °C, 2.62 kJ/g TS) and corresponding US pretreatment (10 min, 60 % amplitude, 2.37 kJ/g TS) were administered to the tWAS of a mixed sludge and fed to anaerobic digesters over sludge retention times (SRTs) of 20, 14, and 7 days to compare effects of feed pretreatment on AD efficiency. The digester utilizing MW-pretreated tWAS (80 °C, 2.62 kJ/g TS) had the greatest fecal coliform removal (73.4 and 69.8 % reduction, respectively), greatest solids removal (44.2 % TS reduction), and highest overall methane production (248.2 L CH4/kg volatile solids) at 14- and 7-day SRTs. However, despite the fourfold to fivefold increases in cell death upon pretreatment, improvements from the digester fed MW-pretreated sludge were marginal (i.e., increases in efficiency of less

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.

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

International Nuclear Information System (INIS)

Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

2016-01-01

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.

Assessment of viability in the bacterial standing stock of the Antarctic Sea from the Indian side

Digital Repository Service at National Institute of Oceanography (India)

LokaBharathi, P.A.; Nair, S.; DeSouza, M.J.B.D.; Chandramohan, D.

designated as section 2 they were one order higher and ranged from 10 sup(9-10) L sup(-1). The percentage of viability in the region was high corresponding to the generally high chlorophyll and primary productivity encountered in the eastern Agulhas bank...

Novel double-isotope technique for enzymatic assay of catecholamines, permitting high precision, sensitivity and plasma sample capacity

International Nuclear Information System (INIS)

Brown, M.J.; Jenner, D.A.

1981-01-01

A novel use of a double-isotope method is described which allows radioenzymatic assays to combine precision and sensitivity. In the catechol O-methyltransferase assay separate portions of each plasma sample are incubated with either S-[ 3 H]- or S-[ 14 C]-adenosyl-L-methionine. Standards of noradrenaline and adrenaline are added to the latter portions and are thus converted into standards of [ 14 C]metadrenalines. These are added to the 3 H-labelled portions after the incubation, where they function as tracers. The final recovery of 14 C radioactivity corrects for (a) the efficiency of methylation in the plasma sample concerned and (b) the recovery of metadrenalines during the extraction procedures. The 3 H/ 14 C ratio is constant in each assay for a given catecholamine concentration and is determined for samples to which standards of noradrenaline and adrenaline are added to the 3 H- (as well as the 14 C-) labelled portions before the initial incubation. The sensitivity of the assay is increased by using high specific radioactivity S-[ 3 H]adenosyl-L-methionine, and low backgrounds are maintained by catecholamine depletion in vivo in the rats used for enzyme preparation. Both catecholamines (1.5 pg/ml; 10 pmol/l) may be detected; the coefficients of variation are 3.0 and 3.2% for noradrenaline and adrenaline respectively (intra-assay) and 4.6 and 5.0% (inter-assay). (author)

High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

Science.gov (United States)

Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

2013-01-01

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. Copyright © 2012 Wiley Periodicals, Inc.

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

International Nuclear Information System (INIS)

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang; Liu, Yan-Hong; Li, Yan; Wang, Jia-Ye; Hattori, Toshio; Ling, Hong; Zhang, Feng-Min

2010-01-01

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

Science.gov (United States)

Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

2018-04-01

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

Science.gov (United States)

Magcwebeba, Tandeka Unathi; Riedel, Sylvia; Swanevelder, Sonja; Swart, Pieter; De Beer, Dalene; Joubert, Elizabeth; Andreas Gelderblom, Wentzel Christoffel

2016-11-01

The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo. © 2016 Royal Pharmaceutical Society.

Experimental Calcium Silicate-Based Cement with and without Zirconium Oxide Modulates Fibroblasts Viability.

