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Sample records for survival clonogenic assay

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

    Science.gov (United States)

    Woolston, Caroline; Martin, Stewart

    2011-01-01

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

  2. 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.

  3. 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.

  4. Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.

    Science.gov (United States)

    Pauwels, Bea; Korst, Annelies E C; de Pooter, Christel M J; Pattyn, Greet G O; Lambrechts, Hilde A J; Baay, Marc F D; Lardon, Filip; Vermorken, Jan B

    2003-03-01

    Since there is a growing interest in preclinical research on interactions between radiation and cytotoxic agents, this study focused on the development of an alternative to the very laborious clonogenic assay (CA). The colorimetric sulforhodamine B (SRB) assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines (A549, H292), one colon cancer cell line (HT-29) and one breast cancer cell line (MCF-7). In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both assays. The dose-response curves obtained with the SRB assay and the CA were very similar up to 6 Gy. The radiosensitivity parameters (SF(2), alpha, beta, MID and ID(50)) obtained from the SRB assay and the CA were not significantly different between H292, A549 and MCF-7 cells. The radiation dose-response curves for A549 and H292 cells pretreated with 4 n M gemcitabine for 24 h clearly showed a radiosensitizing effect with both assays. The dose-enhancement factors obtained with the SRB assay and the CA were 1.80 and 1.76, respectively, for A549 cells, and 1.52 and 1.41 for H292 cells. The SRB assay was shown to be as useful as the more traditional CA for research on chemotherapy/radiotherapy interactions in cell lines with moderate radiosensitivity. This assay will be used for more extensive in vitro research on radiosensitizing compounds in these cell lines.

  5. Effect of ultrasound sonication on clonogenic survival and mitochondria of ovarian cancer cells in the presence of methylene blue.

    Science.gov (United States)

    Xiang, Junyan; Leung, Albert Wingnang; Xu, Chuanshan

    2014-10-01

    This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells. © 2014 by the American Institute of Ultrasound in Medicine.

  6. Clonogenic assays measure leukemia stem cell killing not detectable by chromium release and flow cytometric cytotoxicity assays.

    Science.gov (United States)

    Williams, Brent A; Wang, Xing-Hua; Keating, Armand

    2010-11-01

    NK-92, a permanent natural killer (NK) cell line, shows cytotoxicity against a variety of tumors and has been tested in a phase I trial. We tested the toxicity of NK-92 and chemotherapy drugs against the stem cell capacity of the acute leukemia cell line, KG1. While the chromium-release assay is the most common method for assessing cytotoxicity of immune effectors, and flow cytometry is increasingly used, the relationship of either assay to clonogenic readouts remains unknown. KG1 was assessed for stem cell frequency by serial dilution, single-cell sorting and colony growth in methylcellulose. KG1 was sorted into CD34(+) CD38(+) and CD34(+) CD38⁻ populations and recultured in liquid medium or methylcellulose to determine the proliferative capacity of each fraction. Cytotoxicity of NK-92, daunorubicin and cytarabine against KG1 was measured using the chromium-release assay, flow cytometry and clonogenic assays. The culture-initiating cell frequency of whole KG1 was between 1 in 100 to 1000 by serial dilution and single-cell sorting. Although a rare (1-3%) CD34(+) CD38⁻ population could be demonstrated in KG1, both fractions had equivalent proliferative capacity. The cumulative flow cytotoxicity assay was more sensitive than the chromium-release assay in detecting target cell killing. At a 10:1 ratio NK-92 eliminated the clonogenic capacity of KG1, which was not predicted by the chromium-release assay. Clonogenic assays provide a more sensitive means of assessing the effect of a cytotoxic agent against putative cancer stem cells within cell lines, provided that they grow well in liquid culture medium or methylcellulose.

  7. ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays.

    Directory of Open Access Journals (Sweden)

    Camilo Guzmán

    Full Text Available The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin "ColonyArea", which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS on human glioblastoma cells (T98G. The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

  8. Cytotoxicity of artemisinin, a dimer of dihydroartemisinin, artemisitene and eupatoriopicrin as evaluated by the MTT and clonogenic assay

    NARCIS (Netherlands)

    Beekman, AC; Woerdenbag, HJ; Kampinga, HH; Konings, AWT

    Artemisinin and its derivatives possess an endoperoxide bridge, which is thought to lead to the production of free-radical species, The cytotoxicity of some of these agents to a murine Ehrlich ascites (EN19) and a human HeLa S3 cancer cell line was determined using the MTT and the clonogenic assay,

  9. A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells

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    Huber Peter E

    2011-02-01

    Full Text Available Abstract Background The protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2. Methods MRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB, or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay. Results TBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy or foci formation (1 Gy. While DNA fragment rejoining (PFGE was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination, but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival enhanced radiation-induced cell killing in the clonogenic assay. Conclusions The data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.

  10. Assessment of the Radioprotection Efficacy of Antioxidant Substances in Foods in regard to Clonogenic Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyosung; Lee, Minho; Kim, Eunhee [Seoul National Univ., Seoul (Korea, Republic of)

    2014-05-15

    Radiation is perceived as a hazard in spite of its diverse uses. The public are more sensitive than ever to the activities from the nuclear power plant industry since Fukushima accident and the consequential environment contamination. The never-dissolved fear from the public of the nuclear energy costs the nuclear industry too much for safety measures not to mention brings the public uneasy life. Beta carotene, oltipraz and luteolin, which are easily taken from various foods, are substances that may protect cells from damage caused by unstable molecules known as free radicals. The purpose of this study is to inform people of accessible radiation protection measures in everyday lives. Three antioxidant substances were investigated regarding their radioprotection effects counted in terms of clonogenic cell surviving fraction in vitro. The radioprotection efficacy was observed with those substances at concentrations below certain levels. At concentrations beyond those levels, the efficacy was canceled out by their inherent cytotoxicity.

  11. Impact of flattening-filter-free radiation on the clonogenic survival of astrocytic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steenken, Caroline; Fleckenstein, Jens; Kegel, Stefan; Jahnke, Lennart; Simeonova, Anna; Hartmann, Linda; Kuebler, Jens; Veldwijk, Marlon R.; Wenz, Frederik; Herskind, Carsten; Giordano, Frank Anton [Universitaetsmedizin Mannheim (UMM), Medical Faculty Mannheim, Heidelberg University, Department of Radiation Oncology, Mannheim (Germany)

    2015-07-15

    Flattening-filter-free (FFF) beams are increasingly used in radiotherapy as delivery times can be substantially reduced. However, the relative biologic effectiveness (RBE) of FFF may be increased relative to conventional flattened (FLAT) beams due to differences in energy spectra. Therefore, we investigated the effects of FFF and FLAT beams on the clonogenic survival of astrocytoma cells. Three cell lines (U251, U251-MGMT, and U87) were irradiated with 6-MV and 10-MV X-rays from a linear accelerator in FFF- or FLAT-beam modes at dose rates in the range of 0.5-24 Gy/min. The surviving fraction (SF) as function of dose (2-12 Gy) was determined by the colony formation assay and fitted by the linear-quadratic model. For both beams (FFF or FLAT), the cells were pelleted in conical 15-ml centrifuge tubes and irradiated at 2-cm depth in a 1 x 1-cm{sup 2} area on the central axis of a 30 x 30-cm{sup 2} field. Dosimetry was performed with a 0.3-cm{sup 3} rigid ionization chamber. RBE was determined for FFF versus FLAT irradiation. The RBE of FFF at 7.3-11.3 Gy was 1.027 ± 0.013 and 1.063 ± 0.018 relative to FLAT beams for 6- and 10-MV beams, respectively, and was only significantly higher than 1 for 10 MV. Significantly increased survival rates were seen for lower dose rates (0.5 Gy/min FLAT vs. 5 Gy/min FLAT) at higher doses (11.9 Gy), while no differences were seen at dose rates ≥ 1.4 Gy/min (1.4 Gy/min FFF vs. 14 Gy/min FFF and 2.4 Gy/min FFF vs. 24 Gy/min FFF). FFF beams showed only a slightly increased RBE relative to FLAT beams in this experimental set-up, which is unlikely to result in clinically relevant differences in outcome. (orig.) [German] Die Flattening-Filter-freie (FFF) Bestrahlungstechnik findet zunehmend Verwendung, da sich die Applikationsdauer der einzelnen Fraktionen deutlich verkuerzen laesst. Aufgrund der Unterschiede im Spektrum koennte die relative biologische Wirksamkeit (RBW) von FFF jedoch hoeher sein als bei konventioneller Technik (d.h. bei

  12. Survival assays using Caenorhabditis elegans.

    Science.gov (United States)

    Park, Hae-Eun H; Jung, Yoonji; Lee, Seung-Jae V

    2017-02-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans.

  13. No supra-additive effects of goserelin and radiotherapy on clonogenic survival of prostate carcinoma cells in vitro

    Directory of Open Access Journals (Sweden)

    Thelen Paul

    2007-08-01

    Full Text Available Abstract Background Oncological results of radiotherapy for locally advanced prostate cancer (PC are significantly improved by simultaneous application of LHRH analoga (e.g. goserelin. As 85% of PC express LHRH receptors, we investigated the interaction of goserelin incubation with radiotherapy under androgen-deprived conditions in vitro. Methods LNCaP and PC-3 cells were stained for LHRH receptors. Downstream the LHRH receptor, changes in protein expression of c-fos, phosphorylated p38 and phosphorylated ERK1/2 were analyzed by means of Western blotting after incubation with goserelin and irradiation with 4 Gy. Both cell lines were incubated with different concentrations of goserelin in hormone-free medium. 12 h later cells were irradiated (0 – 4 Gy and after 12 h goserelin was withdrawn. Endpoints were clonogenic survival and cell viability (12 h, 36 h and 60 h after irradiation. Results Both tested cell lines expressed LHRH-receptors. Changes in protein expression demonstrated the functional activity of goserelin in the tested cell lines. Neither in LNCaP nor in PC-3 any significant effects of additional goserelin incubation on clonogenic survival or cell viability for all tested concentrations in comparison to radiation alone were seen. Conclusion The clinically observed increase in tumor control after combination of goserelin with radiotherapy in PC cannot be attributed to an increase in radiosensitivity of PC cells by goserelin in vitro.

  14. The cytotoxic activity of cisplatin, carboplatin and teniposide alone and combined determined on four human small cell lung cancer cell lines by the clonogenic assay

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Christensen, I J

    1988-01-01

    Using the clonogenic assay to compare the cytotoxic activity of cisplatin and carboplatin on four human small cell lung cancer cell lines, cisplatin was shown to be equally or more potent than carboplatin at equitoxic doses with 1 h incubation. Increased potency of carboplatin was revealed when...

  15. An evaluation of dose equivalence between synchrotron microbeam radiation therapy and conventional broad beam radiation using clonogenic and cell impedance assays.

    Science.gov (United States)

    Ibahim, Mohammad Johari; Crosbie, Jeffrey C; Yang, Yuqing; Zaitseva, Marina; Stevenson, Andrew W; Rogers, Peter A W; Paiva, Premila

    2014-01-01

    High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments. To develop an in vitro approach to determine biological dose equivalence between MRT and BB using two different cell-based assays. The acute response of tumour and normal cell lines (EMT6.5, 4T1.2, NMuMG, EMT6.5ch, 4T1ch5, SaOS-2) to MRT (50-560 Gy) and BB (1.5-10 Gy) irradiation was investigated using clonogenic and real time cell impedance sensing (RT-CIS)/xCELLigence assays. MRT was performed using a lattice of 25 or 50 µm-wide planar, polychromatic kilovoltage X-ray microbeams with 200 µm peak separation. BB irradiations were performed using a Co60 teletherapy unit or a synchrotron radiation source. BB doses that would generate biological responses similar to MRT were calculated by data interpolation and verified by clonogenic and RT-CIS assays. For a given cell line, MRT equivalent BB doses identified by RT-CIS/xCELLigence were similar to those identified by clonogenic assays. Dose equivalence between MRT and BB were verified in vitro in two cell lines; EMT6.5ch and SaOS-2 by clonogenic assays and RT-CIS/xCELLigence. We found for example, that BB doses of 3.4±0.1 Gy and 4.40±0.04 Gy were radiobiologically equivalent to a peak, microbeam dose of 112 Gy using clonogenic and RT-CIS assays respectively on EMT6.5ch cells. Our data provides the first determination of biological dose equivalence between BB and MRT modalities for different cell lines and identifies RT-CIS/xCELLigence assays as a suitable substitute for clonogenic assays. These results will be useful for the safe selection of MRT doses for future veterinary and clinical trials.

  16. Non-monotonic changes in clonogenic cell survival induced by disulphonated aluminum phthalocyanine photodynamic treatment in a human glioma cell line

    Directory of Open Access Journals (Sweden)

    Muralidhar K

    2010-04-01

    Full Text Available Abstract Background Photodynamic therapy (PDT involves excitation of sensitizer molecules by visible light in the presence of molecular oxygen, thereby generating reactive oxygen species (ROS through electron/energy transfer processes. The ROS, thus produced can cause damage to both the structure and the function of the cellular constituents resulting in cell death. Our preliminary investigations of dose-response relationships in a human glioma cell line (BMG-1 showed that disulphonated aluminum phthalocyanine (AlPcS2 photodynamically induced loss of cell survival in a concentration dependent manner up to 1 μM, further increases in AlPcS2concentration (>1 μM were, however, observed to decrease the photodynamic toxicity. Considering the fact that for most photosensitizers only monotonic dose-response (survival relationships have been reported, this result was unexpected. The present studies were, therefore, undertaken to further investigate the concentration dependent photodynamic effects of AlPcS2. Methods Concentration-dependent cellular uptake, sub-cellular localization, proliferation and photodynamic effects of AlPcS2 were investigated in BMG-1 cells by absorbance and fluorescence measurements, image analysis, cell counting and colony forming assays, flow cytometry and micronuclei formation respectively. Results The cellular uptake as a function of extra-cellular AlPcS2 concentrations was observed to be biphasic. AlPcS2 was distributed throughout the cytoplasm with intense fluorescence in the perinuclear regions at a concentration of 1 μM, while a weak diffuse fluorescence was observed at higher concentrations. A concentration-dependent decrease in cell proliferation with accumulation of cells in G2+M phase was observed after PDT. The response of clonogenic survival after AlPcS2-PDT was non-monotonic with respect to AlPcS2 concentration. Conclusions Based on the results we conclude that concentration-dependent changes in physico

  17. Characterizing responses to gamma radiation by a highly clonogenic fish brain endothelial cell line.

    Science.gov (United States)

    Vo, Nguyen T K; Sokeechand, Bibi S H; Seymour, Colin B; Mothersill, Carmel E

    2017-07-01

    The clonogenic property and radiobiological responses of a fish brain endothelial cell line, eelB, derived from the American eel were studied. Clonogenic assays were performed to determine the plating efficiency of the eelB cells and to evaluate the clonogenic survival fractions after direct irradiation to low-dose low-LET gamma radiation or receiving irradiated cell conditioned medium in the bystander effect experiments. eelB had the second highest plating efficiency ever reported to date for fish cell lines. Large eelB macroscopic colonies could be formed in a short period of time and were easy to identify and count. Unlike with other fish clonogenic cell lines, which had a relatively slow proliferation profile, clonogenic assays with the eelB cells could be completed as early as 12 days in culture. After direct irradiation with gamma rays at low doses ranging from 0.1Gy to 5Gy, the dose-clonogenic survival curve of the eelB cell line showed a linear trend and did not develop a shoulder region. A classical radio-adaptive response was not induced with the clonogenic survival endpoint when the priming dose (0.1 or 0.5Gy) was delivered 6h before the challenge dose (3 or 5Gy). However, a radio-adaptive response was observed in progeny cells that survived 5Gy and developed lethal mutations. eelB appeared to lack the ability to produce damaging radiation-induced bystander signals on both eelB and HaCaT recipient cells. eelB cell line could be a very useful cell model in the study of radiation impacts on the aquatic health. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Radiogenic neoplasia in thyroid and mammary clonogens

    Energy Technology Data Exchange (ETDEWEB)

    Clifton, K.H.

    1992-05-20

    We have developed rat thyroid and mammary clonogen transplantation systems for the study of radiogenic cancer induction at the target cell level in vivo. The epithelial cell populations of both glands contain small subpopulations of cells which are capable of giving rise to monoclonal glandular structures when transplanted and stimulated with appropriate hormones. Previous results indicated that these clonogens are the precursor cells of radiogenic cancer, and that initiation, is common event at the clonegenic cell level. Detailed information on the physiologic control of clonogen proliferation, differentiation, and total numbers is thus essential to an understanding of the carcinogenic process. We report here studies on investigations on the relationships between grafted thyroid cell number and the rapidity and degree of reestablishment of the thyroid-hypothalamus-pituitary feedback axis in thyroidectomized rats maintained on a normal diet or an iodine deficient diet; studies of the persistence of, and the differentiation potential and functional characteristics of, the TSH-(thyrotropin-) responsive sub- population of clonogens during goitrogenesis, the plateau-phase of goiter growth, and goiter involution; studies of changes in the size of the clonogen sub-population during goitrogenesis, goiter involution and the response to goitrogen rechallenge; and a large carcinogenesis experiment on the nature of the grafted thyroid cell number-dependent suppression of promotion/progression to neoplasia in grafts of radiation-initiated thyroid cells. Data from these studies will be used in the design of future carcinogenesis experiments on neoplastic initiation by high and low LET radiations and on cell interactions during the neoplastic process.

  19. The anti-protozoan drug nifurtimox preferentially inhibits clonogenic tumor cells under hypoxic conditions

    OpenAIRE

    Li, Quhuan; Lin, Qun; Kim, Hoon; Yun, Zhong

    2017-01-01

    Tumor hypoxia is an independent prognostic indicator of tumor malignant progression and poor patient survival. Therefore, eradication of hypoxic tumor cells is of paramount importance for successful disease control. In this study, we have made a new discovery that nifurtimox, a clinically approved drug to treat Chagas disease caused by the parasitic protozoan trypanosomes, can function as a hypoxia-activated cytotoxin. We have found that nifurtimox preferentially kill clonogenic tumor cells e...

  20. Cell Proliferation and Cytotoxicity Assays.

    Science.gov (United States)

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

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

  1. Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

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    Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

    2013-10-14

    The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

  2. Multiple MTS Assay as the Alternative Method to Determine Survival Fraction of the Irradiated HT-29 Colon Cancer Cells.

    Science.gov (United States)

    Arab-Bafrani, Zahra; Shahbazi-Gahrouei, Daryoush; Abbasian, Mahdi; Fesharaki, Mehrafarin

    2016-01-01

    A multiple colorimetric assay has been introduced to evaluate the proliferation and determination of survival fraction (SF) of irradiated cells. The estimation of SF based on the cell-growth curve information is the major advantage of this assay. In this study, the utility of multiple-MTS assay for the SF estimation of irradiated HT-29 colon cancer cells, which were plated before irradiation, was evaluated. The SF of HT-29 colon cancer cells under irradiation with 9 MV photon was estimated using multiple-MTS assay and colony assay. Finally, the correlation between two assays was evaluated. Results showed that there are no significant differences between the SF obtained by two assays at different radiation doses (P > 0.05), and the survival curves have quite similar trends. In conclusion, multiple MTS-assay can be a reliable method to determine the SF of irradiated colon cancer cells that plated before irradiation.

  3. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    Science.gov (United States)

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

    2012-10-09

    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

  4. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells.

    Science.gov (United States)

    Vega-Avila, Elisa; Pugsley, Michael K

    2011-01-01

    The aim of this review is to briefly describe some colorimetric methods that are commonly used to evaluate a new chemical entity (NCE) on cell cultures in non-clinical oncology discovery research. These methods have the distinct advantage over other techniques in that they can be applied and used in a cell monolayer or a suspension culture. Both protein assay determination and cell viability assays may be conducted using these culture systems. The viability of cell cultures is routinely assessed by utilizing the metabolic capacity of cells which biochemically convert chemicals (usually color dyes) which can then be conveniently measured at specific wavelengths using a multi-well plate reader. Resazurin (Alamar Blue) is an example of one of these metabolically active compounds. Resazurin is a nontoxic dye that can also be used to measure migration and cellular invasion without resorting to sacrifice of the cells during the test procedure. Another is 5-bromo-2-deoxyuridine (bromodeoxyuridine or BrdU) which is a thymidine analog that incorporates into the DNA of dividing cells during the S-phase of the cell cycle. We will also discuss the colorimetric version of the traditional 3H-thymidine incorporation and immunoenzymatic assay used to measure DNA synthesis and its application to discovery research.