Science.gov (United States)

Slompo, Camila; Peres-Buzalaf, Camila; Gasque, Kellen Cristina da Silva; Damante, Carla Andreotti; Ordinola-Zapata, Ronald; Duarte, Marco Antonio Hungaro; de Oliveira, Rodrigo Cardoso

2015-01-01

The aim of this study was to verify whether the use of zirconium oxide as a radiopacifier of an experimental calcium silicate-based cement (WPCZO) leads to cytotoxicity. Fibroblasts were treated with different concentrations (10 mg/mL, 1 mg/mL, and 0.1 mg/mL) of the cements diluted in Dulbecco's modified Eagle's medium (DMEM) for periods of 12, 24, and 48 h. Groups tested were white Portland cement (WPC), white Portland cement with zirconium oxide (WPCZO), and white mineral trioxide aggregate Angelus (MTA). Control group cells were not treated. The cytotoxicity was evaluated through mitochondrial-activity (MTT) and cell-density (crystal violet) assays. All cements showed low cytotoxicity. In general, at the concentration of 10 mg/mL there was an increase in viability of those groups treated with WPC and WPCZO when compared to the control group (pcement with 20% zirconium oxide as the radiopacifier showed low cytotoxicity as a promising material to be exploited for root-end filling.

Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese.

Science.gov (United States)

Vinderola, C G; Prosello, W; Ghiberto, T D; Reinheimer, J A

2000-09-01

We evaluated the suitability of Argentinian Fresco cheese as a food carrier of probiotic cultures. We used cultures of Bifidobacterium bifidum (two strains), Bifidobacterium longum (two strains), Bifidobacterium sp. (one strain), Lactobacillus acidophilus (two strains), and Lactobacillus casei (two strains) in different combinations, as probiotic adjuncts. Probiotic, lactic starter (Lactococcus lactis and Streptococcus thermophilus), and contaminant (coliforms, yeasts, and molds) organisms were counted at 0, 30, and 60 d of refrigerated storage. Furthermore, the acid resistance of probiotic and starter bacteria was determined from hydrochloric solutions (pH 2 and 3) of Fresco cheese. The results showed that nine different combinations of bifidobacteria and L. acidophilus had a satisfactory viability (count decreases in 60 d casei cultures assayed also showed a satisfactory survival (counts decreased casei). On the other hand, the three combinations of bifidobacteria, L. acidophilus, and L. casei tested adapted well to the Fresco cheese environment. When a cheese homogenate at pH 3 was used to partially simulate the acidic conditions in the stomach, the probiotic cultures had an excellent ability to remain viable up to 3 h. At pH 2, the cell viability was more affected; B. bifidum was the most resistant organism. This study showed that the Argentinian Fresco cheese could be used as an adequate carrier of probiotic bacteria.

Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin.

Science.gov (United States)

Lin, Yueh-Ming; Chen, Chih-I; Hsiang, Yi-Ping; Hsu, Yung-Chia; Cheng, Kung-Chuan; Chien, Pei-Hsuan; Pan, Hsiao-Lin; Lu, Chien-Chang; Chen, Yun-Ju

2018-06-14

Chemotherapeutic 5-fluorouracil (5-FU) combined with oxaliplatin is often used as the standard treatment for colorectal cancer (CRC). The disturbing side effects and drug resistance commonly observed in chemotherapy motivate us to develop alternative optimal therapeutic options for CRC treatment. Chrysin, a natural and biologically active flavonoid abundant in propolis, is reported to have antitumor effects on a few CRCs. However, whether and how chrysin achieves similar effectiveness to the 5-FU combination is not clear. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), western blotting, fluorescence microscopy, and reactive oxygen species (ROS) production were assayed. We found that chrysin exhibited similar inhibition of cell viability as the 5-FU combination in a panel of human CRC cells. Furthermore, the results showed that chrysin significantly increased the levels of LC3-II, an autophagy-related marker, in CRC cells, which was not observed with the 5-FU combination. More importantly, blockage of autophagy induction restored chrysin-attenuated CRC cell viability. Further mechanistic analysis revealed that chrysin, not the 5-FU combination, induced ROS generation, and in turn, inhibited the phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Collectively, these results imply that chrysin may be a potential replacement for the 5-FU and oxaliplatin combination to achieve antitumor activity through autophagy for CRC treatment in the future.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

Science.gov (United States)

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18

International Nuclear Information System (INIS)

Pastrana, Diana V.; Buck, Christopher B.; Pang, Y.-Y. S.; Thompson, Cynthia D.; Castle, Philip E.; FitzGerald, Peter C.; Krueger Kjaer, Susanne; Lowy, Douglas R.; Schiller, John T.