  5. An optimized colony forming assay for low-dose-radiation cell survival measurement

    Energy Technology Data Exchange (ETDEWEB)

    Zhu J.; Sutherland B.; Hu W.; Ding N.; Ye C.; Usikalu M.; Li S.; Hu B.; Zhou G.

    2011-11-01

    The aim of this study is to develop a simple and reliable method to quantify the cell survival of low-dose irradiations. Two crucial factors were considered, the same number of cells plated in each flask and an appropriate interval between cell plating and irradiation. For the former, we optimized cell harvest with trypsin, diluted cells in one container, and directly seeded cells on the bottom of flasks in a low density before irradiation. Reproducible plating efficiency was obtained. For the latter, we plated cells on the bottom of flasks and then monitored the processing of attachment, cell cycle variations, and the plating efficiency after exposure to 20 cGy of X-rays. The results showed that a period of 4.5 h to 7.5 h after plating was suitable for further treatment. In order to confirm the reliability and feasibility of our method, we also measured the survival curves of these M059K and M059J glioma cell lines by following the optimized protocol and obtained consistent results reported by others with cell sorting system. In conclusion, we successfully developed a reliable and simple way to measure the survival fractions of human cells exposed to low dose irradiation, which might be helpful for the studies on low-dose radiation biology.

  6. TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Mark A.; Shekhar, Tanmay M. [Department of Biochemistry and Genetics, La Trobe University, Bundoora, Victoria (Australia); La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia); Hall, Nathan E. [La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia); Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Melbourne, Victoria (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry and Genetics, La Trobe University, Bundoora, Victoria (Australia); La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia)

    2016-05-15

    Highlights: • Treatment with TRAIL or EMS provokes mutations in clonogenically viable TK6 cells. • TRAIL is 2–5-fold less mutagenic than an equivalently lethal concentration of EMS. • EMS mainly causes transition mutations at the HPRT and TK1 loci of TK6 cells. • Most loss-of-function HPRT or TK1 mutations caused by TRAIL treatment are deletions. - Abstract: When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of

  7. Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays

    DEFF Research Database (Denmark)

    Molina, Lazaro; Rodriguez, Cayo Juan Ramos; Ronchel, Maria C.

    1998-01-01

    Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn5 transposon bearing a fusion of the P(lac) promoter to the gef killing gene...... and a fusion of the Pm promoter to the lad gene plus the positive regulator of the Pm promoter, the xylS gene. This mini-Tn5 transposon was transferred to the chromosome of Pseudomonas putida CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide...

  8. Age-dependent decline in mouse lung regeneration with loss of lung fibroblast clonogenicity and increased myofibroblastic differentiation.

    Directory of Open Access Journals (Sweden)

    Julia A Paxson

    Full Text Available While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days, and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a and more alpha smooth muscle actin (αSMA positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.

  9. The anti-protozoan drug nifurtimox preferentially inhibits clonogenic tumor cells under hypoxic conditions.

    Science.gov (United States)

    Li, Quhuan; Lin, Qun; Kim, Hoon; Yun, Zhong

    2017-01-01

    Tumor hypoxia is an independent prognostic indicator of tumor malignant progression and poor patient survival. Therefore, eradication of hypoxic tumor cells is of paramount importance for successful disease control. In this study, we have made a new discovery that nifurtimox, a clinically approved drug to treat Chagas disease caused by the parasitic protozoan trypanosomes, can function as a hypoxia-activated cytotoxin. We have found that nifurtimox preferentially kill clonogenic tumor cells especially under the hypoxic conditions of ≤0.1% O2. Mechanistically, nifurtimox becomes activated after tumor cells enter into a fully hypoxic state, as shown by the stabilization of the Hypoxia-Inducible Factor 1α (HIF-1α). Nifurtimox specifically induces the formation of 53BP1 foci, a hallmark of DNA double-stranded breaks, in hypoxic tumor cells. Hypoxia-dependent activation of nifurtimox involves P450 (cytochrome) oxidoreductase. The anti-protozoan drug nifurtimox holds promise as a new hypoxia-activated cytotoxin with the potential to preferentially eliminates severely hypoxic tumor cells.

  10. Survival

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — These data provide information on the survival of California red-legged frogs in a unique ecosystem to better conserve this threatened species while restoring...

  11. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Chow, Paik Wah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Chan, Kok Meng [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Inayat-Hussain, Salmaan Hussain [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Rajab, Nor Fadilah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia)

    2015-04-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and

  12. The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of (111)In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin).

    Science.gov (United States)

    Cornelissen, Bart; McLarty, Kristin; Kersemans, Veerle; Reilly, Raymond M

    2008-08-01

    Our objective was to define the relationships between tumor uptake of [(111)In]-IGF-1 and [(111)In]-IGF-1(E3R), an analogue which does not bind insulin growth factor-1 (IGF-1) binding proteins (i.e., IGFBP-3), and the level of IGF-1 receptor (IGF-1R) expression on human breast cancer (BC) xenografts in athymic mice, as well as the feasibility for tumor imaging. A second objective was to correlate IGF-1R (and HER2 density) with the cytotoxicity of trastuzumab in the absence/presence of IGFBP-3 or the IGF-1R tyrosine kinase inhibitor, AG1024. The tumor and normal tissue uptake of [(111)In]-IGF-1 and [(111)In]-IGF-1(E3R) were determined at 4 h postinjection in mice implanted subcutaneously with MDA-MB-231, H2N, HR2 or MCF-7/HER2-18 human BC xenografts (8.5x10(4), 1.4x10(4), 4.0x10(4) and 1.0x10(5) IGF-1R/cell, respectively). The effect of co-injection of IGF-1 (50 microg) or IGFBP-3 (2 or 25 microg) was studied. The relationship between tumor uptake of [(111)In]-IGF-1(E3R) and IGF-1R density was examined. MicroSPECT/CT imaging was performed on mice with MCF-7/HER2-18 tumors injected with [(111)In]-IGF-1(E3R). The surviving fraction of BC cells exposed to trastuzumab (67.5 mug/ml) in the absence/presence of IGFBP-3 (1 microg/ml) or the IGF-1R kinase inhibitor, AG1024 (1 or 5 microg/ml), was determined. [(111)In]-IGF-1 was specifically taken up by MCF-7/HER2-18 xenografts; tumor uptake was decreased twofold when co-injected with IGF-1 (1.9+/-0.1 vs. 1.0+/-0.1 %ID/g). Co-injection of IGBP-3 decreased kidney uptake of [(111)In]-IGF-1 up to twofold and increased circulating radioactivity threefold. There was a strong linear correlation (r(2)=0.99) between the tumor uptake of (111)In-IGF-1(E3R) and IGF-1R density. Tumor uptake ranged from 0.4+/-0.05 %ID/g for H2N to 2.5+/-0.5 %ID/g for MCF-7/HER2-18 xenografts. MCF-7/HER2-18 tumors were visualized by microSPECT/CT. Resistance of BC cells to trastuzumab was directly associated with IGF-1R expression, despite co-expression of

  13. The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of {sup 111}In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin)

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart; McLarty, Kristin; Kersemans, Veerle [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Reilly, Raymond M. [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Toronto General Research Institute, University Health Network Toronto, Toronto, ON, M5S 3M2 (Canada)], E-mail: raymond.reilly@utoronto.ca

    2008-08-15

    Introduction: Our objective was to define the relationships between tumor uptake of [{sup 111}In]-IGF-1 and [{sup 111}In]-IGF-1(E3R), an analogue which does not bind insulin growth factor-1 (IGF-1) binding proteins (i.e., IGFBP-3), and the level of IGF-1 receptor (IGF-1R) expression on human breast cancer (BC) xenografts in athymic mice, as well as the feasibility for tumor imaging. A second objective was to correlate IGF-1R (and HER2 density) with the cytotoxicity of trastuzumab in the absence/presence of IGFBP-3 or the IGF-1R tyrosine kinase inhibitor, AG1024. Methods: The tumor and normal tissue uptake of [{sup 111}In]-IGF-1 and [{sup 111}In]-IGF-1(E3R) were determined at 4 h postinjection in mice implanted subcutaneously with MDA-MB-231, H2N, HR2 or MCF-7/HER2-18 human BC xenografts (8.5x10{sup 4}, 1.4x10{sup 4}, 4.0x10{sup 4} and 1.0x10{sup 5} IGF-1R/cell, respectively). The effect of co-injection of IGF-1 (50 {mu}g) or IGFBP-3 (2 or 25 {mu}g) was studied. The relationship between tumor uptake of [{sup 111}In]-IGF-1(E3R) and IGF-1R density was examined. MicroSPECT/CT imaging was performed on mice with MCF-7/HER2-18 tumors injected with [{sup 111}In]-IGF-1(E3R). The surviving fraction of BC cells exposed to trastuzumab (67.5 {mu}g/ml) in the absence/presence of IGFBP-3 (1 {mu}g/ml) or the IGF-1R kinase inhibitor, AG1024 (1 or 5 {mu}g/ml), was determined. Results: [{sup 111}In]-IGF-1 was specifically taken up by MCF-7/HER2-18 xenografts; tumor uptake was decreased twofold when co-injected with IGF-1 (1.9{+-}0.1 vs. 1.0{+-}0.1 %ID/g). Co-injection of IGBP-3 decreased kidney uptake of [{sup 111}In]-IGF-1 up to twofold and increased circulating radioactivity threefold. There was a strong linear correlation (r{sup 2}=0.99) between the tumor uptake of {sup 111}In-IGF-1(E3R) and IGF-1R density. Tumor uptake ranged from 0.4{+-}0.05 %ID/g for H2N to 2.5{+-}0.5 %ID/g for MCF-7/HER2-18 xenografts. MCF-7/HER2-18 tumors were visualized by microSPECT/CT. Resistance of BC

  14. Comparable cell survival between high dose rate flattening filter free and conventional dose rate irradiation.

    Science.gov (United States)

    Verbakel, Wilko F A R; van den Berg, Jaap; Slotman, Ben J; Sminia, Peter

    2013-04-01

    Investigation of clonogenic cell survival and cell proliferation following single dose and fractionated delivery of high dose rate flattening filter free (FFF) irradiation compared to conventional dose rates. The human astrocytoma D384, glioma T98 and lung carcinoma SW1573 cell lines were irradiated using either a single dose (0-12 Gy) or a fractionated protocol of 5 daily fractions of 2 Gy (D384) or 3 Gy (SW1573). Cells were irradiated inside a phantom using fixed gantry beams of a linear accelerator. A sliding window technique created homogeneous dose distributions over the surface of the cell cultures. Irradiations using standard beams (6 MV, 600 MU/min.) and high dose rate FFF beams (10 MV, 2400 MU/min.) were compared. Cell survival was determined by clonogenic assay. In the fractionated irradiation set-up, the number of clonogenic cells was estimated by including tumor cell proliferation during the overall treatment time in the analysis. All cell lines showed equal cell survival following irradiation using either the FFF beams or conventional flattened (FF) beams. This was observed after single dose exposure (0-12 Gy) as well as after fractionated irradiation (p = 0.08 for D384 and 0.20 for SW1373 cell lines). FFF irradiation with a dose rate of 2400 MU/min and four times higher dose per pulse compared to irradiation with FF beams did not change cell survival for three human cancer cell lines up to a fraction dose of 12 Gy compared to irradiation using FF beams.

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

    Science.gov (United States)

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

    2001-01-01

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

  16. Radiogenic neoplasia in thyroid and mammary clonogens. Progress report, January 1, 1990--December 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Clifton, K.H.

    1992-05-20

    We have developed rat thyroid and mammary clonogen transplantation systems for the study of radiogenic cancer induction at the target cell level in vivo. The epithelial cell populations of both glands contain small subpopulations of cells which are capable of giving rise to monoclonal glandular structures when transplanted and stimulated with appropriate hormones. Previous results indicated that these clonogens are the precursor cells of radiogenic cancer, and that initiation, is common event at the clonegenic cell level. Detailed information on the physiologic control of clonogen proliferation, differentiation, and total numbers is thus essential to an understanding of the carcinogenic process. We report here studies on investigations on the relationships between grafted thyroid cell number and the rapidity and degree of reestablishment of the thyroid-hypothalamus-pituitary feedback axis in thyroidectomized rats maintained on a normal diet or an iodine deficient diet; studies of the persistence of, and the differentiation potential and functional characteristics of, the TSH-(thyrotropin-) responsive sub- population of clonogens during goitrogenesis, the plateau-phase of goiter growth, and goiter involution; studies of changes in the size of the clonogen sub-population during goitrogenesis, goiter involution and the response to goitrogen rechallenge; and a large carcinogenesis experiment on the nature of the grafted thyroid cell number-dependent suppression of promotion/progression to neoplasia in grafts of radiation-initiated thyroid cells. Data from these studies will be used in the design of future carcinogenesis experiments on neoplastic initiation by high and low LET radiations and on cell interactions during the neoplastic process.

  17. Telomerase inhibition targets clonogenic multiple myeloma cells through telomere length-dependent and independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Sarah K Brennan

    2010-09-01

    Full Text Available Plasma cells constitute the majority of tumor cells in multiple myeloma (MM but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC. These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.Human MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1 as revealed by quantitative real-time PCR.Telomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

  18. Phenotypic identification of subclones in multiple myeloma with different chemoresistant, cytogenetic and clonogenic potential.

    Science.gov (United States)

    Paíno, T; Paiva, B; Sayagués, J M; Mota, I; Carvalheiro, T; Corchete, L A; Aires-Mejía, I; Pérez, J J; Sanchez, M L; Barcena, P; Ocio, E M; San-Segundo, L; Sarasquete, M E; García-Sanz, R; Vidriales, M-B; Oriol, A; Hernández, M-T; Echeveste, M-A; Paiva, A; Blade, J; Lahuerta, J-J; Orfao, A; Mateos, M-V; Gutiérrez, N C; San-Miguel, J F

    2015-05-01

    Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

  19. Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling.

    Science.gov (United States)

    Booth, D; Haley, J D; Bruskin, A M; Potten, C S

    2000-04-01

    Damage to the normal replacing tissues of the body, specifically the gastro-intestinal tract, limits the treatment and hence, cure rate of cancer patients. Here, we investigate the possibility that the sensitivity of the gastro-intestinal tract can be manipulated by transforming growth factor beta3 (TGF-beta3), making it more resistant to radiation in a murine model. The effects of TGF-beta3 were assessed using the crypt microcolony assay, a test of crypt stem-cell functional competence, in animal survival studies examining diarrhoea severity, labelling index and crypt size. Prior treatment with TGF-beta3 can result in a 3- to 4-fold increase (protection factor, PF) in surviving crypts, whilst longer exposure can raise the PF to almost 12. Protection of intestinal clonogenic stem cells results in marked protection of survival with a corresponding reduction in the duration and level of diarrhoea and ultimate restoration of normal histology in surviving mice. Inhibition of proliferation can be demonstrated when sufficient TGF-beta3 exposure is studied. Crypt size is also reduced. In conclusion, TGF-beta3 protects small intestinal clonogenic stem cells from radiation damage, reducing diarrhoea and animal mortality. The mode of action is believed to be specific inhibition of stem-cell proliferation.

  20. Evaluation of a MTT assay in measurement of radiosensitizing effect

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, Keiko; Mitsuhashi, Norio; Saitoh, Jun-ichi; Maebayashi, Katsuya; Sakurai, Hideyuki; Akimoto, Tetsuo; Niibe, Hideo [Gunma Univ., Maebashi (Japan). School of Medicine

    1999-11-01

    The usefulness of a MTT assay by measuring the radiosensitizing effect of caffeine on rat yolk sac tumor cell line with a mutant-type p53 in vitro was evaluated. A rat yolk sac tumor cell line with a mutant-type p53, NMT-1R, was used in this study. The radiosensitivity of NMT-1R with or without caffeine was measured with a MTT assay. The results were compared with those by a clonogenic assay. Caffeine at a concentration of 2.0 mM which released radiation-induced G{sub 2} block demonstrated a radiosensitizing effect, but caffeine at a concentration of 0.5 mM did not. The radiosensitizing effect of caffeine measured by a MTT assay correlated with that measured by a clonogenic assay. A MTT assay was useful to measure radiosensitivity and/or a radiosensitizing effect in vitro. (author)

  1. CDKN3 expression is negatively associated with pathological tumor stage and CDKN3 inhibition promotes cell survival in hepatocellular carcinoma.

    Science.gov (United States)

    Dai, Wei; Miao, Huilai; Fang, Shuo; Fang, Tao; Chen, Nianping; Li, Mingyi

    2016-08-01

    Aberrant expression of CDKN3 may be involved in carcinogenesis of liver cancer. The effect of CDKN3 on tumorigenesis and the molecular mechanisms involved have not been fully elucidated. Immunohistochemistry was performed to detect CDKN3 expression levels in tumor tissues. CDKN3 siRNA was used to knockdown CDKN3 in QGY7701 hepatocellular carcinoma (HCC) cells. Colony formation assay was used to measure the clonogenic capacity of the tumor cells. Cell viability was determined by MTT assay. Logistic regression was performed to analyze the association between CDKN3 expression level and the HCC clinical pathology index. The CDKN3 expression level was significantly decreased in HCC tumor tissues compared with normal liver tissue and liver cirrhosis tissue. Additionally, CDKN3 expression was negatively‑associated with the pathological stage of the tumor. Inhibition of CKDN3 promoted the clonogenic capacity and chemotherapeutic tolerance in HCC tissues compared with controls. Knockdown of CDKN3 resulted in downregulation of p53 and p21 protein levels, whereas, AKT serine/threonine kinase 1 expression was upregulated. Thus, CDKN3 expression may reduce the survival of tumor cells and alter the sensitivity to therapeutic agents via the AKT/P53/P21 signaling pathway. Therefore, CDKN3 may be involved in tumor differentiation and self-renewal.

  2. Effects of propranolol in combination with radiation on apoptosis and survival of gastric cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    Chaudhary Prakash

    2010-10-01

    Full Text Available Abstract Background The National Comprehensive Cancer Network (NCCN guidelines recommend radiotherapy as a standard treatment for patients with a high risk of recurrence in gastric cancer. Because gastric cancer demonstrates limited sensitivity to radiotherapy, a radiosensitizer might therefore be useful to enhance the radiosensitivity of patients with advanced gastric carcinoma. In this study, we evaluated if propranolol, a β-adrenoceptor (β-AR antagonist, could enhance radiosensitivity and explored its precise molecular mechanism in gastric cancer cells. Methods Human gastric adenocarcinoma cell lines (SGC-7901 and BGC-823 were treated with or without propranolol and exposed to radiation. Cell viability and clonogenic survival assays were performed, and cell apoptosis was evaluated with flow cytometry. In addition, the expression of nuclear factor κB (NF-κB, vascular endothelial growth factor (VEGF, cyclooxygenase 2 (COX-2, and epidermal growth factor receptor (EGFR were detected by western blot and real-time reverse transcription polymerase chain reaction (PCR. Results Propranolol combined with radiation decreased cell viability and clonogenic survivability. Furthermore, it also induced apoptosis in both cell lines tested, as determined by Annexin V staining. In addition, treatment with propranolol decreased the level of NF-κB and, subsequently, down-regulated VEGF, COX-2, and EGFR expression. Conclusions Taken together, these results suggested that propranolol enhanced the sensitivity of gastric cancer cells to radiation through the inhibition of β-ARs and the downstream NF-κB-VEGF/EGFR/COX-2 pathway.

  3. In vitro clonogenic capacity of periodontal ligament fibroblasts cultured with Emdogain.

    Science.gov (United States)

    Ashkenazi, Malka; Shaked, Ilanit

    2006-02-01

    The aim of the present study was to evaluate the efficiency of Emdogain (EMD) in preserving the size of the periodontal ligament progenitor pool (clonogenic capacity) and in promoting their proliferation. Periodontal ligament fibroblasts (PDLF) were obtained from explants of young permanent healthy tooth. After initial outgrowth (10 days to 2 weeks following explantation), the culture medium of experimental flasks was replaced with medium supplemented with 100 microg ml(-1) EMD, whereas the other served as controls and were fed with regular medium. Following 5 weeks, the cells were washed (3x), harvested (trypsin + EDTA), and evaluated for their viability. Viable cells from each group were inoculated into six 96-well plates at a concentration of one viable cell per two wells and were allowed to grow for 5 weeks. The percentage of cells with clonogenic capacity was determined as the number of colonies formed/number of cells seeded x 100 in the experimental and control groups. Three degrees of dish area coverage were utilized: up to 25%, between 25% and 75% and higher than 75%. This experiment was repeated four times from four different donors. A total of 2328 cells were evaluated, half of which, were cultured with EMD. The mean percentage of cells (from all donors) who exhibited any clonogenic capacity in the presence of EMD was comparable with that of cells cultured in the absence of EMD: 26.6 +/- 14.3% when compared with 34.6 +/- 20.6% respectively (P = 0.186). Similarly, the percentage of clones that proliferated to cover up to 25% of the well area was comparable in the two groups 7.5 +/- 8.6 for EMD-treated clones and 7.1 +/- 7.8 for untreated clones (P = 0.674). The percentage of clones that proliferated to cover 25% up to 75% of the well area was greater EMD-treated clones as compared with the untreated cells: 8.1 +/- 6.7% vs 3.8 +/- 3%. However this difference was not statistically significant (P = 0.277). In contrast, the percentage of clones that covered

  4. Identification of type 1 von Willebrand disease patients with reduced von Willebrand factor survival by assay of the VWF propeptide in the European study: molecular and clinical markers for the diagnosis and management of type 1 VWD (MCMDM-1VWD)

    DEFF Research Database (Denmark)

    Haberichter, S.L.; Castaman, G.; Budde, U.

    2008-01-01

    VWF survival. In this study, we report the assay of VWFpp and VWF:Ag in 19 individuals recruited from 6 European centers within the MCMDM-1VWD study. Eight individuals had a VWF:Ag level less than 30 IU/dL. Seven of these patients had a robust desmopressin response and significantly reduced VWF half...

  5. ATM is required for the prolactin-induced HSP90-mediated increase in cellular viability and clonogenic growth after DNA damage.

    Science.gov (United States)

    Karayazi Atici, Ödül; Urbanska, Anna; Gopal Gopinathan, Sesha; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

    2017-11-24

    Prolactin acts as a survival factor for breast cancer cells, but the prolactin signaling pathway and the mechanism is unknown. Previously, we identified the master chaperone, heat shock protein 90α (HSP90α), as a prolactin-Janus-Kinase-(JAK2)-signal-transducer-and-activator-of-transcription-5-(STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of prolactin-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase protein (ATM) plays a critical role in the cellular response to double strand DNA damage. Prolactin increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the prolactin receptor, as the prolactin receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxy geldanamycin and BIIB021, reduced the prolactin-mediated increase in cell viability of doxorubicin treated cells, and led to a decrease in JAK2, ATM and phospho-ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the prolactin-mediated increase in cell viability of DNA damaged cells, supporting the involvement of each, as well as the cross-talk of ATM with the prolactin pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor, KU55933, and doxorubicin, and also between the HSP90 inhibitor, BIIB021, and doxorubicin. Short interfering RNA, directed against ATM, prevented the PRL-mediated increase in cell survival in both 2D and 3D collagen gel cultures, and in clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the prolactin-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA damaging agents. Copyright © 2017 Endocrine Society.

  6. XIAP inhibitors induce differentiation and impair clonogenic capacity of acute myeloid leukemia stem cells.

    Science.gov (United States)

    Moreno-Martínez, Daniel; Nomdedeu, Meritxell; Lara-Castillo, María Carmen; Etxabe, Amaia; Pratcorona, Marta; Tesi, Niccolò; Díaz-Beyá, Marina; Rozman, María; Montserrat, Emili; Urbano-Ispizua, Alvaro; Esteve, Jordi; Risueño, Ruth M

    2014-06-30

    Acute myeloid leukemia (AML) is a neoplasia characterized by the rapid expansion of immature myeloid blasts in the bone marrow, and marked by poor prognosis and frequent relapse. As such, new therapeutic approaches are required for remission induction and prevention of relapse. Due to the higher chemotherapy sensitivity and limited life span of more differentiated AML blasts, differentiation-based therapies are a promising therapeutic approach. Based on public available gene expression profiles, a myeloid-specific differentiation-associated gene expression pattern was defined as the therapeutic target. A XIAP inhibitor (Dequalinium chloride, DQA) was identified in an in silico screening searching for small molecules that induce similar gene expression regulation. Treatment with DQA, similarly to Embelin (another XIAP inhibitor), induced cytotoxicity and differentiation in AML. XIAP inhibition differentially impaired cell viability of the most primitive AML blasts and reduced clonogenic capacity of AML cells, sparing healthy mature blood and hematopoietic stem cells. Taken together, these results suggest that XIAP constitutes a potential target for AML treatment and support the evaluation of XIAP inhibitors in clinical trials.

  7. Upregulation of mitochondrial NAD(+) levels impairs the clonogenicity of SSEA1(+) glioblastoma tumor-initiating cells.

    Science.gov (United States)

    Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

    2017-06-09

    Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD(+)) metabolism. However, the functional role of NAD(+) metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD(+) levels affect the characteristics of glioma-driven SSEA1(+) TICs, including clonogenic growth potential. An increase in the mitochondrial NAD(+) levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD(+) levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

  8. Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells

    Science.gov (United States)

    Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

    2017-01-01

    Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors. PMID:28604662

  9. Assessment of Radiation-Equivalency of Favorite Foods Based on Apoptotic and Clonogenic Cell Death

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seunghee; Park, Hyosung; Lee, Minho; Kim, Eunhee [Seoul National Univ., Seoul (Korea, Republic of)

    2013-05-15

    In this study, the cytotoxicity of favorite foods was investigated to provide chemical doses causing bio-effects comparable with those by radiation exposure. Extracts from alcohol, cigarette and coffee have been investigated for their cytotoxicity in terms of apoptosis induction and clonogenic cell killing. The comparison of familiar favorite food items with radiation in cellular toxicity might help the public have balanced judgment on the severity of radiation exposure. Future study pursues estimating dose-rate effect and the investigation will be continued with other cell lines. People fear that ionizing radiation, despite of its usefulness, bears significant risk factor. In particular, after Fukushima nuclear accident in March of 2011, people become over-sensitive to radiation exposure and nuclear power. Such fear by the public of nuclear power inevitably costs the nuclear industry a high expense for safety measures, not to mention brings the public uneasy life. It is important that the consequence of radiation exposure is conveyed to the public in a friendly way with actual data.

  10. Sulforhodamine B assay and chemosensitivity.

    Science.gov (United States)

    Voigt, Wieland

    2005-01-01

    The sulforhodamine B (SRB) assay was developed by Skehan and colleagues to measure drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications. Its principle is based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic acid-fixed cells. Under mild acidic conditions it binds to and under mild basic conditions it can be extracted from cells and solubilized for measurement. Results of the SRB assay were linear with cell number and cellular protein measured at cellular densities ranging from 1 to 200% of confluence. Its sensitivity is comparable with that of several fluorescence assays and superior to that of Lowry or Bradford. The signal-to-noise ratio is favorable and the resolution is 1000-2000 cells/well. It performed similarly compared to other cytotoxicity assays such as MTT or clonogenic assay. The SRB assay possesses a colorimetric end point and is nondestructive and indefinitely stable. These practical advances make the SRB assay an appropriate and sensitive assay to measure drug-induced cytotoxicity even at large-scale application.

  11. Low drug resistance to both platinum and taxane chemotherapy on an in vitro drug resistance assay predicts improved survival in patients with advanced epithelial ovarian, fallopian and peritoneal cancer.

    Science.gov (United States)

    Matsuo, Koji; Bond, Virginia K; Eno, Michele L; Im, Dwight D; Rosenshein, Neil B

    2009-12-01

    The objective of this study was to evaluate the role of an in vitro drug resistance assay to platinum and taxane in the management of advanced epithelial ovarian, fallopian and primary peritoneal cancer. All patients with FIGO Stage IIIc and IV who received postoperative chemotherapy with platinum and taxane for more than 4 courses after the initial cytoreductive surgery between 1995 and 2008 were evaluated. Patients who received neoadjuvant chemotherapy were not included. An in vitro drug resistance assay (EDR Assay, Oncotech, Tustin, CA) was used to determine drug resistance for each patient's tumor tissue. Level of drug resistance was described as extreme (EDR), intermediate (IDR), or low (LDR). Response to chemotherapy and survival were correlated to the EDR Assay. Of the 335 patients who underwent primary cytoreductive surgery, 173 cases met the criteria for statistical evaluation. The 58 patients (33.5%) whose tumors had LDR to both platinum and taxane had statistically improved progression-free survival and overall survival (OS) compared with the 115 patients (66.5%) who demonstrated IDR or EDR to platinum and/or taxane (5-year OS rates, 41.1% vs. 30.9%, p = 0.014). The 5-year OS rates for the 28 (16.2%) cases that had optimal cytoreduction with LDR to both platinum and taxane was significantly improved over the 62 (35.8%) cases that were suboptimally cytoreduced with IDR or EDR to platinum and/or taxane (54.1% vs. 20.4%, respectively, p chemotherapy, as determined by an in vitro drug resistance assay, independently predicts improved survival in patients with advanced epithelial ovarian, fallopian and peritoneal cancer, especially in those patients who undergo optimal primary cytoreduction.

  12. Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations.

    Science.gov (United States)

    Duru, Valentine; Khim, Nimol; Leang, Rithea; Kim, Saorin; Domergue, Anais; Kloeung, Nimol; Ke, Sopheakvatey; Chy, Sophy; Eam, Rotha; Khean, Chanra; Loch, Kaknika; Ken, Malen; Lek, Dysoley; Beghain, Johann; Ariey, Frédéric; Guerin, Philippe J; Huy, Rekol; Mercereau-Puijalon, Odile; Witkowski, Benoit; Menard, Didier

    2015-12-22

    The declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies. The piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594). All parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10%, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2% vs. 0.17%, P <1 × 10(-7)). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10% were associated with 32-fold higher risk of recrudescence (95% CI, 4.5-224; P = 0.0005). PSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.

  13. Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

    Science.gov (United States)

    Ma, Dongrui; Chua, Alvin Wen Choong; Yang, Ennan; Teo, Peiyun; Ting, Yixin; Song, Colin; Lane, Ellen Birgitte; Lee, Seng Teik

    2015-03-24

    There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

  14. Successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential.

    Science.gov (United States)

    Anagnostakis, Ioannis; Papassavas, Andreas C; Michalopoulos, Efstathios; Chatzistamatiou, Theofanis; Andriopoulou, Sofia; Tsakris, Athanassios; Stavropoulos-Giokas, Catherine

    2014-01-01

    Cord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150°C on critical quality control variables of CB collections to investigate the possible use of mechanical freezers at -150°C as an alternative to storage in liquid- (or vapor-) phase nitrogen. A total of 105 CB units were thawed and washed at different time intervals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recoveries were calculated. Also, total absolute colony-forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. Significant differences (p freezer at -150°C may represent an alternative cryostorage condition for CB cryopreservation. © 2013 American Association of Blood Banks.

  15. Toward computer simulation of high-LET in vitro survival curves

    Science.gov (United States)

    Heuskin, A.-C.; Michiels, C.; Lucas, S.

    2013-09-01

    We developed a Monte Carlo based computer program called MCSC (Monte Carlo Survival Curve) able to predict the survival fraction of cells irradiated in vitro with a broad beam of high linear energy transfer particles. Three types of cell responses are studied: the usual high dose response, the bystander effect and the low-dose hypersensitivity (HRS). The program models the broad beam irradiation and double strand break distribution following Poisson statistics. The progression of cells through the cell cycle is taken into account while the repair takes place. Input parameters are experimentally determined for A549 lung carcinoma cells irradiated with 10 and 20 keV µm-1 protons, 115 keV µm-1 alpha particles and for EAhy926 endothelial cells exposed to 115 keV µm-1 alpha particles. Results of simulations are presented and compared with experimental survival curves obtained for A549 and EAhy296 cells. Results are in good agreement with experimental data for both cell lines and all irradiation protocols. The benefits of MCSC are several: the gain of time that would have been spent performing time-consuming clonogenic assays, the capacity to estimate survival fraction of cell lines not forming colonies and possibly the evaluation of radiosensitivity parameters of given individuals.

  16. SU-E-T-253: Open-Source Automatic Software for Quantifying Biological Assays of Radiation Effects

    Energy Technology Data Exchange (ETDEWEB)

    Detappe, A [University of Lyon (France); Dana-Farber Cancer Institute, Boston, MA (United States); Korideck, H [Dana-Farber Cancer Institute, Boston, MA (United States); Makrigiorgos, G; Berbeco, R [Dana-Farber Cancer Institute, Boston, MA (United States); Brigham and Women' s Hospital, Boston, MA (United States)

    2014-06-01

    Purpose: Clonogenic cell survival is a common assay for quantifying the effect of drugs and radiation. Manual counting of surviving colonies can take 30–90seconds per plate, a major limitation for large studies. Currently available automatic counting tools are not easily modified for radiation oncology research. Our goal is to provide an open-source toolkit for precise, accurate and fast analysis of biological assays in radiation oncology. Methods: As an example analysis, we used HeLa cells incubated with gadolinium nanoparticles prior to irradiation. After treatment, the cells are grown for 14days to allow for colony formation. To analyze the colony growth, we capture images of each dish for archiving and automatic computer-based analysis. A FujifilmX20 camera is placed at the top of a box setup, 20cm above the sample, which is backlit by a LED lamp placed at the bottom of the box. We use a Gaussian filter (width=1.3mm) and color threshold (19–255). The minimum size for a colony to be counted is 1mm. For this example, 20 dishes with a large range of colonies were analyzed. Each dish was counted 3 times manually by 3 different users and then compared to our counter. Results: Automatic counting of cell colonies takes an average of 7seconds, enabling the analysis process to be accelerated 4–12 times. The average precision of the automatic counter was 1.7%. The Student t-test demonstrated the non-significant differences between the two counting methods (p=0.64). The ICC demonstrated the reliability of each method with ICC>0.999 (automatic) and ICC=0.95 (manual). Conclusion: We developed an open-source automatic toolkit for the analysis of biological assays in radiation oncology and demonstrated the accuracy, precision and effort savings for clonogenic cell survival quantification. This toolkit is currently being used in two laboratories for routine experimental analysis and will be made freely available on our departmental website.

  17. Kinetics of mammary clonogenic cells and rat mammary cancer induction by X-rays or fission neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Kenji [Hiroshima Univ. (Japan). Research Inst. for Radiation Biology and Medicine; Higgins, P.D.; Tanner, M.A.; Gould, M.N.; Clifton, K.H.

    1999-12-01

    Following the hormonal treatment of rats with high prolactin levels and glucocorticoid deficiency (Prl+/Glc-) for 48 days (Day +48), total recoverable mammary DNA was increased by more than sevenfold, tritiated thymidine uptake by nearly fourfold, and total mammary clonogens by about fivefold. Irradiation with 4, 40, and 80 cGy X-rays on Day +48 increased total mammary carcinomas per rat-day-at-risk linearly with dose, and 40 and 80 cGy significantly decreased first carcinoma latency. A dose of 40 cGy X-rays before hormone treatment (Day -1) yielded tumor latencies and frequencies insignificantly different from unirradiated controls but significantly different from those when the dose was given on Day +48. Total carcinomas per rat-day-at-risk were fitted better by a function of dose to the power 0.4 than by a linear function after exposure to 1, 10. and 20 cGy fission neutrons, and 10 and 20 cGy significantly shortened the time to appearance of the first cancer. In contrast to results with X-rays, 10 cGy neutrons on Day -1 yielded tumor frequencies and latencies insignificantly different from 10 cGy neutrons on Day +48. The carcinogenic action of X-rays, but not of neutrons, was thus influenced by total clonogen numbers and/or proliferation rates. (author)

  18. Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes.

    Science.gov (United States)

    Schroll, Monica M; LaBonia, Gabriel J; Ludwig, Katelyn R; Hummon, Amanda B

    2017-08-04

    Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug-resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition, and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up-regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy

  19. Mitochondrial-Targeted Decyl-Triphenylphosphonium Enhances 2-Deoxy-D-Glucose Mediated Oxidative Stress and Clonogenic Killing of Multiple Myeloma Cells.

    Directory of Open Access Journals (Sweden)

    Jeanine Schibler

    Full Text Available Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266, using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG induced ER stress as detected by qPCR (BiP, ATF4 and immunoblotting (BiP, CHOP and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM

  20. Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

    Directory of Open Access Journals (Sweden)

    Sandhya S Buchanan

    2010-09-01

    Full Text Available Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001, 170+/-70 BFU-E (approximately 40%, p<0.0001, and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171, and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0

  1. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    Energy Technology Data Exchange (ETDEWEB)

    Gulliya, K.S.; Pervaiz, S.

    1989-03-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested.

  2. The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

    Science.gov (United States)

    Maurelli, Riccardo; Tinaburri, Lavinia; Gangi, Fabio; Bondanza, Sergio; Severi, Anna Lisa; Scarponi, Claudia; Albanesi, Cristina; Mesiti, Giuseppe; Guerra, Liliana; Capogrossi, Maurizio C; Dellambra, Elena

    2016-03-01

    The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis. © 2016. Published by The Company of Biologists Ltd.

  3. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    Science.gov (United States)

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  4. Enzyme assays

    OpenAIRE

    Bisswanger, Hans

    2014-01-01

    The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Neverthele...

  5. Activity of daptomycin or linezolid in combination with rifampin or gentamicin against biofilm-forming Enterococcus faecalis or E. faecium in an in vitro pharmacodynamic model using simulated endocardial vegetations and an in vivo survival assay using Galleria mellonella larvae.

    Science.gov (United States)

    Luther, Megan K; Arvanitis, Marios; Mylonakis, Eleftherios; LaPlante, Kerry L

    2014-08-01

    Enterococci are the third most frequent cause of infective endocarditis. A high-inoculum stationary-phase in vitro pharmacodynamic model with simulated endocardial vegetations was used to simulate the human pharmacokinetics of daptomycin at 6 or 10 mg/kg of body weight/day or linezolid at 600 mg every 12 h (q12h), alone or in combination with gentamicin at 1.3 mg/kg q12h or rifampin at 300 mg q8h or 900 mg q24h. Biofilm-forming, vancomycin-susceptible Enterococcus faecalis and vancomycin-resistant Enterococcus faecium (vancomycin-resistant enterococcus [VRE]) strains were tested. At 24, 48, and 72 h, all daptomycin-containing regimens demonstrated significantly more activity (decline in CFU/g) than any linezolid-containing regimen against biofilm-forming E. faecalis. The addition of gentamicin to daptomycin (at 6 or 10 mg/kg) in the first 24 h significantly improved bactericidal activity. In contrast, the addition of rifampin delayed the bactericidal activity of daptomycin against E. faecalis, and the addition of rifampin antagonized the activities of all regimens against VRE at 24 h. Also, against VRE, the addition of gentamicin to linezolid at 72 h improved activity and was bactericidal. Rifampin significantly antagonized the activity of linezolid against VRE at 72 h. In in vivo Galleria mellonella survival assays, linezolid and daptomycin improved survival. Daptomycin at 10 mg/kg improved survival significantly over that with linezolid against E. faecalis. The addition of gentamicin improved the efficacy of daptomycin against E. faecalis and those of linezolid and daptomycin against VRE. We conclude that in enterococcal infection models, daptomycin has more activity than linezolid alone. Against biofilm-forming E. faecalis, the addition of gentamicin in the first 24 h causes the most rapid decline in CFU/g. Of interest, the addition of rifampin decreased the activity of daptomycin against both E. faecalis and VRE. Copyright © 2014, American Society for

  6. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation.

    LENUS (Irish Health Repository)

    Gill, Catherine

    2009-01-01

    BACKGROUND: Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. METHODS: cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. RESULTS: PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. CONCLUSION: Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

  7. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation

    Directory of Open Access Journals (Sweden)

    Dowling Catherine

    2009-06-01

    Full Text Available Abstract Background Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. Methods cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. Results PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. Conclusion Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

  8. Bortezomib reduces the tumorigenicity of multiple myeloma via downregulation of upregulated targets in clonogenic side population cells.