2004-01-01

Sensitive high-throughput neutralization assays, based upon pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene, were developed and validated for human papillomavirus (HPV)16, HPV18, and bovine papillomavirus 1 (BPV1). SEAP pseudoviruses were produced by transient transfection of codon-modified papillomavirus structural genes into an SV40 T antigen expressing line derived from 293 cells, yielding sufficient pseudovirus from one flask for thousands of titrations. In a 96-well plate format, in this initial characterization, the assay was reproducible and appears to be as sensitive as, but more specific than, a standard papillomavirus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). The neutralization assay detected type-specific HPV16 or HPV18 neutralizing antibodies (titers of 160-10240) in sera of the majority of a group of women infected with the corresponding HPV type, but not in virgin women. Sera from HPV16 VLP vaccinees had high anti-HPV16 neutralizing titers (mean: 45000; range: 5120-163840), but no anti-HPV18 neutralizing activity. The SEAP pseudovirus-based neutralization assay should be a practical method for quantifying potentially protective antibody responses in HPV natural history and prophylactic vaccine studies

Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

Science.gov (United States)

Kim, Wan-Joong; Cho, Hyo Young; Jeong, Bongjin; Byun, Sangwon; Huh, JaeDoo; Kim, Young Jun

2017-01-01

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min. PMID:29278402

Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

Directory of Open Access Journals (Sweden)

Khadija G. Syeda

2017-10-01

Full Text Available Background/Aims: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin B undergo posttranslational modifications (e.g., isoprenylation at their C-terminal cysteine residues. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Using human islets, rodent islets, and INS-1 832/13 cells, we recently reported significant metabolic defects under glucotoxic and endoplasmic reticulum (ER stress conditions, including caspase 3 activation and lamin B degradation. The current study is aimed at further understanding the regulatory roles of protein prenylation in the induction of the aforestated metabolic defects. Methods: Subcellular phase partitioning assay was done using Triton X-114. Cell morphology and metabolic cell viability assays were carried out using standard methodologies. Results: We report that exposure of pancreatic β-cells to Simvastatin, an inhibitor of mevalonic acid (MVA biosynthesis, and its downstream isoprenoid derivatives, or FTI-277, an inhibitor of farnesyltransferase that mediates farnesylation of lamins, leads to activation of caspase 3 and lamin B degradation. Furthermore, Simvastatin-treatment increased activation of p38MAPK (a stress kinase and inhibited ERK1/2 (regulator of cell proliferation. Inhibition of farnesylation also resulted in the release of degraded lamin B into the cytosolic fraction and promoted loss in metabolic cell viability. Conclusion: Based on these findings we conclude that protein prenylation plays key roles in islet β-cell function. These findings affirm further support to the hypothesis that defects in prenylation pathway induce caspase-3 activation and nuclear lamin degradation in pancreatic β-cells under the duress of metabolic stress (e.g., glucotoxicity.

Pollen viability of Salix myrtilloides L. – an endangered species in Poland

Directory of Open Access Journals (Sweden)