    Directory of Open Access Journals (Sweden)

    Miho Nara

    Full Text Available Side population (SP cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11 express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3 express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM, polycomb (e.g., EZH2, EPC1 and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5 more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680 and a proteasome inhibitor (bortezomib with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3 and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.

  9. Kynetic resazurin assay (KRA) for bacterial quantification of foodborne pathogens

    Science.gov (United States)

    Arenas, Yaxal; Mandel, Arkady; Lilge, Lothar

    2012-03-01

    Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

  10. In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania chagasi

    Directory of Open Access Journals (Sweden)

    Tafuri Washington L

    2007-05-01

    Full Text Available Abstract Background There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi. Results In the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18. In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages. Conclusion These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

  11. SU-E-T-352: Why Is the Survival Rate Low in Oropharyngeal Squamous Cell Carcinoma?

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Z; Feng, Y; Rasmussen, K; Rice, J; Stephenson, S; Ferreira, Maria C [East Carolina University, Greenville, NC (United States); Liu, T [Baylor College of Medicine, Houston, TX (United States); Yuh, K [California Institute of Technology, Pasadena, CA (United States); Wang, R; Grecula, J [Case Western Reserve University, Cleveland, OH (United States); Lo, S [The Ohio State University, Columbus, OH (United States); Mayr, N; Yuh, W [University of Washington, Seattle, WA (United States)

    2014-06-01

    Purpose: Tumors are composed of a large number of clonogens that have the capability of indefinite reproduction. Even when there is complete clinical or radiographic regression of the gross tumor mass after treatment, tumor recurrence can occur if the clonogens are not completely eradicated by radiotherapy. This study was to investigate the colonogen number and its association with the tumor control probability (TCP) in oropharyngeal squamous cell carcinoma (OSCCA). Methods: A literature search was conducted to collect clinical information of patients with OSCCA, including the prescription dose, tumor volume and survival rate. The linear-quadratic (LQ) model was incorporated into TCP model for clinical data analysis. The total dose ranged from 60 to 70 Gy and tumor volume ranged from 10 to 50 cc. The TCP was calculated for each group according to tumor size and dose. The least χ{sup 2} method was used to fit the TCP calculation to clinical data while other LQ model parameters (α, β) were adopted from the literature, due to the limited patient data. Results: A total of 190 patients with T2–T4 OSCCA were included. The association with HPV was not available for all the patients. The 3-year survival rate was about 82% for T2 squamous cell carcinoma and 40% for advanced tumors. Fitting the TCP model to the survival data, the average clonogen number was 1.56×10{sup 12}. For the prescription dose of 70 Gy, the calculated TCP ranged from 40% to 90% when the tumor volume varied from 10 to 50 cc. Conclusion: Our data suggests variation between the clonogen number and TCP in OSCCA. Tumors with larger colonogen number tend to have lower TCP and therefore dose escalation above 70 Gy may be indicated in order to improve the TCP and survival rate. Our result will require future confirmation with a large number of patients.

  12. Improvement of interleukin 2 production, clonogenic capability and restoration of stromal cell function in human immunodeficiency virus-type-1 patients after highly active antiretroviral therapy.

    Science.gov (United States)

    Isgrò, Antonella; Aiuti, Alessandro; Mezzaroma, Ivano; Addesso, Maria; Riva, Elisabetta; Giovannetti, Antonello; Mazzetta, Francesca; Alario, Cecilia; Mazzone, Annamaria; Ruco, Luigi; Aiuti, Fernando

    2002-09-01

    Haematological abnormalities frequently occur in patients infected by human immunodeficiency virus-type 1 (HIV-1). Increasing evidence indicates that bone marrow suppression (BM) results from viral infection of accessory cells, with impaired stromal function and alteration of haematopoietic growth factor network. We have investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells was observed before treatment, characterized by decreased interleukin 2 (IL-2) and elevated tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell-expressed and secreted) levels, along with a defective BM clonogenic activity. Antiretroviral therapy showed increased BM clonogenic capability, associated with normalization of IL-2 production and chemokine receptors expression on CD34+ cells. Pre-therapy, BM accessory cells were represented by macrophage-like cells, in some cases positive for HIV-1 DNA, suggesting that these cells are the main target of HIV-1 infection. During therapy, the stromal cells became predominantly fibroblastoid-like, as observed in normal controls, and were negative for HIV-1 DNA. Controlling HIV-1 replication may produce amelioration of stem cell activity, and restoration of stromal cell pattern and functions, with increased IL-2 production at BM level.

  13. ATRA and the specific RARα agonist, NRX195183, have opposing effects on the clonogenicity of pre-leukemic murine AML1-ETO bone marrow cells.

    Science.gov (United States)

    Chee, L C Y; Hendy, J; Purton, L E; McArthur, G A

    2013-06-01

    All-trans retinoic acid (ATRA) is used successfully in the treatment of acute promyelocytic leukemia (APL). ATRA enhances hematopoietic stem cell self-renewal through retinoic acid receptor (RAR)γ activation while promoting differentiation of committed myeloid progenitors through RARα activation. Its lack of success in the treatment of non-APL acute myeloid leukemia (AML) may be related to ATRA's non-selectivity for the RARα and RARγ isotypes, and specific RARα activation may be more beneficial in promoting myeloid differentiation. To investigate this hypothesis, the effects of ATRA and the specific RARα agonist NRX195183 was assessed in AML1-ETO (AE)-expressing murine bone marrow (BM) progenitors. ATRA potentiated the in vitro clonogenicity of these cells while NRX195183 had the opposite effect. Morphological and flow cytometric analysis confirmed a predominantly immature myeloid population in the ATRA-treated AE cells while the NRX195183-treated cells demonstrated an increase in the mature myeloid population. Similarly, NRX195183 treatment promoted myeloid differentiation in an AE9a in vivo murine model. In the ATRA-treated AE cells, gene expression analyses revealed functional networks involving SERPINE1 and bone morphogenetic protein 2; AKT phosphorylation was upregulated. Collectively, these findings confirm the contrasting roles of specific RARα and RARγ activation in the clonogenicity and differentiation of AE cells with potential significant implications in the treatment of non-APL AML using a specific RARα agonist.

  14. Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

    Science.gov (United States)

    Alexaline, Maia M.; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T.; Doucet, Christelle; Fortunel, Nicolas O.

    2015-01-01

    Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. Significance This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. PMID:25848122

  15. Autophagy in response to photodynamic therapy: cell survival vs. cell death

    Science.gov (United States)

    Oleinick, Nancy L.; Xue, Liang-yan; Chiu, Song-mao; Joseph, Sheeba

    2009-02-01

    Autophagy (or more properly, macroautophagy) is a pathway whereby damaged organelles or other cell components are encased in a double membrane, the autophagosome, which fuses with lysosomes for digestion by lysosomal hydrolases. This process can promote cell survival by removing damaged organelles, but when damage is extensive, it can also be a mechanism of cell death. Similar to the Kessel and Agostinis laboratories, we have reported the vigorous induction of autophagy by PDT; this was found in human breast cancer MCF-7 cells whether or not they were able to efficiently induce apoptosis. One way to evaluate the role of autophagy in PDT-treated cells is to silence one of the essential genes in the pathway. Kessel and Reiners silenced the Atg7 gene of murine leukemia L1210 cells using inhibitory RNA and found sensitization to PDT-induced cell death at a low dose of PDT, implying that autophagy is protective when PDT damage is modest. We have examined the role of autophagy in an epithelium-derived cancer cell by comparing parental and Atg7-silenced MCF-7 cells to varying doses of PDT with the phthalocyanine photosensitizer Pc 4. In contrast to L1210 cells, autophagy-deficient MCF-7 cells were more resistant to the lethal effects of PDT, as judged by clonogenic assays. A possible explanation for the difference in outcome for L1210 vs. MCF-7 cells is the greatly reduced ability of the latter to undergo apoptosis, a deficiency that may convert autophagy into a cell-death process even at low PDT doses. Experiments to investigate the mechanism(s) responsible are in process.

  16. Deletion of Ptp4a3 reduces clonogenicity and tumor-initiation ability of colitis-associated cancer cells in mice

    Directory of Open Access Journals (Sweden)

    Julie M. Cramer

    2014-07-01

    Full Text Available The PTP4A3 gene is highly expressed in human colon cancer and often associates with enhanced metastatic potential. Genetic disruption of the mouse Ptp4a3 gene reduces the frequency of colon tumor formation in mice treated in a colitis-associated cancer model. In the current study, we have examined the role of Ptp4a3 in the tumor-initiating cell population of mouse colon tumors using an in vitro culture system. Tumors generated in vivo following AOM/DSS treatment were isolated, dissociated, and expanded on a feeder layer resulting in a CD133+ cell population, which expressed high levels of Ptp4a3. Tumor cells deficient for Ptp4a3 exhibited reduced clonogenicity and growth potential relative to WT cells as determined by limiting dilution analysis. Importantly, expanded tumor cells from WT mice readily formed secondary tumors when transplanted into nude mice, while tumor cells without Ptp4a3 expression failed to form secondary tumors and thus were not tumorigenic. These results demonstrate that Ptp4a3 contributes to the malignant phenotype of tumor-initiating cells and supports its role as a potential therapeutic target to inhibit tumor self-renewal and metastasis.

  17. Combined in utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors.

    Science.gov (United States)

    Cimino-Reale, Graziella; Ferrario, Daniele; Casati, Barbara; Brustio, Roberta; Diodovich, Cristina; Collotta, Angelo; Vahter, Marie; Gribaldo, Laura

    2008-07-30

    The effects of arsenate (As) and atrazine (Atr) on myeloid progenitors (colony-forming unit-granulocyte/macrophage, CFU-GM) cells derived from bone marrow were studied in male and female mice after combined in utero and juvenile exposure. Female adult mice were treated with arsenate in drinking water during gestation. Then, separate groups of males and females' offspring were exposed for 4 months to atrazine, to additional arsenate or to co-exposure of atrazine and arsenate together in drinking water. In male mice, arsenate and the combined exposure did not modulate the percentage of CFU-GM progenitors, whereas atrazine significantly decreases the clonogenicity of myeloid cells. In females, the percentage of CFU-GM significantly decreased after atrazine exposure did not change with arsenate treatment, but dramatically increased after the combined exposure. The expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in bone marrow cells was investigated, and an up-regulation of receptor beta was observed in both genders. A gene expression profile was generated using nylon membranes spotted with 1185 cancer-related genes. Results from microarrays indicate that atrazine alone did not stimulate the expression of any of the genes analysed in both male and female. Arsenic induced gene expression modulation only in female. Major significant changes on the gene expression resulted following the co-exposure to arsenic and atrazine in both male and female.

  18. Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Skovrind, Ida; Christensen, Marlene Louise

    2013-01-01

    Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment...... and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent...... tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation...

  19. CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component.

    Science.gov (United States)

    Kahlert, Ulf D; Bender, Noemi O; Maciaczyk, Donata; Bogiel, Tomasz; Bar, Eli E; Eberhart, Charles G; Nikkhah, Guido; Maciaczyk, Jarosław

    2012-01-01

    Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133(-)/CD15(-) and CD133(+)/CD15(+) but not in CD133(-)/CD15(+) cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.

  20. WE-EF-BRA-08: Cell Survival in Modulated Radiation Fields and Altered DNA-Repair at Field Edges

    Energy Technology Data Exchange (ETDEWEB)

    Bartzsch, S; Oelfke, U [The Institute of Cancer Research, London (United Kingdom); Eismann, S [University of Heidelberg, Heidelberg, DE (Germany)

    2015-06-15

    Purpose: Tissue damage prognoses in radiotherapy are based on clonogenic assays that provide dose dependent cell survival rates. However, recent work has shown that apart from dose, systemic reactions and cell-cell communication crucially influence the radiation response. These effects are probably a key in understanding treatment approaches such as microbeam radiation therapy (MRT). In this study we tried to quantify the effects on a cellular level in spatially modulated radiation fields. Methods: Pancreas carcinoma cells were cultured, plated and irradiated by spatially modulated radiation fields with an X-ray tube and at a synchrotron. During and after treatment cells were able to communicate via the intercellular medium. Afterwards we stained for DNA and DNA damage and imaged with a fluorescence microscope. Results: Intriguingly we found that DNA damage does not strictly increase with dose. Two cell entities appear that have either a high or a low amount of DNA lesions, indicating that DNA damage is also a cell stress reaction. Close to radiation boundaries damage-levels became alike; they were higher than expected at low and lower than expected at high doses. Neighbouring cells reacted similarly. 6 hours after exposure around 40% of the cells resembled in their reactions neighbouring cells more than randomly chosen cells that received the same dose. We also observed that close to radiation boundaries the radiation induced cell-cycle arrest disappeared and the size of DNA repair-centres increased. Conclusion: Cell communication plays an important role in the radiation response of tissues and may be both, protective and destructive. These effects may not only have the potential to affect conventional radiotherapy but may also be exploited to spare organs at risk by intelligently designing irradiation geometries. To that end intensive work is required to shed light on the still obscure processes in cell-signalling and radiation biology.

  1. Survival Analysis

    CERN Document Server

    Miller, Rupert G

    2011-01-01

    A concise summary of the statistical methods used in the analysis of survival data with censoring. Emphasizes recently developed nonparametric techniques. Outlines methods in detail and illustrates them with actual data. Discusses the theory behind each method. Includes numerous worked problems and numerical exercises.

  2. Modelling survival

    DEFF Research Database (Denmark)

    Ashauer, Roman; Albert, Carlo; Augustine, Starrlight

    2016-01-01

    well GUTS, calibrated with short-term survival data of Gammarus pulex exposed to four pesticides, can forecast effects of longer-term pulsed exposures. Thirdly, we tested the ability of GUTS to estimate 14-day median effect concentrations of malathion for a range of species and use these estimates...

  3. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma

    Science.gov (United States)

    Pal, Rekha; Greene, Stephanie

    2015-01-01

    This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR). Two medulloblastoma cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5) and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival. PMID:26394044

  4. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Rekha Pal

    Full Text Available This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR. Two medulloblastoma cell lines (DAOY and UW228 were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5 and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival.

  5. Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Nilgun Gurbuz

    Full Text Available Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA, induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT, concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D-mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification

  6. Innovations’ Survival

    Directory of Open Access Journals (Sweden)

    Jakub Tabas

    2016-01-01

    Full Text Available Innovations currently represent a tool of maintaining the going concern of a business entity and its competitiveness. However, effects of innovations are not infinite and if an innovation should constantly preserve a life of business entity, it has to be a continual chain of innovations, i.e. continual process. Effective live of a single innovation is limited while the limitation is derived especially from industry. The paper provides the results of research on innovations effects in the financial performance of small and medium-sized enterprises in the Czech Republic. Objective of this paper is to determine the length and intensity of the effects of technical innovations in company’s financial performance. The economic effect of innovations has been measured at application of company’s gross production power while the Deviation Analysis has been applied for three years’ time series. Subsequently the Survival Analysis has been applied. The analyses are elaborated for three statistical samples of SMEs constructed in accordance to the industry. The results obtained show significant differences in innovations’ survival within these three samples of enterprises then. The results are quite specific for the industries, and are confronted and discussed with the results of authors’ former research on the issue.

  7. Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line.

    Science.gov (United States)

    Nooter, K.; Boersma, A. W.; Oostrum, R. G.; Burger, H.; Jochemsen, A. G.; Stoter, G.

    1995-01-01

    Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment. PMID:7880739

  8. 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.

  9. Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field.

    LENUS (Irish Health Repository)

    Shields, L

    2014-10-31

    Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells are not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci\\/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant

  10. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  11. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  12. A bioluminescence assay for aldehyde dehydrogenase activity.

    Science.gov (United States)

    Duellman, Sarah J; Valley, Michael P; Kotraiah, Vinayaka; Vidugiriene, Jolanta; Zhou, Wenhui; Bernad, Laurent; Osterman, Jean; Kimball, Joshua J; Meisenheimer, Poncho; Cali, James J

    2013-03-15

    The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Lateral flow assays.

    Science.gov (United States)

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  14. Lateral flow assays

    Science.gov (United States)

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  15. Tube-Forming Assays.

    Science.gov (United States)

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  16. Modelling the comet assay.

    Science.gov (United States)

    McArt, Darragh G; McKerr, George; Howard, C Vyvyan; Saetzler, Kurt; Wasson, Gillian R

    2009-08-01

    The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

  17. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

    Science.gov (United States)

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  18. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  19. Dual effects of radiation bystander signaling in urothelial cancer: purinergic-activation of apoptosis attenuates survival of urothelial cancer and normal urothelial cells.

    Science.gov (United States)

    Bill, Malgorzata A; Srivastava, Kirtiman; Breen, Conor; Butterworth, Karl T; McMahon, Stephen J; Prise, Kevin M; McCloskey, Karen D

    2017-11-14

    Radiation therapy (RT) delivers tumour kill, directly and often via bystander mechanisms. Bladder toxicity is a dose limiting constraint in pelvic RT, manifested as radiation cystitis and urinary symptoms. We aimed to investigate the impact of radiation-induced bystander signaling on normal/cancer urothelial cells. Human urothelial cancer cells T24, HT1376 and normal urothelial cells HUC, SV-HUC were used. Cells were irradiated and studied directly, or conditioned medium from irradiated cells (CM) was transferred to naïve, cells. T24 or SV-HUC cells in the shielded half of irradiated flasks had increased numbers of DNA damage foci vs non-irradiated cells. A physical barrier blocked this response, indicating release of transmitters from irradiated cells. Clonogenic survival of shielded T24 or SV-HUC was also reduced; a physical barrier prevented this phenomenon. CM-transfer increased pro-apoptotic caspase-3 activity, increased cleaved caspase-3 and cleaved PARP expression and reduced survival protein XIAP expression. This effect was mimicked by ATP. ATP or CM evoked suramin-sensitive Ca 2+ -signals. Irradiation increased [ATP] in CM from T24. The CM-inhibitory effect on T24 clonogenic survival was blocked by apyrase, or mimicked by ATP. We conclude that radiation-induced bystander signaling enhances urothelial cancer cell killing via activation of purinergic pro-apoptotic pathways. This benefit is accompanied by normal urothelial damage indicating RT bladder toxicity is also bystander-mediated.

  20. Dual effects of radiation bystander signaling in urothelial cancer: purinergic-activation of apoptosis attenuates survival of urothelial cancer and normal urothelial cells

    Science.gov (United States)

    Bill, Malgorzata A.; Srivastava, Kirtiman; Breen, Conor; Butterworth, Karl T.; McMahon, Stephen J.; Prise, Kevin M.; McCloskey, Karen D.

    2017-01-01

    Radiation therapy (RT) delivers tumour kill, directly and often via bystander mechanisms. Bladder toxicity is a dose limiting constraint in pelvic RT, manifested as radiation cystitis and urinary symptoms. We aimed to investigate the impact of radiation-induced bystander signaling on normal/cancer urothelial cells. Human urothelial cancer cells T24, HT1376 and normal urothelial cells HUC, SV-HUC were used. Cells were irradiated and studied directly, or conditioned medium from irradiated cells (CM) was transferred to naïve, cells. T24 or SV-HUC cells in the shielded half of irradiated flasks had increased numbers of DNA damage foci vs non-irradiated cells. A physical barrier blocked this response, indicating release of transmitters from irradiated cells. Clonogenic survival of shielded T24 or SV-HUC was also reduced; a physical barrier prevented this phenomenon. CM-transfer increased pro-apoptotic caspase-3 activity, increased cleaved caspase-3 and cleaved PARP expression and reduced survival protein XIAP expression. This effect was mimicked by ATP. ATP or CM evoked suramin-sensitive Ca2+-signals. Irradiation increased [ATP] in CM from T24. The CM-inhibitory effect on T24 clonogenic survival was blocked by apyrase, or mimicked by ATP. We conclude that radiation-induced bystander signaling enhances urothelial cancer cell killing via activation of purinergic pro-apoptotic pathways. This benefit is accompanied by normal urothelial damage indicating RT bladder toxicity is also bystander-mediated. PMID:29228614

  1. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R [Danville, CA; Benett, William J [Livermore, CA; Coleman, Matthew A [Oakland, CA; Pearson, Francesca S [Livermore, CA; Nasarabadi, Shanavaz L [Livermore, CA

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  2. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  3. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  4. Global assays of fibrinolysis.

    Science.gov (United States)

    Ilich, A; Bokarev, I; Key, N S

    2017-10-01

    Fibrinolysis is an important and integral part of the hemostatic system. Acting as a balance to blood coagulation, the fibrinolytic system protects the body from unwanted thrombus formation and occlusion of blood vessels. As long as blood coagulation and fibrinolysis remain in equilibrium, response to injury, such as vessel damage, is appropriately regulated. However, alterations in this balance may lead to thrombosis or bleeding. A variety of methods have been proposed to assess fibrinolytic activity in blood or its components, but due to the complexity of the system, the design of a "gold standard" assay that reflects overall fibrinolysis has remained an elusive goal. In this review, we describe the most commonly used methods that have been described, such as thromboelastography (TEG and ROTEM), global fibrinolytic capacity in plasma and whole blood, plasma turbidity methods, simultaneous thrombin and plasmin generation assays, euglobulin clot lysis time and fibrin plate methods. All of these assays have strengths and limitations. We suggest that some methods may be preferable for detecting hypofibrinolytic conditions, whereas others may be better for detecting hyperfibrinolytic states. © 2017 John Wiley & Sons Ltd.

  5. Evaluation of boron neutron capture effects in cell culture using sulforhodamine-B assay and a colony assay.

    Science.gov (United States)

    Wittig, A; Sauerwein, W; Pöller, F; Fuhrmann, C; Hideghéty, K; Streffer, C

    1998-06-01

    The purpose of this study was to find an in vitro method for determining the cytotoxicity of boronated drugs as well as their potential suitability for neutron capture therapy. The survival of human melanoma cells has been determined by a colony assay and the sulforhodamine-B assay after X-irradiation and irradiation with fast d(14) + Be-neutrons using the boronated compound borocaptate sodium (BSH). The cytotoxic effects of BSH have been studied using both methods. Under well-defined experimental conditions, and after a sufficient amount of time for the expression of radiation damage, the results of the sulforhodamine-B assay are qualitatively comparable with the results of the colony assay. The sulforhodamine-B assay is suitable for the screening of compounds for potential use in neutron capture therapy because it is a fast and efficient method that is reproducible and technically advantageous.

  6. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  7. Multinationals and plant survival

    DEFF Research Database (Denmark)

    Bandick, Roger

    2010-01-01

    The aim of this paper is twofold: first, to investigate how different ownership structures affect plant survival, and second, to analyze how the presence of foreign multinational enterprises (MNEs) affects domestic plants’ survival. Using a unique and detailed data set on the Swedish manufacturing...... sector, I am able to separate plants into those owned by foreign MNEs, domestic MNEs, exporting non-MNEs, and purely domestic firms. In line with previous findings, the result, when conditioned on other factors affecting survival, shows that foreign MNE plants have lower survival rates than non......-MNE plants. However, separating the non-MNEs into exporters and non-exporters, the result shows that foreign MNE plants have higher survival rates than non-exporting non-MNEs, while the survival rates of foreign MNE plants and exporting non-MNE plants do not seem to differ. Moreover, the simple non...

  8. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention...... is adapted to receive one or more replaceable solid support(s) (40) onto which chemical entities (41) are attached, said device comprising a base (1, 60, 80, 300, 400, 10, 70, 140, 20, 90, 120, 150, 30, 100), one or more inlet(s) (5), one or more outlet(s) (6). The base and the solid support (40) defines......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  9. Clonogenicity: holoclones and meroclones contain stem cells.

    Directory of Open Access Journals (Sweden)

    Charlotte M Beaver

    Full Text Available When primary cultures of normal cells are cloned, three types of colony grow, called holoclones, meroclones and paraclones. These colonies are believed to be derived from stem cells, transit-amplifying cells and differentiated cells respectively. More recently, this approach has been extended to cancer cell lines. However, we observed that meroclones from the prostate cancer cell line DU145 produce holoclones, a paradoxical observation as meroclones are thought to be derived from transit-amplifying cells. The purpose of this study was to confirm this observation and determine if both holoclones and meroclones from cancer cell lines contain stem cells. We demonstrated that both holoclones and meroclones can be serially passaged indefinitely, are highly proliferative, can self-renew to form spheres, are serially tumorigenic and express stem cell markers. This study demonstrates that the major difference between holoclones and meroclones derived from a cancer cell line is the proportion of stem cells within each colony, not the presence or absence of stem cells. These findings may reflect the properties of cancer as opposed to normal cells, perhaps indicating that the hierarchy of stem cells is more extensive in cancer.

  10. In vitro cytotoxicity of {sup 211}At-labeled trastuzumab in human breast cancer cell lines: effect of specific activity and HER2 receptor heterogeneity on survival fraction

    Energy Technology Data Exchange (ETDEWEB)

    Akabani, Gamal [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Carlin, Sean [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Welsh, Phil [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Zalutsky, Michael R. [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States)]. E-mail: zalut001@mc.duke.edu

    2006-04-15

    Introduction: Radioimmunotherapy with anti-HER2 monoclonal antibodies (mAbs) such as trastuzumab is a promising strategy for treating HER2-positive breast and ovarian carcinoma patients. The objective of this study was to determine the cytotoxic effectiveness of trastuzumab labeled with the 7.2-h half-life {alpha}-particle emitter {sup 211}At. Methods: Experiments were performed on SKBr-3, BT-474 and the transfected MCF7/HER2-18 human breast carcinoma cell lines. Intrinsic radiosensitivity was determined after exposure to external beam irradiation. The cytotoxicity of {sup 211}At-labeled trastuzumab was measured by clonogenic assays. The distribution of HER2 receptor expression on the cell lines was measured using fluorescence-activated cell sorting. A pharmacokinetic (PK)/microdosimetric model was established to assess the effects of specific activity (SA), HER2 receptor expression and absorbed dose on survival fraction (SF). Results: With external beam irradiation, the 2-Gy SF for BT-474, SKBr-3 and MCF7/HER2-18 cells was 0.78, 0.53 and 0.64 Gy, respectively. Heterogeneous HER2 expression was observed, with a subpopulation of cells lacking measurable receptor (14.5%, SKBr-3; 0.34%, MCF-7/HER2; 1.73%, BT-474). When plotted as a function of activity concentration, SF curves were biphasic and inversely proportional to SA; however, when the model was applied and absorbed doses calculated, the SF curve was monoexponential independent of SA. Thus, the PK model was able to demonstrate the effects of competition between cold and labeled mAb. These studies showed that the relative biological effectiveness of {sup 211}At-labeled trastuzaumab was about 10 times higher than that of external beam therapy. Conclusion: These in vitro studies showed that {sup 211}At-labeled trastuzumab mAb is an effective cytotoxic agent for the treatment of HER2-positive tumor cells. The SA of the labeled mAb and the homogeneity of HER2 receptor expression are important variables influencing

  11. Timp1 Promotes Cell Survival by Activating the PDK1 Signaling Pathway in Melanoma

    Directory of Open Access Journals (Sweden)

    Mariana Toricelli

    2017-04-01

    Full Text Available High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and β1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5-trisphosphate (PIP3, generated under phosphatidylinositol-3-kinase (PI3K activation, enables the recruitment and activation of protein kinase B (PKB/AKT and phosphoinositide-dependent kinase 1 (PDK1 at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCβIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.

  12. On differences in radiosensitivity estimation: TCP experiments versus survival curves. A theoretical study.

    Science.gov (United States)

    Stavrev, Pavel; Stavreva, Nadejda; Ruggieri, Ruggero; Nahum, Alan

    2015-08-07

    We have compared two methods of estimating the cellular radiosensitivity of a heterogeneous tumour, namely, via cell-survival and via tumour control probability (TCP) pseudo-experiments. It is assumed that there exists intra-tumour variability in radiosensitivity and that the tumour consists predominantly of radiosensitive cells and a small number of radio-resistant cells.Using a multi-component, linear-quadratic (LQ) model of cell kill, a pseudo-experimental cell-survival versus dose curve is derived. This curve is then fitted with a mono-component LQ model describing the response of a homogeneous cell population. For the assumed variation in radiosensitivity it is shown that the composite pseudo-experimental survival curve is well approximated by the survival curve of cells with uniform radiosensitivity.For the same initial cell radiosensitivity distribution several pseudo-experimental TCP curves are simulated corresponding to different fractionation regimes. The TCP model used accounts for clonogen proliferation during a fractionated treatment. The set of simulated TCP curves is then fitted with a mono-component TCP model. As in the cell survival experiment the fit with a mono-component model assuming uniform radiosensitivity is shown to be highly acceptable.However, the best-fit values of cellular radiosensitivity produced via the two methods are very different. The cell-survival pseudo-experiment yields a high radiosensitivity value, while the TCP pseudo-experiment shows that the dose-response is dominated by the most resistant sub-population in the tumour, even when this is just a small fraction of the total.

  13. Assay for calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  14. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay.

    Science.gov (United States)

    Boe-Hansen, Gry B; Morris, Ian D; Ersbøll, Annette K; Greve, Torben; Christensen, Preben

    2005-04-01

    During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.

  15. Network ties and survival

    DEFF Research Database (Denmark)

    Acheampong, George; Narteh, Bedman; Rand, John

    2017-01-01

    Poultry farming has been touted as one of the major ways by which poverty can be reduced in low-income economies like Ghana. Yet, anecdotally there is a high failure rate among these poultry farms. This current study seeks to understand the relationship between network ties and survival chances...... of small commercial poultry farms (SCPFs). We utilize data from a 2-year network survey of SCPFs in rural Ghana. The survival of these poultry farms are modelled using a lagged probit model of farms that persisted from 2014 into 2015. We find that network ties are important to the survival chances...... but this probability reduces as the number of industry ties increases but moderation with dynamic capability of the firm reverses this trend. Our findings show that not all network ties aid survival and therefore small commercial poultry farmers need to be circumspect in the network ties they cultivate and develop....

  16. Aircraft Survivability: Rotorcraft Survivability. Summer 2010

    Science.gov (United States)

    2010-01-01

    protect those who serve to protect us?” The answer is a mixed bag. I am fortunate to have joined a group of dedicated men and women who represent this...and Service subject matter experts on rotorcraft safety and survivability to complete the study and report the results to the Joint Chiefs of...Operations and Support CDD TEMP DT DT/OT LUT IOT &E BLRIP TEMP TEMP LRIP Acquisition & LFT Strategies B C LFT&E Review Requirements Approve TEMPs

  17. Digital Assays Part II: Digital Protein and Cell Assays.

    Science.gov (United States)

    Basu, Amar S

    2017-08-01

    A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

  18. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive...... on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women....

  19. Biological assays in microfabricated structures

    Science.gov (United States)

    Fishman, Daniel M.; Fare, Thomas L.; Dong, Qianping; Fan, Z. Hugh; Davis, Timothy J.; Kumar, Rajan

    1999-04-01

    Microfluidic control in microfabricated glass channels enables miniaturized, fast, and multianalyte assays. We are applying this technology in several areas, including real- time environmental monitoring for airborne biological agents. Two complementary approaches are being used in parallel. the first is assaying for the presence of nucleotide sequences that are markers for specific hazardous, engineered bacteria. The second is an assay that monitors the functionality of an in vitro biochemical pathway, in which the pathway that is chosen is sensitive to the presence of the class of toxins to be detected. The first approach is discussed here. The detected signal from the nucleic-acid-based assay is from fluorescently labeled probes that hybridize to bead-bound amplified DNA sequences. Detection approaches and their benefits will be discussed.

  20. Microtiter dish biofilm formation assay.

    Science.gov (United States)

    O'Toole, George A

    2011-01-30

    Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes

  1. Proof-Carrying Survivability

    Science.gov (United States)

    2013-01-15

    pp.289-302 ( Impact factor : 2.09). 2. Julic, J. and Zuo, Y. (2012). “An RFID Survivability Impact Model in the Military Domain”, Proc. of 18 th...Availability, Reliability and Security, 40(4), pp. 406-418 ( Impact factor : 2.016). 10. Zuo, Y. (2010). “A Holistic Approach for Specification of Security... Impact factor : 1.596). 20. Zuo, Y., Pimple, M. and Lande, S. (2009). “A Framework for RFID Survivability Requirement Analysis and Specification”, Proc

  2. N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays.

    Science.gov (United States)

    Nguyen, Hong P T; Xiao, L; Deane, James A; Tan, Ker-Sin; Cousins, Fiona L; Masuda, Hirotaka; Sprung, Carl N; Rosamilia, Anna; Gargett, Caroline E

    2017-11-01

    Is there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays? N-cadherin isolates clonogenic, self-renewing human endometrial epithelial progenitor cells with high proliferative potential that differentiate into cytokeratin+ gland-like structures in vitro and identifies their location in some cells of gland profiles predominantly in basalis endometrium adjacent to the myometrium. Human endometrium contains a small population of clonogenic, self-renewing epithelial cells with high proliferative potential that differentiate into large gland-like structures, but their identity and location is unknown. Stage-specific embryonic antigen-1 (SSEA-1) distinguishes the epithelium of basalis from functionalis and is a marker of human post-menopausal (Post-M) endometrial epithelium. Prospective observational study of endometrial epithelial cells obtained from hysterectomy samples taken from 50 pre-menopausal (Pre-M) and 24 Post-M women, of which 4 were from women who had taken daily estradiol valerate 2 mg/day for 8 weeks prior. Gene profiling was used to identify differentially expressed surface markers between fresh EpCAM (Epithelial Cell Adhesion Molecule)-magnetic bead-selected basalis-like epithelial cells from Post-M endometrium compared with predominantly functionalis epithelial cells from Pre-M endometrium and validated by qRT-PCR. In vitro clonogenicity and self-renewal assays were used to assess the stem/progenitor cell properties of magnetic bead-sorted N-cadherin+ and N-cadherin- epithelial cells. The cellular identity, location and phenotype of N-cadherin+ cells was assessed by dual colour immunofluorescence and confocal microscopy for cytokeratin, proliferative status (Ki-67), ERα, SSEA-1, SOX9 and epithelial mesenchymal transition (EMT) markers on full thickness human endometrium. CDH2 (N-cadherin gene) was one of 11 surface molecules highly expressed in Post-M compared to

  3. Survivability via Control Objectives

    Energy Technology Data Exchange (ETDEWEB)

    CAMPBELL,PHILIP L.

    2000-08-11

    Control objectives open an additional front in the survivability battle. A given set of control objectives is valuable if it represents good practices, it is complete (it covers all the necessary areas), and it is auditable. CobiT and BS 7799 are two examples of control objective sets.

  4. Artists’ Survival Rate

    DEFF Research Database (Denmark)

    Bille, Trine; Jensen, Søren

    2017-01-01

    The literature of cultural economics generally finds that an artistic education has no significant impact on artists’ income and careers in the arts. In our research, we have readdressed this question by looking at the artists’ survival in the arts occupations. The results show that an artistic...... education has a significant impact on artists’ careers in the arts and we find important industry differences....

  5. Education for Survival

    Science.gov (United States)

    Aldrich, Richard

    2010-01-01

    This article provides a brief overview of current approaches to education and concludes that none of these is sufficient to meet the challenges that now face the human race. It argues instead for a new concept of education for survival. (Contains 1 note.)

  6. Flexible survival regression modelling

    DEFF Research Database (Denmark)

    Cortese, Giuliana; Scheike, Thomas H; Martinussen, Torben

    2009-01-01

    Regression analysis of survival data, and more generally event history data, is typically based on Cox's regression model. We here review some recent methodology, focusing on the limitations of Cox's regression model. The key limitation is that the model is not well suited to represent time-varyi...

  7. Seeds to survive

    NARCIS (Netherlands)

    Groot, S.P.C.

    2002-01-01

    Seeds are important for man, either as propagation material of crops or directly for the production of foods, fodder and drinks. The natural function of seeds is dispersal of its genes to successive generations. Survival mechanisms seed have evolved sometimes interfere with those preferred by

  8. Survival After Retirement.

    Science.gov (United States)

    Holloway, Clark; Youngblood, Stuart A.

    1986-01-01

    Examined survival rates after retirement in a large corporation. A regression analysis was performed to control for age, sex, job status, and type of work differences that may influence longevity. Short-term suvivors seemed to undergo a different adjustment process than long-term survivors. (Author/ABL)

  9. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  10. Activity Assays for Rhomboid Proteases.

    Science.gov (United States)

    Arutyunova, E; Strisovsky, K; Lemieux, M J

    2017-01-01

    Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein. © 2017 Elsevier Inc. All rights reserved.

  11. Surviving Sepsis Campaign

    DEFF Research Database (Denmark)

    Rhodes, Andrew; Evans, Laura E; Alhazzani, Waleed

    2017-01-01

    OBJECTIVE: To provide an update to "Surviving Sepsis Campaign Guidelines for Management of Sepsis and Septic Shock: 2012". DESIGN: A consensus committee of 55 international experts representing 25 international organizations was convened. Nominal groups were assembled at key international meetings......, and to formulate recommendations as strong or weak, or best practice statement when applicable. RESULTS: The Surviving Sepsis Guideline panel provided 93 statements on early management and resuscitation of patients with sepsis or septic shock. Overall, 32 were strong recommendations, 39 were weak recommendations...... of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for these critically ill patients with high mortality....

  12. Surviving Sepsis Campaign

    DEFF Research Database (Denmark)

    Rhodes, Andrew; Evans, Laura E; Alhazzani, Waleed

    2017-01-01

    OBJECTIVE: To provide an update to "Surviving Sepsis Campaign Guidelines for Management of Sepsis and Septic Shock: 2012." DESIGN: A consensus committee of 55 international experts representing 25 international organizations was convened. Nominal groups were assembled at key international meetings......, and to formulate recommendations as strong or weak, or best practice statement when applicable. RESULTS: The Surviving Sepsis Guideline panel provided 93 statements on early management and resuscitation of patients with sepsis or septic shock. Overall, 32 were strong recommendations, 39 were weak recommendations...... of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for these critically ill patients with high mortality....

  13. Cracking the survival code

    Science.gov (United States)

    Füllgrabe, Jens; Heldring, Nina; Hermanson, Ola; Joseph, Bertrand

    2014-01-01

    Modifications of histones, the chief protein components of the chromatin, have emerged as critical regulators of life and death. While the “apoptotic histone code” came to light a few years ago, accumulating evidence indicates that autophagy, a cell survival pathway, is also heavily regulated by histone-modifying proteins. In this review we describe the emerging “autophagic histone code” and the role of histone modifications in the cellular life vs. death decision. PMID:24429873

  14. Artillery Survivability Model

    Science.gov (United States)

    2016-06-01

    experiment mode also enables users to set their own design of experiment by manipulating an editable CSV file. The second one is a real-time mode that...renders a 3D virtual environment of a restricted battlefield where the survivability movements of an artillery company are visualized . This mode...provides detailed visualization of the simulation and enables future experimental uses of the simulation as a training tool. 14. SUBJECT TERMS

  15. Survival analysis models and applications

    CERN Document Server

    Liu, Xian

    2012-01-01

    Survival analysis concerns sequential occurrences of events governed by probabilistic laws.  Recent decades have witnessed many applications of survival analysis in various disciplines. This book introduces both classic survival models and theories along with newly developed techniques. Readers will learn how to perform analysis of survival data by following numerous empirical illustrations in SAS. Survival Analysis: Models and Applications: Presents basic techniques before leading onto some of the most advanced topics in survival analysis.Assumes only a minimal knowledge of SAS whilst enablin

  16. Worldwide interest in the comet assay: a bibliometric study.

    Science.gov (United States)

    Neri, Monica; Milazzo, Daniele; Ugolini, Donatella; Milic, Mirta; Campolongo, Alessandra; Pasqualetti, Patrizio; Bonassi, Stefano

    2015-01-01

    The comet assay is a rapid, sensitive and relatively simple method for measuring DNA damage. A bibliometric study was performed to evaluate temporal and geographical trends, research quality and main areas of interest in scientific production in this field. A PubMed search strategy was developed and 7674 citations were retrieved in the period 1990-2013. Notably, the MeSH (Medical Subject Headings) term 'comet assay', officially introduced in 2000, is used by indexers only in two thirds of papers retrieved. Articles on the comet assay were published in 78 countries, spread over the 5 continents. The EU contributed the greatest output, producing >2900 articles with IF (42.0%) and totalling almost 10000 IF points, and was followed by USA. In the new millennium, research with this assay reached a plateau or slow decline in the most industrialised areas (USA, Germany, UK, Italy), while its use has boomed in emerging countries, with increases of 5- to 7-fold in the last 10 years in China, India and Brazil, for instance. This transition resulted in a slow decrease of scientific production quality, as the countries that increased their relative weight typically had lower mIFs. The most common MeSH terms used in papers using the comet assay referred to wide areas of interest, such as DNA damage and repair, cell survival and apoptosis, cancer and oxidative stress, occupational and environmental health. Keywords related to humans, rodents and cell culture were also frequently used. The top journal for the comet assay articles was found to be Mutation Research, followed by Mutagenesis. Most papers using the comet assay as a biomarker were published in genetic and toxicology journals, with a stress on environmental and occupational disciplines. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  18. Applied survival analysis using R

    CERN Document Server

    Moore, Dirk F

    2016-01-01

    Applied Survival Analysis Using R covers the main principles of survival analysis, gives examples of how it is applied, and teaches how to put those principles to use to analyze data using R as a vehicle. Survival data, where the primary outcome is time to a specific event, arise in many areas of biomedical research, including clinical trials, epidemiological studies, and studies of animals. Many survival methods are extensions of techniques used in linear regression and categorical data, while other aspects of this field are unique to survival data. This text employs numerous actual examples to illustrate survival curve estimation, comparison of survivals of different groups, proper accounting for censoring and truncation, model variable selection, and residual analysis. Because explaining survival analysis requires more advanced mathematics than many other statistical topics, this book is organized with basic concepts and most frequently used procedures covered in earlier chapters, with more advanced topics...