Magdalena Pogorzelec

2016-12-01

Full Text Available Salix myrtilloides L. (swamp willow is the most endangered species among the boreal Salix species in Poland. The number and size of its population have been decreasing constantly since the 1990s. The main aim of the study was to determine the viability of collected S. myrtilloides pollen and optimal conditions for its in vitro germination. The pollen of S. myrtilloides was collected from 25 male individuals from a population growing in the mid-forest peat bog Dekowina (Sobibór Landscape Park in May 2014. Two methods were applied to estimate the viability of fresh and stored pollen: staining pollen with 2% acetocarmine solution and in vitro germinability. Various temperature (11°C, 23°C and light conditions as well as different concentrations of glucose (1%, 2.5%, 5%, or 7.5% were tested for the optimization of in vitro germination. We documented relatively high S. myrtilloides pollen viability. Pollen tube growth was found to be largely affected by both glucose content in the medium and thermal conditions during germination. Fresh pollen germinated most effectively on the medium with 2.5% glucose (stored pollen – in 5% glucose, at 23°C and in the presence of light. We conclude that pollen viability of S. myrtilloides does not seem to be a limiting factor for reproductive success. Moreover, the pollen is not sterile even after storage for 12 months. The S. myrtilloides individuals from the Dekowina peat bog produce viable pollen grains that are able to germinate and therefore it can be used to pollinate other populations present in the Polesie Lubelskie region for gene pool enrichment.

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.

MAML1 regulates cell viability via the NF-κB pathway in cervical cancer cell lines

International Nuclear Information System (INIS)

Kuncharin, Yanin; Sangphech, Naunpun; Kueanjinda, Patipark; Bhattarakosol, Parvapan; Palaga, Tanapat

2011-01-01

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and β-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-κB pathway was investigated, CaSki cells overexpressing DN

MAML1 regulates cell viability via the NF-{kappa}B pathway in cervical cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Kuncharin, Yanin [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Sangphech, Naunpun [Biotechnology Program, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Kueanjinda, Patipark [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Bhattarakosol, Parvapan [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Department of Microbiology, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand)

2011-08-01

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and {beta}-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-{kappa}B pathway was investigated, CaSki cells overexpressing

Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability

Science.gov (United States)

Luo, Haitao; Jiang, Bingbing; Li, Bingyun; Li, Zhaoliang; Jiang, Bing-Hua; Chen, Yi Charlie

2012-01-01

Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

Science.gov (United States)

Cousins, Matthew M; Swan, David; Magaret, Craig A; Hoover, Donald R; Eshleman, Susan H

2012-01-01

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

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

Science.gov (United States)

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

2012-02-01

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

Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

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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

Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

Science.gov (United States)

Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

2007-01-01

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-PCR, SSOP-ELISA, PCR Dot

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

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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.

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

Science.gov (United States)

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

2015-03-01

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

Anthropogenic Impacts on Mortality and Population Viability of the Monarch Butterfly.

Science.gov (United States)

Malcolm, Stephen B

2018-01-07

Monarch butterflies (Danaus plexippus) are familiar herbivores of milkweeds of the genus Asclepias, and most monarchs migrate each year to locate these host plants across North American ecosystems now dominated by agriculture. Eastern migrants overwinter in high-elevation forests in Mexico, and western monarchs overwinter in trees on the coast of California. Both populations face three primary threats to their viability: (a) loss of milkweed resources for larvae due to genetically modified crops, pesticides, and fertilizers; (b) loss of nectar resources from flowering plants; and (c) degraded overwintering forest habitats due to commercially motivated deforestation and other economic activities. Secondary threats to population viability include (d) climate change effects on milkweed host plants and the dynamics of breeding, overwintering, and migration; (e) the influence of invasive plants and natural enemies; (f) habitat fragmentation and coalescence that promote homogeneous, species-depleted landscapes; and (g) deliberate culture and release of monarchs and invasive milkweeds.

Space mutagenic effects on cistanche deserticola seed viability and parasitic condition

International Nuclear Information System (INIS)