  19. Survival after blood transfusion

    DEFF Research Database (Denmark)

    Kamper-Jørgensen, Mads; Ahlgren, Martin; Rostgaard, Klaus

    2008-01-01

    of transfusion recipients in Denmark and Sweden followed for up to 20 years after their first blood transfusion. Main outcome measure was all-cause mortality. RESULTS: A total of 1,118,261 transfusion recipients were identified, of whom 62.0 percent were aged 65 years or older at the time of their first...... the SMR remained significantly 1.3-fold increased. CONCLUSION: The survival and relative mortality patterns among blood transfusion recipients were characterized with unprecedented detail and precision. Our results are relevant to assessments of the consequences of possible transfusion-transmitted disease...... as well as for cost-benefit estimation of new blood safety interventions....

  20. Nuclear War Survival Skills

    Energy Technology Data Exchange (ETDEWEB)

    Kearny, C.H.

    2002-06-24

    The purpose of this book is to provide Americans with information and instructions that will significantly increase their chances of surviving a possible nuclear attack. It brings together field-tested instructions that, if followed by a large fraction of Americans during a crisis that preceded an attack, could save millions of lives. The author is convinced that the vulnerability of our country to nuclear threat or attack must be reduced and that the wide dissemination of the information contained in this book would help achieve that objective of our overall defense strategy.

  1. Design of survivable networks

    CERN Document Server

    Stoer, Mechthild

    1992-01-01

    The problem of designing a cost-efficient network that survives the failure of one or more nodes or edges of the network is critical to modern telecommunications engineering. The method developed in this book is designed to solve such problems to optimality. In particular, a cutting plane approach is described, based on polyhedral combinatorics, that is ableto solve real-world problems of this type in short computation time. These results are of interest for practitioners in the area of communication network design. The book is addressed especially to the combinatorial optimization community, but also to those who want to learn polyhedral methods. In addition, interesting new research problemsare formulated.

  2. Salicylate activates AMPK and synergizes with metformin to reduce the survival of prostate and lung cancer cells ex vivo through inhibition of de novo lipogenesis.

    Science.gov (United States)

    O'Brien, Andrew J; Villani, Linda A; Broadfield, Lindsay A; Houde, Vanessa P; Galic, Sandra; Blandino, Giovanni; Kemp, Bruce E; Tsakiridis, Theodoros; Muti, Paola; Steinberg, Gregory R

    2015-07-15

    Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers of the colon, lung and prostate and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) by binding at the A-769662 drug binding site on the AMPK β1-subunit, a mechanism that is distinct from metformin which disrupts the adenylate charge of the cell. A hallmark of many cancers is high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is currently unknown whether targeting the AMPK-ACC-lipogenic pathway using salicylate and/or metformin may be effective for inhibiting cancer cell survival. Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations achievable following the ingestion of aspirin (novo lipogenesis and these effects were enhanced with the addition of clinical concentrations of metformin (100 μM) and eliminated in mouse embryonic fibroblasts (MEFs) deficient in AMPK β1. Supplementation of media with fatty acids and/or cholesterol reverses the suppressive effects of salicylate and metformin on cell survival indicating the inhibition of de novo lipogenesis is probably important. Pre-clinical studies evaluating the use of salicylate based drugs alone and in combination with metformin to inhibit de novo lipogenesis and the survival of prostate and lung cancers are warranted. © 2015 Authors; published by Portland Press Limited.

  3. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival.

    Science.gov (United States)

    Kamarajan, Pachiyappan; Hayami, Takayuki; Matte, Bibiana; Liu, Yang; Danciu, Theodora; Ramamoorthy, Ayyalusamy; Worden, Francis; Kapila, Sunil; Kapila, Yvonne

    2015-01-01

    The use of small antimicrobial peptides or bacteriocins, like nisin, to treat cancer is a new approach that holds great promise. Nisin exemplifies this new approach because it has been used safely in humans for many years as a food preservative, and recent laboratory studies support its anti-tumor potential in head and neck cancer. Previously, we showed that nisin (2.5%, low content) has antitumor potential in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. The current studies explored a naturally occurring variant of nisin (nisin ZP; 95%, high content) for its antitumor effects in vitro and in vivo. Nisin ZP induced the greatest level of apoptosis in HNSCC cells compared to low content nisin. HNSCC cells treated with increasing concentrations of nisin ZP exhibited increasing levels of apoptosis and decreasing levels of cell proliferation, clonogenic capacity, and sphere formation. Nisin ZP induced apoptosis through a calpain-dependent pathway in HNSCC cells but not in human oral keratinocytes. Nisin ZP also induced apoptosis dose-dependently in human umbilical vein endothelial cells (HUVEC) with concomitant decreases in vascular sprout formation in vitro and reduced intratumoral microvessel density in vivo. Nisin ZP reduced tumorigenesis in vivo and long-term treatment with nisin ZP extended survival. In addition, nisin treated mice exhibited normal organ histology with no evidence of inflammation, fibrosis or necrosis. In summary, nisin ZP exhibits greater antitumor effects than low content nisin, and thus has the potential to serve as a novel therapeutic for HNSCC.

  4. Oxygen levels do not determine radiation survival of breast cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Chann Lagadec

    Full Text Available For more than a century oxygen has been known to be one of the most powerful radiosensitizers. However, despite decades of preclinical and clinical research aimed at overcoming tumor hypoxia, little clinical progress has been made so far. Ionizing radiation damages DNA through generation of free radicals. In the presence of oxygen these lesions are chemically modified, and thus harder to repair while hypoxia protects cells from radiation (Oxygen enhancement ratio (OER. Breast cancer stem cells (BSCSs are protected from radiation by high levels of free radical scavengers even in the presence of oxygen. This led us to hypothesize that BCSCs exhibit an OER of 1. Using four established breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SUM159PT and primary breast cancer samples, we determined the number of BCSCs using cancer stem cell markers (ALDH1, low proteasome activity, compared radiation clonogenic survival and mammosphere formation under normoxic and hypoxic conditions, and correlated these results to the expression levels of key members of the free radical scavenging systems. The number of BCSCs increased with increased aggressiveness of the cancer. This correlated with increased radioresistance (SF(8Gy, and decreasing OERs. When cultured as mammospheres, breast cancer cell lines and primary samples were highly radioresistant and not further protected by hypoxia (OER∼1.We conclude that because BCSCs are protected from radiation through high expression levels of free radical scavengers, hypoxia does not lead to additional radioprotection of BCSCs.

  5. Survival, DNA Integrity, and Ultrastructural Damage in Antarctic Cryptoendolithic Eukaryotic Microorganisms Exposed to Ionizing Radiation.

    Science.gov (United States)

    Pacelli, Claudia; Selbmann, Laura; Zucconi, Laura; Raguse, Marina; Moeller, Ralf; Shuryak, Igor; Onofri, Silvano

    2017-02-01

    Life dispersal between planets, planetary protection, and the search for biosignatures are main topics in astrobiology. Under the umbrella of the STARLIFE project, three Antarctic endolithic microorganisms, the melanized fungus Cryomyces antarcticus CCFEE 515, a hyaline strain of Umbilicaria sp. (CCFEE 6113, lichenized fungus), and a Stichococcus sp. strain (C45A, green alga), were exposed to high doses of space-relevant gamma radiation (60Co), up to 117.07 kGy. After irradiation survival, DNA integrity and ultrastructural damage were tested. The first was assessed by clonogenic test; viability and dose responses were reasonably described by the linear-quadratic formalism. DNA integrity was evaluated by PCR, and ultrastructural damage was observed by transmission electron microscopy. The most resistant among the tested organisms was C. antarcticus both in terms of colony formation and DNA preservation. Besides, results clearly demonstrate that DNA was well detectable in all the tested organisms even when microorganisms were dead. This high resistance provides support for the use of DNA as a possible biosignature during the next exploration campaigns. Implication in planetary protection and contamination during long-term space travel are put forward. Key Words: Biosignatures-Ionizing radiation-DNA integrity-Eukaryotic microorganisms-Fingerprinting-Mars exploration. Astrobiology 17, 126-135.

  6. Comet Assay in Cancer Chemoprevention.

    Science.gov (United States)

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  7. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  8. Ecotoxicological applications and significance of the comet assay.

    Science.gov (United States)

    Jha, Awadhesh N

    2008-05-01

    Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the

  9. Earthworm dispersal assay for rapidly evaluating soil quality.

    Science.gov (United States)

    Kim, Shin Woong; Kim, Dokyung; Moon, Jongmin; Chae, Yooeun; Kwak, Jin Il; Park, Younsu; Jeong, Seung-Woo; An, Youn-Joo

    2017-10-01

    Earthworms enhance soil functioning and are therefore key species in the soil. Their presence is generally a positive sign for a terrestrial ecosystem, because these species serve as important biomarkers in soil quality evaluations. We describe a novel bioassay, the "dispersal assay," that is a simple and rapid technique for field-based soil quality evaluations. It is based on the premise that earthworms prefer optimal soils if given the choice. Thus, assay tubes containing a reference soil were inserted in target sites, and earthworms were placed into these tubes. According to their soil preference, the earthworms dispersed into the surrounding soil, remained in the initial soil within the tubes, avoided both by crawling up the tube, or died. Furthermore, sensitivity responses to metal concentrations, electrical conductivity, and soil pH were observed in field tests. Although the dispersal assay did not completely match traditional toxicity endpoints such as earthworm survival, we found that it can serve as an in situ screening test for assessing soil quality. Overall, our dispersal assay was relatively rapid (within 24 h), had low levels of variation, and showed high correlations between earthworm behavior and soil physicochemical properties. Environ Toxicol Chem 2017;36:2766-2772. © 2017 SETAC. © 2017 SETAC.

  10. Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours

    NARCIS (Netherlands)

    von Bueren, A. O.; Bacolod, M. D.; Hagel, C.; Heinimann, K.; Fedier, A.; Kordes, U.; Pietsch, T.; Koster, J.; Grotzer, M. A.; Friedman, H. S.; Marra, G.; Kool, M.; Rutkowski, S.

    2012-01-01

    BACKGROUND: Tumours are responsive to temozolomide (TMZ) if they are deficient in O-6-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient. METHODS: The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA

  11. Improved testing for microsatellite instability in colorectal cancer using a simplified 3-marker assay.

    Science.gov (United States)

    Esemuede, Iyare; Forslund, Ann; Khan, Sajid A; Qin, Li-Xuan; Gimbel, Mark I; Nash, Garrett M; Zeng, Zhaoshi; Rosenberg, Shoshana; Shia, Jinru; Barany, Francis; Paty, Philip B

    2010-12-01

    In colorectal cancer (CRC), microsatellite instability (MSI) is a valuable marker of defective DNA mismatch repair that identifies cancers with distinct phenotypic properties, including favorable survival. However, the optimal assay for MSI status is unknown. We have evaluated a simplified 3-marker assay for MSI and compared it with the 5-marker (NCI) assay to see if technical variations in MSI testing are important. DNA samples from 357 CRCs were evaluated for MSI using the 5 microsatellite markers recommended for the NCI assay (BAT 25, BAT26, D2S123, D5S346, and D17S250). Results were compared with a simplified 3-marker assay (BAT25, BAT26, and D2S123). CRCs identified as MSI were evaluated for their clinical, pathological, and genetic characteristics. The 5-marker assay identified 96 cancers as MSI. Only 56 of these were MSI by the 3-marker assay (3-marker+ group), leaving 40 cases identified as MSI only by NCI criteria (3-marker- group). The remaining 261 cancers were microsatellite stable (MSS). The 3-marker+ MSI tumors had features characteristic of MSI tumors: more proximal, poorly differentiated, associated with hereditary nonpolyposis colorectal cancer (HNPCC), more BRAF mutations, fewer KRAS mutations, better 5-year disease-specific survival, more frequent mismatch repair (MMR) protein loss, and less likely to be metastatic on presentation (P cancers (P colorectal cancer.

  12. OBESITY IN CANCER SURVIVAL

    Science.gov (United States)

    Parekh, Niyati; Chandran, Urmila; Bandera, Elisa V.

    2013-01-01

    Although obesity is a well known risk factor for several cancers, its role on cancer survival is poorly understood. We conducted a systematic literature review to assess the current evidence evaluating the impact of body adiposity on the prognosis of the three most common obesity-related cancers: prostate, colorectal, and breast. We included 33 studies of breast cancer, six studies of prostate cancer, and eight studies of colorectal cancer. We note that the evidence over-represents breast cancer survivorship research and is sparse for prostate and colorectal cancers. Overall, most studies support a relationship between body adiposity and site-specific mortality or cancer progression. However, most of the research was not specifically designed to study these outcomes and, therefore, several methodological issues should be considered before integrating their results to draw conclusions. Further research is urgently warranted to assess the long-term impact of obesity among the growing population of cancer survivors. PMID:22540252

  13. Surviving relatives after suicide

    DEFF Research Database (Denmark)

    Nørrelykke, Helle; Cohrt, Pernille

    suicide in Denmark. This means that at least 400 people undergo the trauma it is when one of their near relatives commits suicide. We also know that the loss from suicide involves a lot of conflicting feelings - like anger, shame, guilt and loss and that the lack of therapy/treatment of these difficult...... and conflicting feelings may result in pathological expansion of grief characterized by extremely reduced quality of life involving severe psychical and social consequences. Suicide a subject of taboo In the 1980s WHO drafted a health policy document (‘Health for all year 2000’) with 38 targets for attaining......We would like to focus on the surviving relatives after suicides, because it is generally accepted that it is especially difficult to recover after the loss from suicide and because we know as a fact that one suicide affects five persons on average. Every year approximately 700 people commit...

  14. Obesity in cancer survival.

    Science.gov (United States)

    Parekh, Niyati; Chandran, Urmila; Bandera, Elisa V

    2012-08-21

    Although obesity is a well-known risk factor for several cancers, its role on cancer survival is poorly understood. We conducted a systematic literature review to assess the current evidence evaluating the impact of body adiposity on the prognosis of the three most common obesity-related cancers: prostate, colorectal, and breast. We included 33 studies of breast cancer, six studies of prostate cancer, and eight studies of colo-rectal cancer. We note that the evidence overrepresents breast cancer survivorship research and is sparse for prostate and colorectal cancers. Overall, most studies support a relationship between body adiposity and site-specific mortality or cancer progression. However, most of the research was not specifically designed to study these outcomes and, therefore, several methodological issues should be considered before integrating their results to draw conclusions. Further research is urgently warranted to assess the long-term impact of obesity among the growing population of cancer survivors.

  15. Candida survival strategies.

    Science.gov (United States)

    Polke, Melanie; Hube, Bernhard; Jacobsen, Ilse D

    2015-01-01

    Only few Candida species, e.g., Candida albicans, Candida glabrata, Candida dubliniensis, and Candida parapsilosis, are successful colonizers of a human host. Under certain circumstances these species can cause infections ranging from superficial to life-threatening disseminated candidiasis. The success of C. albicans, the most prevalent and best studied Candida species, as both commensal and human pathogen depends on its genetic, biochemical, and morphological flexibility which facilitates adaptation to a wide range of host niches. In addition, formation of biofilms provides additional protection from adverse environmental conditions. Furthermore, in many host niches Candida cells coexist with members of the human microbiome. The resulting fungal-bacterial interactions have a major influence on the success of C. albicans as commensal and also influence disease development and outcome. In this chapter, we review the current knowledge of important survival strategies of Candida spp., focusing on fundamental fitness and virulence traits of C. albicans. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Psychology and survival.

    Science.gov (United States)

    Phillips, D P; Ruth, T E; Wagner, L M

    1993-11-06

    We examined the deaths of 28,169 adult Chinese-Americans, and 412,632 randomly selected, matched controls coded "white" on the death certificate. Chinese-Americans, but not whites, die significantly earlier than normal (1.3-4.9 yr) if they have a combination of disease and birthyear which Chinese astrology and medicine consider ill-fated. The more strongly a group is attached to Chinese traditions, the more years of life are lost. Our results hold for nearly all major causes of death studied. The reduction in survival cannot be completely explained by a change in the behaviour of the Chinese patient, doctor, or death-registrar, but seems to result at least partly from psychosomatic processes.

  17. Norovirus surrogate survival on spinach during preharvest growth.

    Science.gov (United States)

    Hirneisen, Kirsten A; Kniel, Kalmia E

    2013-04-01

    Produce can become contaminated with human viral pathogens in the field through soil, feces, or water used for irrigation; through application of manure, biosolids, pesticides, and fertilizers; and through dust, insects, and animals. The objective of this study was to assess the survival and stability of human noroviruses and norovirus surrogates (Murine norovirus [MNV] and Tulane virus [TV]) on foliar surfaces of spinach plants in preharvest growth conditions. Spinach plants were housed in a biocontrol chamber at optimal conditions for up to 7 days and infectivity was determined by plaque assay. Virus inoculation location had the largest impact on virus survival as viruses present on adaxial leaf surfaces had lower decimal reduction time (D values) than viruses present on abaxial leaf surfaces. Under certain conditions, spinach type impacted virus survival, with greater D values observed from survival on semi-savoy spinach leaves. Additional UVA and UVB exposure to mimic sunlight affected virus survival on adaxial surfaces for both semi-savoy and smooth spinach plants for both viruses. Human GII norovirus inoculated onto semi-savoy spinach had an average D value that was not statistically significant from MNV and TV, suggesting that these surrogates may have similar survival on spinach leaves compared with human noroviruses. An understanding of the behavior of enteric viruses on spinach leaves can be used to enhance growers' guidelines and for risk assessment with certain growing conditions.

  18. Antioxidants and the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

    It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

  19. SNO+ Scintillator Purification and Assay

    Science.gov (United States)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  20. Reporter Gene Assays in Ecotoxicology.

    Science.gov (United States)

    Elad, Tal; Belkin, Shimshon

    The need for simple and rapid means for evaluating the potential toxic effects of environmental samples has prompted the development of reporter gene assays, based on tester cells (bioreporters) genetically engineered to report on sample toxicity by producing a readily quantifiable signal. Bacteria are especially suitable to serve as bioreporters owing to their fast responses, low cost, convenient preservation, ease of handling, and amenability to genetic manipulations. Various bacterial bioreporters have been introduced for general toxicity and genotoxicity assessment, and the monitoring of endocrine disrupting and dioxin-like compounds has been mostly covered by similarly engineered eukaryotic cells. Some reporter gene assays have been validated, standardized, and accredited, and many others are under constant development. Efforts are aimed at broadening detection spectra, lowering detection thresholds, and combining toxicity identification capabilities with characterization of the toxic effects. Taking advantage of bacterial robustness, attempts are also being made to incorporate bacterial bioreporters into field instrumentation for online continuous monitoring or on-site spot checks. However, key hurdles concerning test validation, cell preservation, and regulatory issues related to the use of genetically modified organisms still remain to be overcome.