Xu Rong; Zhou Feng; Yu Jing; Chen Jun; Sun Suqin; Liu Yougang; Liu Tongning

2009-01-01

The seeds of Cistanche deserticola which from a single plant with fine properties were carried to the space by the recoverable experiment satellite 'Shijian No.8'. After space loading, the seed viability and characters of infrared spectroscopy were analyzed and then planted to observe and investigate the variation and heredity. The results showed that compared to the control group, the seed viability and germination rate increased observably after space loading. As well, the plants grew much healthier at the seedling stage. The results indicated that space loading has obvious promote effects on the seed viability, germination rate and disease resistance of Cistanche deserticola. The analysis results of infrared spectroscopy showed the contents of protein and carbohydrate were increased distinctly and the contents of oil or fat reduced somewhat in the seeds after space loading. The intensity ratio between characteristic absorption peak of protein and characteristic absorption peak of fat (I 1625 /I 1745 ) were increased from 1.07 to 1.16, which could be related to the enhancement of seed viability and the reduction of germination restraint substances. It could be concluded that microgravity and intense radiation in the space caused seed viability and material metabolism change. (authors)

Population viability analysis on domestic horse breeds (Equus caballus)

DEFF Research Database (Denmark)

Thirstrup, Janne Pia; Bach, Lars; Loeschcke, Volker

2009-01-01

simulation package was used for the population viability analysis. First, we investigated the future viability of these breeds based on present demographic and environmental parameters. Second, a sensitivity analysis revealed the most important variables for the viability of these breeds. Third, we examined...... concerning reproduction of the mares had the greatest impact, with the number of mares actively breeding being the most influential on the population forecasts. The results suggest that closing the Knabstrupper studbooks can be done only if increasing the number of mares actively breeding counteracts...... the loss of genetic variation attributable to such a management strategy. It is recommended, based on these results, that the number of Frederiksborg and Knabstrupper mares actively breeding must be increased to approximately 30% in the 2 breeds that are presently using only 13%, while leaving the third...

Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

Science.gov (United States)

Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

2016-02-01

We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

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

2018-06-01

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.

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

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

Turnover of Glycerolipid Metabolite Pool and Seed Viability

Directory of Open Access Journals (Sweden)

Xiao-Long Hu

2018-05-01

Full Text Available Hydration–dehydration cycles can frequently cause stress to seeds, but can also be used to improve germination. However, the molecular basis of the stress caused is poorly understood. Herein, we examine the effects of hydration–dehydration cycles on seed viability and profile the membrane glycerolipid molecular species. We find that seed viability was not affected during the first two cycles, but significantly decreased as further cycles were applied, until all viability was lost. The abundances of seven glycerolipid classes increased and decreased through hydration and dehydration, respectively, but the phosphatidic acid and diacylglycerol abundances changed in the opposite sense, while total glycerolipid contents remained constant. This suggests that during hydration–dehydration cycles, turnover of glycerolipid metabolite pools take place, while no significant lipid synthesis or degradation is involved. As further hydration–dehydration cycles occurred, lipid unsaturation increased, plastidic lipids decreased, and phosphatidylserine acyl chains lengthened. The latter two could be lethal for seeds. Our findings reveal a novel model of membrane lipid changes, and provide new insights into the responses of seeds to hydration–dehydration cycles.

Ca-Lignosulphonate and sclerotial viability of Sclerotinia sclerotiorum

Directory of Open Access Journals (Sweden)

MATTEO MONTANARI

2012-01-01

Full Text Available Lignosulphonates, low cost by-products of the pulping process, have shown suppressive effects against some diseases caused by soil-borne pathogens. In this study, the effect of 1.5% v/v calcium lignosulphonate (Ca-Ls amendment to two commercial potting mixes (peat + coconut fibres; PC; and municipal compost + peat + pumice; MCPP on the viability of Sclerotinia sclerotiorum sclerotia was investigated. Sclerotia were buried in the Ca-Ls amended substrates for 30 days. Non-amended PC and MCPP, sterile sand and sterile PC with and without Ca-Ls were used as controls. The viability of sclerotia recovered from PC and MCPP amended with Ca-Ls was reduced by 50 and 42% respectively compared to control treatments. Ca-Ls amendment decreased sclerotial viability by enhancing the activity of the indigenous mycoparasitic fungi, Fusarium oxysporum, Mucor spp. and Trichoderma spp. The biocontrol ability of Ca-Ls against sclerotia was due to the stimulation of microbial activity and is, therefore, strictly dependent on the microbial composition of the substrate.

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

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