  1. Surviving a Suicide Attempt

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Harrasi

    2016-09-01

    Full Text Available Suicide is a global phenomenon in all regions of the world affecting people of all age groups. It has detrimental consequences on patients, their families, and the community as a whole. There have been numerous risk factors described for suicide including mental illness, stressful life situations, loss of social support, and general despair. The association of suicide with Islam has not been extensively studied. The common impression from clinical practice is that being a practicing Muslim reduces the risk of suicide. Another factor associated with suicide is starting a patient on antidepressants. However, this has been questioned recently. This report describes a middle-aged man with depression and multiple social stressors who survived a serious suicide attempt. The discussion will focus on the factors that lead him to want to end his life and the impact of the assumed protective factors such as religious belief and family support on this act of self-harm. Such patients can be on the edge when there is an imbalance between risk factors (such as depression, insomnia, and psychosocial stressors and protective factors (like religious affiliation and family support. All physicians are advised to assess the suicide risk thoroughly in patients with depression regardless of any presumed protective factor.

  2. Surviving a Suicide Attempt.

    Science.gov (United States)

    Al-Harrasi, Ahmed; Al Maqbali, Mandhar; Al-Sinawi, Hamed

    2016-09-01

    Suicide is a global phenomenon in all regions of the world affecting people of all age groups. It has detrimental consequences on patients, their families, and the community as a whole. There have been numerous risk factors described for suicide including mental illness, stressful life situations, loss of social support, and general despair. The association of suicide with Islam has not been extensively studied. The common impression from clinical practice is that being a practicing Muslim reduces the risk of suicide. Another factor associated with suicide is starting a patient on antidepressants. However, this has been questioned recently. This report describes a middle-aged man with depression and multiple social stressors who survived a serious suicide attempt. The discussion will focus on the factors that lead him to want to end his life and the impact of the assumed protective factors such as religious belief and family support on this act of self-harm. Such patients can be on the edge when there is an imbalance between risk factors (such as depression, insomnia, and psychosocial stressors) and protective factors (like religious affiliation and family support). All physicians are advised to assess the suicide risk thoroughly in patients with depression regardless of any presumed protective factor.

  3. Will the olympics survive?.

    Science.gov (United States)

    Khosla, T.

    1977-01-01

    The United States of America dominated 58 events in athletics, field and swimming, which between them accounted for 35 per cent of all events in the Munich Olympiad. 1972; these events favour taller individuals. But, in 25 per cent of other events (1) cycling, (2) fencing, (3) gymnastics, (4) judo, (5) weightlifting and (6) Graeco Roman wrestling the U.S.A. did not win a single medal. The failure of the U.S.A. to maintain her lead in Munich was largely due to weaknesses in these other events in many of which the potential medallists can be derived from the lower half of the height distribution (events 3 to 6). These weaknesses are Russia's strength and they continued to remain unstrengthened at Montreal. Also, the domination held by the U.S.A. in swimming was seriously challenged by East Germany. The present trends indicate that the U.S.A.'s ranking is likely to slip further to the third position in Moscow 1980. Factors inhibiting the survival of the Olympics are pointed. PMID:861436

  4. Arcobacter butzleri survives within trophozoite of Acanthamoeba castellanii.

    Science.gov (United States)

    Villanueva, María P; Medina, Gustavo; Fernández, Heriberto

    2016-01-01

    The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. In vivo transgenic mutation assays.

    Science.gov (United States)

    Thybaud, Véronique; Dean, Stephen; Nohmi, Takehiko; de Boer, Johan; Douglas, George R; Glickman, Barry W; Gorelick, Nancy J; Heddle, John A; Heflich, Robert H; Lambert, Iain; Martus, Hans-Jörg; Mirsalis, Jon C; Suzuki, Takayoshi; Yajima, Nobuhiro

    2003-10-07

    Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when

  6. Cell survival in a simulated Mars environment

    Science.gov (United States)

    Todd, Paul; Kurk, Michael Andy; Boland, Eugene; Thomas, David

    2016-07-01

    were introduced on the first day (less than 1 hour). All Samples were mixed into Mars regolith simulant for this test. Biological samples consisting of Cyanobacteria: Anabena sp., Chroococcidiopsis CCMEE171, Plectonema boryanum; Eubacteria: Bacillus subtilis, Pseudomonas aeruginosa, and Eukaryota: Chlorella ellipsoidia were maintained in the simulator under the above-described conditions. The exposed specimens were tested for intracellular esterase activity (fluorescein diacetate (FDA) hydrolysis), chlorophyll content (where appropriate) and reproductive survival (colony formation on nutrient plates). These tests all yielded low-level positive results indicating some survival in all cases. Three control populations of each species were simultaneously exposed to -80 C dark storage, +4 C dark storage, and +25 C diurnal cycles in the same Mars regolith simulant (Orbital Technologies, Madison, WI). The survival hierarchy based on intracellular esterase assay, in decreasing order of survival was Anabena > Chroococcidiopsis > Pseudomonas > Bacillus subtilis > Chlorella > Plectonema, and the range of survival based on this test was 8% - 50%. The survival hierarchy based on post-exposure colony growth was Plectonema > Chroococcidiopsis = Chlorella > Anabena, and Pseudomonas exhibited higher survival than Bacillus subtilis. These results indicate a need for longer-term high-fidelity planetary simulation studies of a wider variety of microbial species including extremophiles, such as psychrophilic strains like Psychrobacter spp., Planococcus halocryophilus, Rhodococcus sp. and the yeast Rhodotorula sp. that could be found in human environments. This research was supported by NASA NIAC Phase I Grant "Mars Ecopoiesis Testbed" NNX14AM97G.

  7. Survival After Relapse of Medulloblastoma.

    Science.gov (United States)

    Koschmann, Carl; Bloom, Karina; Upadhyaya, Santhosh; Geyer, J Russell; Leary, Sarah E S

    2016-05-01

    Survival after recurrence of medulloblastoma has not been reported in an unselected cohort of patients in the contemporary era. We reviewed 55 patients diagnosed with medulloblastoma between 2000 and 2010, and treated at Seattle Children's Hospital to evaluate patterns of relapse treatment and survival. Fourteen of 47 patients (30%) over the age of 3 experienced recurrent or progressive medulloblastoma after standard therapy. The median time from diagnosis to recurrence was 18.0 months (range, 3.6 to 62.6 mo), and site of recurrence was metastatic in 86%. The median survival after relapse was 10.3 months (range, 1.3 to 80.5 mo); 3-year survival after relapse was 18%. There were trend associations between longer survival and having received additional chemotherapy (median survival 12.8 vs. 1.3 mo, P=0.16) and radiation therapy (15.4 vs. 5.9 mo, P=0.20). Isolated local relapse was significantly associated with shorter survival (1.3 vs. 12.8 mo, P=0.009). Recurrence of medulloblastoma is more likely to be metastatic than reported in previous eras. Within the limits of our small sample, our data suggest a potential survival benefit from retreatment with cytotoxic chemotherapy and radiation even in heavily pretreated patients. This report serves as a baseline against which to evaluate novel therapy combinations.

  8. Predictive Assay For Cancer Targets

    Energy Technology Data Exchange (ETDEWEB)

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  9. Predictive assay for cancer targets

    Science.gov (United States)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  10. Advanced amperometric respiration assay for antimicrobial susceptibility testing.

    Science.gov (United States)

    Chotinantakul, Kamonnaree; Suginta, Wipa; Schulte, Albert

    2014-10-21

    A ferricyanide-based electrochemical cell respiration assay was adapted for use in broad-spectrum antimicrobial susceptibility testing (AST). Total bacterial respiration was converted into faradaic current by electro-oxidation of ferrocyanide, produced when ferricyanide is reduced by bacterial electron-transport. For Escherichia coli (E. coli), the signal was linear with 5-13 × 10(8) colony-forming units in measuring buffer. For AST, test cells were treated with drugs before ferricyanide addition; cell counts from the amperometric assay provided a measure of drug-induced cell death. Initial trials with six antimicrobial agents produced incorrect susceptibility classifications for drugs that were electroactive at the potential used to detect ferrocyanide or which affected cellular respiration rates. We therefore changed the procedure from drug-treatment and assay in the same buffer to sequential drug exposure in treatment buffer, centrifugal separation of surviving cells, cell resuspension, incubation in the presence of ferricyanide and finally ferrocyanide amperometry in drug-free buffer. Data analysis with E. coli led to an activity classification that agreed with cell culture-based ASTs, obtained by a quicker, more convenient procedure. The potential of this approach was confirmed by trials with the highly virulent bacterium Burkholderia pseudomallei, a particularly antimicrobial-resistant pathogen that is the cause of lethal melioidosis in tropical climates and is currently of concern as a potential bioterrorism agent.

  11. Marketing child survival.

    Science.gov (United States)

    Grant, J P

    1984-01-01

    Growth monitoring charts, packets of oral rehydration salts (ORS), and vaccines, are inexpensive, life-saving, growth-protecting technologies which can enable parents to protect their children against the worst effects of poverty. Similarly, a matrix of current and easily understandable information about pregnancy, breast feeding, weaning, feeding during and immediately after illness, child spacing, and preparing and using home-made oral rehydration solutions, also could empower parents to protect the lives and the health of their children. The question arises as to how can these technologies and this information be put at the disposal of millions of families in the low-income world. The initial task of the Child Survival and Development Revolution is the communication of what is now possible, yet little is known about how to communicate information whose principal value is to the poor. There are 2 large-scale precedents: the Green Revolution, which in many instances succeeded in putting into the hands of thousands of small and large farmers the techniques and the knowledge which enabled them to double and treble the yields from their lands; and the campaign to put the knowledge and the means of family planning at the disposal of many millions of people. There are 2 lessons to be learned from these precedents: they have shown that the way to promote a people's technology and to put information at the disposal of the majority is by mobilizing all possible resources and working through all possible channels both to create the demand and to meet it; and neither the Green Revolution nor the family planning movement rally took off until they were viewed as political and economic priorities and given the full support of the nation's political leadership. Nowhere are these 2 lessons more clearly illustrated than in present-day Indonesia. Because the campaign for family planning was given high personal and political priority by the President, and because 85% of all family

  12. Monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, S.S.; Gudi, R.D.; Athwal, R.S.

    1988-12-01

    The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the loss of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.

  13. The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence

    DEFF Research Database (Denmark)

    Ignatiadis, Michail; Azim, Hatem A; Desmedt, Christine

    2016-01-01

    % (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%). Conclusions and Relevance: Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade...... was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required. Objective: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared...... and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2...

  14. Microelectronic DNA assay for the detection of BRCA1 gene mutations

    Science.gov (United States)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

    2004-01-01

    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  15. Global Activities and Plant Survival

    DEFF Research Database (Denmark)

    Bandick, Roger

    2014-01-01

    This chapter provides an extensive review of the empirical evidence found for Sweden concerning plant survival. The result reveals that foreign MNE plants and exporting non-MNE plants have the lowest exit rates, followed by purely domestic-oriented plants, and that domestic MNE plants have...... the highest exit rates. Moreover, the exit rates of globally engaged plants seem to be unaffected by increased foreign presence, whereas there appears to be a negative impact on the survival rates of non-exporting non-MNE plants. Finally, the result reveals that the survival ratio of plants of acquired...... exporters, but not other types of plants, improves post acquisition....

  16. Cardiovascular disease incidence and survival

    DEFF Research Database (Denmark)

    Byberg, Stine; Agyemang, Charles; Zwisler, Ann Dorthe

    2016-01-01

    Studies on cardiovascular disease (CVD) incidence and survival show varying results between different ethnic groups. Our aim was to add a new dimension by exploring the role of migrant status in combination with ethnic background on incidence of-and survival from-CVD and more specifically acute...... of some types of cardiovascular disease compared to Danish-born. Family-reunified migrants on the other hand had lower rates of CVD. All migrants had better survival than Danish-born indicating that migrants may not always be disadvantaged in health....

  17. Detailed review of transgenic rodent mutation assays.

    Science.gov (United States)

    Lambert, Iain B; Singer, Timothy M; Boucher, Sherri E; Douglas, George R

    2005-09-01

    Induced chromosomal and gene mutations play a role in carcinogenesis and may be involved in the production of birth defects and other disease conditions. While it is widely accepted that in vivo mutation assays are more relevant to the human condition than are in vitro assays, our ability to evaluate mutagenesis in vivo in a broad range of tissues has historically been quite limited. The development of transgenic rodent (TGR) mutation models has given us the ability to detect, quantify, and sequence mutations in a range of somatic and germ cells. This document provides a comprehensive review of the TGR mutation assay literature and assesses the potential use of these assays in a regulatory context. The information is arranged as follows. (1) TGR mutagenicity models and their use for the analysis of gene and chromosomal mutation are fully described. (2) The principles underlying current OECD tests for the assessment of genotoxicity in vitro and in vivo, and also nontransgenic assays available for assessment of gene mutation, are described. (3) All available information pertaining to the conduct of TGR assays and important parameters of assay performance have been tabulated and analyzed. (4) The performance of TGR assays, both in isolation and as part of a battery of in vitro and in vivo short-term genotoxicity tests, in predicting carcinogenicity is described. (5) Recommendations are made regarding the experimental parameters for TGR assays, and the use of TGR assays in a regulatory context.

  18. [Physical activity and cancer survival].

    Science.gov (United States)

    Romieu, Isabelle; Touillaud, Marina; Ferrari, Pietro; Bignon, Yves-Jean; Antoun, Sami; Berthouze-Aranda, Sophie; Bachmann, Patrick; Duclos, Martine; Ninot, Grégory; Romieu, Gilles; Sénesse, Pierre; Behrendt, Jan; Balosso, Jacques; Pavic, Michel; Kerbrat, Pierre; Serin, Daniel; Trédan, Olivier; Fervers, Béatrice

    2012-10-01

    Physical activity has been shown in large cohort studies to positively impact survival in cancer survivors. Existing randomized controlled trials showed a beneficial effect of physical activity on physical fitness, quality of life, anxiety and self-esteem; however, the small sample size, the short follow-up and the lack of standardization of physical activity intervention across studies impaired definite conclusion in terms of survival. Physical activity reduces adiposity and circulating estrogen levels and increases insulin sensitivity among other effects. A workshop was conducted at the International Agency for Research on Cancer in April 2011 to discuss the role of physical activity on cancer survival and the methodology to develop multicentre randomized intervention trials, including the type of physical activity to implement and its association with nutritional recommendations. The authors discuss the beneficial effect of physical activity on cancer survival with a main focus on breast cancer and report the conclusions from this workshop.

  19. An ultrafiltration assay for lysyl oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Shackleton, D.R.; Hulmes, D.J. (Univ. of Edinburgh Medical School (England))

    1990-03-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a (4,5-3H)-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.

  20. Development and application of assays for serotonin

    Energy Technology Data Exchange (ETDEWEB)

    Gow, I.F.

    1987-01-01

    In this thesis, two assays for serotonin were developed, validated, and used to investigate the relationship between platelet aggregation, serotonin levels and sodium status and serotonin levels and platelet function in patients with cardiovascular disease. A radioimmunoassay (RIA) using an (/sup 125/I)-labelled tracer was developed and validated for the measurement of serotonin in human platelet-rich plasma (PRP) and rat serum. Antisera were raised against N-succinamylserotonin conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. An assay for serotonin using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) was developed, and used to validate the RIA. The RIA can be used to assay up to 100 samples/day compared with 10-20/day by the HPLC-ECD assay.

  1. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert

    2016-01-01

    OBJECTIVE: Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab......) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...... with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative...

  2. Operational slack and venture survival

    OpenAIRE

    Azadegan, Arash; Patel, Pankaj; Parida, Vinit

    2013-01-01

    Slack can act as a double-edged sword. While it can buffer against environmental threats to help ensure business continuity, slack canalso be costly and reduce profitability. In this study, we focus on operational slack, the form related to the firm’s production processes. We investigate the role of operational slack on firm survival during its venture stage, when its survival is significantly challenged by environmental threats. Specifically, we explore how change in three types of environme...

  3. Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

    Directory of Open Access Journals (Sweden)

    Susan Richter

    Full Text Available Zinc finger nucleases (ZFN are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK, in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116. All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002 and 4.3±0.8% (p = 0.001 for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

  4. A lateral electrophoretic flow diagnostic assay

    OpenAIRE

    Lin, R; Skandarajah, A; Gerver, RE; Neira, HD; Fletcher, DA; Herr, AE

    2015-01-01

    © 2015 The Royal Society of Chemistry. Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to ...

  5. Effects of all Trans Retinoic Acid Combined with Cisplatin on Survival of Gastric Cancer Cell Line (AGS

    Directory of Open Access Journals (Sweden)

    N. Najafzadeh

    2013-10-01

    Full Text Available Introduction & Objective: All-trans retinoic acid, a derivative of retinoids, is widely used to in-duce prolifferation, differentiation and apoptosis in normal, precancareous and cancerous cells. Cisplatin, an effective drug for cancer treatment, induces apoptosis via cross-linking to DNA. Previous studies on ovarian and melanoma cancer cells have showed synergistic ef-fects of cisplatin and retinoic acid. Our aim is to study such synergistic effect on gastric de-rived cell line, AGS. Materials & Methods: In this experimental study gastric cancer cell line was cultured with dif-ferent concentration of retinoic acid and cisplatin and their combination. The cell death was evaluated with clonogenic assay and Acridine Orange/ Ethidium Bromide staining. Results: The results showed that all-trans retinoic acid had not significant effect on cell death in gastric cancer. The results showed that high doses of retinoic acid and cisplatin can cause cell death via necrosis and early apoptosis, respectively. The plates were treated with the combination of 10 µM retinoic acid and 5, 10 µg cisplatin, and more cell death were ob-served (P<0.001. It seems that, suseptability of this cell line to retinoic acid is dose depend-ent. Conclusion: In this study, we concluded that the combination of retinoic acid and cisplatin was more effective on cell death than cisplatin and retinoic acid alone. (Sci J Hamadan Univ Med Sci 2013; 20 (3:207-214

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

    Science.gov (United States)

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

    2015-02-01

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

  7. METHODOLOGICAL ASPECTS OF QUANTITATIVE RECEPTOR ASSAYS

    NARCIS (Netherlands)

    SMISTEROVA, J; ENSING, K; DEZEEUW, RA

    Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand

  8. A lateral electrophoretic flow diagnostic assay.

    Science.gov (United States)

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  9. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.

    2001-08-01

    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  10. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    DEFF Research Database (Denmark)

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred

    2016-01-01

    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  11. Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species.

    Science.gov (United States)

    Smith, T; Wharton, D A; Marshall, C J

    2008-01-01

    Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5 degrees C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.

  12. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival.

    Directory of Open Access Journals (Sweden)

    Pachiyappan Kamarajan

    Full Text Available The use of small antimicrobial peptides or bacteriocins, like nisin, to treat cancer is a new approach that holds great promise. Nisin exemplifies this new approach because it has been used safely in humans for many years as a food preservative, and recent laboratory studies support its anti-tumor potential in head and neck cancer. Previously, we showed that nisin (2.5%, low content has antitumor potential in head and neck squamous cell carcinoma (HNSCC in vitro and in vivo. The current studies explored a naturally occurring variant of nisin (nisin ZP; 95%, high content for its antitumor effects in vitro and in vivo. Nisin ZP induced the greatest level of apoptosis in HNSCC cells compared to low content nisin. HNSCC cells treated with increasing concentrations of nisin ZP exhibited increasing levels of apoptosis and decreasing levels of cell proliferation, clonogenic capacity, and sphere formation. Nisin ZP induced apoptosis through a calpain-dependent pathway in HNSCC cells but not in human oral keratinocytes. Nisin ZP also induced apoptosis dose-dependently in human umbilical vein endothelial cells (HUVEC with concomitant decreases in vascular sprout formation in vitro and reduced intratumoral microvessel density in vivo. Nisin ZP reduced tumorigenesis in vivo and long-term treatment with nisin ZP extended survival. In addition, nisin treated mice exhibited normal organ histology with no evidence of inflammation, fibrosis or necrosis. In summary, nisin ZP exhibits greater antitumor effects than low content nisin, and thus has the potential to serve as a novel therapeutic for HNSCC.

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

    National Research Council Canada - National Science Library

    Gilberg Erik; Stumpfe Dagmar; Bajorath Jürgen

    2017-01-01

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

  14. Comet assay on mice testicular cells

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar

    2015-01-01

    for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity...... of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided...... with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014). An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster...

  15. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation...... of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...... International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c...

  16. Pharmacologically active microcarriers for endothelial progenitor cell support and survival.

    Science.gov (United States)

    Musilli, Claudia; Karam, Jean-Pierre; Paccosi, Sara; Muscari, Claudio; Mugelli, Alessandro; Montero-Menei, Claudia N; Parenti, Astrid

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Survival and reproduction of small hive beetle (Coleoptera: Nitidulidae) on commercial pollen substitutes

    Science.gov (United States)

    An assay was developed to investigate the small hive beetle’s (Aethina tumida) potential for survival and reproduction when providing artificial food resources in managed European honey bees (Apis mellifera). Supplemental feeding is done to maintain the health of the hive, initiate comb building, ex...

  18. Survival Processing Eliminates Collaborative Inhibition.

    Science.gov (United States)

    Reysen, Matthew B; Bliss, Heather; Baker, Melissa A

    2017-04-11

    The present experiments examined the effect of processing words for their survival value, relevance to moving, and pleasantness on participants' free recall scores in both nominal groups (non-redundant pooled individual scores) and collaborative dyads. Overall, participants recalled more words in the survival processing conditions than in the moving and pleasantness processing conditions. Furthermore, nominal groups in both the pleasantness condition (Experiment 1) and the moving and pleasantness conditions (Experiment 2) recalled more words than collaborative groups, thereby replicating the oft-observed effect of collaborative inhibition. However, processing words for their survival value appeared to eliminate the deleterious effects of collaborative remembering in both Experiments 1 and 2. These results are discussed in the context of the retrieval strategy disruption hypothesis and the effects of both expertise and collaborative skill on group remembering.

  19. Survival in Women with NSCLC

    Science.gov (United States)

    Katcoff, Hannah; Wenzlaff, Angela S.; Schwartz, Ann G.

    2014-01-01

    Introduction Although lung cancer is the leading cause of cancer death in women, few studies have investigated the hormonal influence on survival after a lung cancer diagnosis and results have been inconsistent. We evaluated the role of reproductive and hormonal factors in predicting overall survival in women with non–small-cell lung cancer (NSCLC). Methods Population-based lung cancer cases diagnosed between November 1, 2001 and October 31, 2005 were identified through the Metropolitan Detroit Surveillance, Epidemiology, and End Results Registry. Interview and follow-up data were collected for 485 women. Cox proportional hazard regression models were used to determine hazard ratios (HRs) for death after an NSCLC diagnosis associated with reproductive and hormonal variables. Results Use of hormone therapy (HT) was associated with improved survival (HR, 0.69; 95% confidence interval, 0.54–0.89), adjusting for stage, surgery, radiation, education level, pack-years of smoking, age at diagnosis, race, and a multiplicative interaction between stage and radiation. No other reproductive or hormonal factor was associated with survival after an NSCLC diagnosis. Increased duration of HT use before the lung cancer diagnosis (132 months or longer) was associated with improved survival (HR, 0.54; 95% confidence interval, 0.37–0.78), and this finding remained significant in women taking either estrogen alone or progesterone plus estrogen, never smokers, and smokers. Conclusion These findings suggest that HT use, in particular use of estrogen plus progesterone, and long-term HT use are associated with improved survival of NSCLC. PMID:24496005

  20. Survival of Sami cancer patients

    Directory of Open Access Journals (Sweden)

    Leena Soininen

    2012-07-01

    Full Text Available Objectives. The incidence of cancer among the indigenous Sami people of Northern Finland is lower than among the Finnish general population. The survival of Sami cancer patients is not known, and therefore it is the object of this study. Study design. The cohort consisted of 2,091 Sami and 4,161 non-Sami who lived on 31 December 1978 in the two Sami municipalities of Inari and Utsjoki, which are located in Northern Finland and are 300–500 km away from the nearest central hospital. The survival experience of Sami and non-Sami cancer patients diagnosed in this cohort during 1979–2009 was compared with that of the Finnish patients outside the cohort. Methods. The Sami and non-Sami cancer patients were matched to other Finnish cancer patients for gender, age and year of diagnosis and for the site of cancer. An additional matching was done for the stage at diagnosis. Cancer-specific survival analyses were made using the Kaplan–Meier method and Cox regression modelling. Results. There were 204 Sami and 391 non-Sami cancer cases in the cohort, 20,181 matched controls without matching with stage, and 7,874 stage-matched controls. In the cancer-specific analysis without stage variable, the hazard ratio for Sami was 1.05 (95% confidence interval 0.85–1.30 and for non-Sami 1.02 (0.86–1.20, indicating no difference between the survival of those groups and other patients in Finland. Likewise, when the same was done by also matching the stage, there was no difference in cancer survival. Conclusion. Long distances to medical care or Sami ethnicity have no influence on the cancer patient survival in Northern Finland.

  1. Survival following spinal cord infarction.

    Science.gov (United States)

    New, P W; McFarlane, C L

    2013-06-01

    Retrospective open cohort. To calculate the survival of patients with spinal cord infarction and to compare the cause of death in patients with different mechanisms of ischaemic injury. Spinal Rehabilitation Unit, Melbourne, Victoria, Australia. Consecutive admissions between 1 January 1995 and 31 December 2008 with recent onset of spinal cord infarction. Linkage to the Registry of Births, Deaths and Marriages (Victoria) was used to determine survival following discharge from in-patient rehabilitation and cause of death. A total of 44 patients were admitted (males=26, 59%), with a median age of 72 years (interquartile range (IQR) 62-79). One patient died during their in-patient rehabilitation programme. In all, 14 patients (n=14/44; 33%) died during the follow-up period. The median survival after diagnosis was 56 months (IQR 28-85) and after discharge from in-patient rehabilitation was 46 months (IQR 25-74). The 1- and 5-year mortality rates were 7.0% (n=3/43; 95% confidence interval (CI)=2.4-18.6%) and 20.9% (n=9/43; 95% CI=11.4-35.2%). There was no statistically significant difference in survival between patients with the different aetiologies of spinal cord infarction (other vs idiopathic: χ(2)=0.6, P=0.7; other vs vascular: χ(2)=1.9, P=0.3). There was no relationship between survival and gender (χ(2)=0.2, P=0.6), age (χ(2)=3.0, P=0.08), level of injury (χ(2)=0.0, P=1) or American Spinal Cord Society Impairment Scale grade of spinal cord injury (χ(2)=0.02, P=0.9). Patients with spinal cord infarction appear to have a fair survival after discharge from in-patient rehabilitation, not withstanding the occurrence of risk factors of vascular disease in many patients.

  2. Genetic aspects of piglet survival

    OpenAIRE

    Knol, E.F.

    2001-01-01

    Piglet mortality is high. In the USA nearly 20% of the piglets do not survive between late gestation and weaning; 7% of the piglets die during farrowing and some 13% are lost during lactation. These statistics from the USA are no exception to the norm. Selection for increased piglet survival, if possible, could have an important economic impact.

    Litters and sows

    Data on some 33.000 litters and some 400.000 piglets from a commercial breeding progr...

  3. Frailty Models in Survival Analysis

    CERN Document Server

    Wienke, Andreas

    2010-01-01

    The concept of frailty offers a convenient way to introduce unobserved heterogeneity and associations into models for survival data. In its simplest form, frailty is an unobserved random proportionality factor that modifies the hazard function of an individual or a group of related individuals. "Frailty Models in Survival Analysis" presents a comprehensive overview of the fundamental approaches in the area of frailty models. The book extensively explores how univariate frailty models can represent unobserved heterogeneity. It also emphasizes correlated frailty models as extensions of

  4. DNA Methyltransferase Activity Assays: Advances and Challenges.

    Science.gov (United States)

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice.

  5. Automation of a chloramphenicol acetyltransferase assay.

    Science.gov (United States)

    Chauchereau, A; Astinotti, D; Bouton, M M

    1990-08-01

    Accurate quantification of chloramphenicol acetyltransferase (CAT) enzyme activity in a large number of samples has been achieved through robotization of a CAT assay on a laboratory workstation (Biomek 1000). The basic principle of this CAT assay relies on the selective diffusion of [3H]acetylchloramphenicol into a water-immiscible liquid scintillation cocktail. This methodology gives unique characteristics to this robotized protocol by allowing complete control over the kinetics of the CAT enzymatic reaction which is a critical parameter in the CAT assay. Thus it has been possible to optimize the CAT assay for every processed sample, through real time monitoring of the enzymatic reaction, and to achieve maximum accuracy in CAT quantification. Moreover the sensitivity of this automated assay is high (detection threshold; 10(-4) CAT unit), and the sample processing is fast (approximately 125 samples per hour). Compared to other CAT assay protocols currently used, our robotized technique offers major advantages in terms of CAT quantification, and sets new standards for CAT assay productivity.

  6. Nano-immunosafety: issues in assay validation

    Energy Technology Data Exchange (ETDEWEB)

    Boraschi, Diana; Italiani, Paola [Institute of Biomedical Technologies, National Research Council, Via G. Moruzzi 1, 56124 Pisa (Italy); Oostingh, Gertie J; Duschl, Albert [Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Casals, Eudald; Puntes, Victor F [Institut Catala de Nanotecnologia, Campus de la UAB - Facultat de Ciencies, Edifici CM7, 08193 Bellaterra (Spain); Nelissen, Inge, E-mail: diana.boraschi@itb.cnr.it [VITO NV, Boeretang 200, BE-2400 Mol (Belgium)

    2011-07-06

    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  7. The comet assay in nanotoxicology research.

    Science.gov (United States)

    Karlsson, Hanna L

    2010-09-01

    Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

  8. Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6- and 24-well miniaturized plate incorporation assays and the Ames II™ assay.

    Science.gov (United States)

    Pant, Kamala; Bruce, Shannon; Sly, Jamie; Klug Laforce, Michelle; Springer, Sandra; Cecil, Mark; Andrus, Edgar; Dakoulas, Emily; Wagner, Valentine O; Hewitt, Nicola J; Kulkarni, Rohan

    2016-07-01

    Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Antifungal chemical compounds identified using a C. elegans pathogenicity assay.

    Directory of Open Access Journals (Sweden)

    Julia Breger

    2007-02-01

    Full Text Available There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2% that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.

  10. Reporter gene assays for investigating GPCR signaling.

    Science.gov (United States)

    Azimzadeh, Pedram; Olson, John A; Balenga, Nariman

    2017-01-01

    Luciferase-based assays are applied to evaluate various cellular processes due to their sensitivity and feasibility. The field of GPCR research has also benefited from this enzymatic reaction both in deorphanization campaigns and in delineation of the signaling pathways. Here, we describe the details of this assay in GPCR studies in 96-well format and will provide examples where the assay can show constitutive activity of an orphan GPCR and demonstrate the impact of cell type on the efficacy and potency of ligands. © 2017 Elsevier Inc. All rights reserved.

  11. Passive neutron assay of irradiated nuclear fuels

    Energy Technology Data Exchange (ETDEWEB)

    Hsue, S.T.; Stewart, J.E.; Kaieda, K.; Halbig, J.K.; Phillips, J.R.; Lee, D.M.; Hatcher, C.R.

    1979-02-01

    Passive neutron assay of irradiated nuclear fuel has been investigated by calculations and experiments as a simple, complementary technique to the gamma assay. From the calculations it was found that the neutron emission arises mainly from the curium isotopes, the neutrons exhibit very good penetrability of the assemblies, and the neutron multiplication is not affected by the burnup. From the experiments on BWR and PWR assemblies, the neutron emission rate is proportional to burnup raised to 3.4 power. The investigations indicate that the passive neutron assay is a simple and useful technique to determine the consistency of burnups between assemblies.

  12. School Leadership: Handbook for Survival.

    Science.gov (United States)

    Smith, Stuart C., Ed.; And Others

    Based on the assumption that the survival of the nation's schools and their leaders depends on these leaders having real influence over the quality of schooling, this volume draws from the work of many authorities to look at leadership from three perspectives: the person, the structure, and the skills. Chapters focusing on the person who holds the…

  13. Long-term haemodialysis survival

    DEFF Research Database (Denmark)

    Heaf, James; Nielsen, Arne Høj; Hansen, Henrik Post

    2012-01-01

    Haemodialysis (HD) treatment for end-stage renal disease bears a poor prognosis. We present a case of a patient who, apart from two transplant periods lasting 8 months in all, was treated with conventional in-centre HD three times a week and who survived for 41 years. Patients should be aware tha...

  14. Characteristics of Patients Who Survived

    NARCIS (Netherlands)

    Verlaan, Jorrit-Jan; Choi, David; Versteeg, Anne; Albert, Todd; Arts, Mark; Balabaud, Laurent; Bunger, Cody; Buchowski, Jacob Maciej; Chung, Chung Kee; Coppes, Maarten Hubert; Crockard, Hugh Alan; Depreitere, Bart; Fehlings, Michael George; Harrop, James; Kawahara, Norio; Kim, Eun Sang; Lee, Chong-Suh; Leung, Yee; Liu, Zhongjun; Martin-Benlloch, Antonio; Massicotte, Eric Maurice; Mazel, Christian; Meyer, Bernhard; Peul, Wilco; Quraishi, Nasir A.; Tokuhashi, Yasuaki; Tomita, Katsuro; Ulbricht, Christian; Wang, Michael; Oner, F. Cumhur

    2016-01-01

    Purpose Survival after metastatic cancer has improved at the cost of increased presentation with metastatic spinal disease. For patients with pathologic spinal fractures and/or spinal cord compression, surgical intervention may relieve pain and improve quality of life. Surgery is generally

  15. Genetic aspects of piglet survival

    NARCIS (Netherlands)

    Knol, E.F.

    2001-01-01

    Piglet mortality is high. In the USA nearly 20% of the piglets do not survive between late gestation and weaning; 7% of the piglets die during farrowing and some 13% are lost during lactation. These statistics from the USA are no exception to the norm. Selection for increased piglet

  16. Cool echidnas survive the fire

    Science.gov (United States)

    Nowack, Julia; Cooper, Christine Elizabeth; Geiser, Fritz

    2016-01-01

    Fires have occurred throughout history, including those associated with the meteoroid impact at the Cretaceous–Palaeogene (K–Pg) boundary that eliminated many vertebrate species. To evaluate the recent hypothesis that the survival of the K–Pg fires by ancestral mammals was dependent on their ability to use energy-conserving torpor, we studied body temperature fluctuations and activity of an egg-laying mammal, the echidna (Tachyglossus aculeatus), often considered to be a ‘living fossil’, before, during and after a prescribed burn. All but one study animal survived the fire in the prescribed burn area and echidnas remained inactive during the day(s) following the fire and substantially reduced body temperature during bouts of torpor. For weeks after the fire, all individuals remained in their original territories and compensated for changes in their habitat with a decrease in mean body temperature and activity. Our data suggest that heterothermy enables mammals to outlast the conditions during and after a fire by reducing energy expenditure, permitting periods of extended inactivity. Therefore, torpor facilitates survival in a fire-scorched landscape and consequently may have been of functional significance for mammalian survival at the K–Pg boundary. PMID:27075255

  17. Modelling survival and connectivity of

    NARCIS (Netherlands)

    van der Molen, J.; van Beek, J.; Augustine, S.; Vansteenbrugge, L.; van Walraven, L.; van Langenberg, V.; van der Veer, H.W.; Hostens, K.; Pitois, S.; Robbens, J.

    2015-01-01

    Three different models were applied to study the reproduction, survival and dispersal of Mnemiopsis leidyi in the Scheldt estuaries and the southern North Sea: a high-resolution particle tracking model with passive particles, a low-resolution particle tracking model with a reproduction model

  18. Survivability of SCADA Control Loop

    NARCIS (Netherlands)

    Camacho, José; de Boer, Pieter-Tjerk; Remke, Anne Katharina Ingrid

    2009-01-01

    The endorsement of information technologies for critical infrastructures control introduces new threats in their security and surveillance. Along with certain level of protection against attacks, it is desirable for critical processes to survive even if they succeed. A stochastic Petri Nets-based

  19. Parathyroid carcinoma survival: improvements in the era of intact parathyroid hormone monitoring?

    Directory of Open Access Journals (Sweden)

    Steve R. Martinez

    2013-02-01

    Full Text Available The intact parathyroid hormone (iPTH assay is a critical test in the diagnosis and management of PTH-mediated hypercalcemia, including parathyroid carcinoma (PCa. We hypothesized that the survival of patients diagnosed with PCa has improved since adoption of the iPTH assay into clinical practice. We identified all confirmed cases of PCa within the Surveillance, Epidemiology and End Results database from 1973 to 2006. Patients were categorized into two eras based upon introduction of the iPTH assay: 1973 to 1997 (era I and 1997 to 2006 (era II, when the iPTH assay was in standard use. We estimated overall survival (OS and disease-specific survival (DSS using the Kaplan-Meier method, with differences among survival curves assessed via log rank. Multivariate Cox proportional hazards models compared the survival rates between treatment eras while controlling for patient age, sex, race/ethnicity, tumor size, nodal status, extent of disease, and type of surgery. Multivariate models included patients undergoing potentially curative surgery and excluded those with dis- tant metastases. Risks of overall and disease-specific mortality were reported as hazard ratios with 95% confidence intervals. Study criteria were met by 370 patients. Median survival was 15.6 years. Five-year rates of OS and DSS were 78% and 88% for era I and 82% and 96% for era II. On multivariate analysis, age, black race, and unknown extent of disease predicted an increased risk of death from any cause. Treatment era did not predict OS. No factor predicted PCa-specific mortality. In multivariate analysis, neither OS nor DSS have improved in the current era that utilizes iPTH for the detection and management of PCa.

  20. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a... intended for use in conjunction with other laboratory findings and clinical assessment of the patient to...

  1. NEW INDIRECT IMMUNOFLUORESCENCE ASSAY AS A ...

    African Journals Online (AJOL)

    hi-tech

    2004-05-05

    HIV-1) infection is based on the detection of antibodies to HIV-1 in plasma or serum. Antibodies against various viral structure proteins are measured by a number of simple and sensitive screening tests. These assays include ...

  2. Validation of a new automated renin assay.

    NARCIS (Netherlands)

    Bruin, R.A. de; Bouhuizen, A.; Diederich, S.; Perschel, F.H.; Boomsma, F.; Deinum, J.

    2004-01-01

    BACKGROUND: Measurement of plasma renin is important for the treatment of patients with congenital adrenal hyperplasia (CAH) and in the evaluation of patients with suspected hyperaldosteronism. Immunologic assays for plasma renin offer easier implementation and standardization than enzyme-kinetic

  3. LINE-1 Cultured Cell Retrotransposition Assay

    Science.gov (United States)

    Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

    2016-01-01

    Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

  4. Automated optical sensing system for biochemical assays

    Science.gov (United States)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  5. The comet assay in nanotoxicology research

    OpenAIRE

    Karlsson, Hanna L

    2010-01-01

    Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The ar...

  6. Nuclear Resonance Fluorescence for Materials Assay

    OpenAIRE

    Quiter, Brian

    2010-01-01

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code M...

  7. Assessment of contaminated sediments with an indoor freshwater/sediment microcosm assay.

    Science.gov (United States)

    Triffault-Bouchet, Gaëlle; Clément, Bernard; Blake, Gérard

    2005-09-01

    This study was conducted to assess the feasibility of using a 2-L, indoor microcosm assay to evaluate five contaminated sediments (A, B, C, D, and E). Toxic potential was deduced in the light of general contamination of sediments, pollutant partitioning in microcosms, and biological responses of species (Pseudokirchneriella subcapitata, Lemna minor, Daphnia magna, Hyalella azteca, Chironomus riparius): E > A > B > C > D. Sediments mainly were contaminated by metals (lead and zinc). Organic pollutant contents varied among the sediments. The major polycyclic aromatic hydrocarbon (PAH) compounds were pyrene, fluoranthene, and phenanthrene. Sediments A, B, and C highly stimulated duckweed growth (> 700%) and impaired daphnid (< 20%) and amphipod survival (< 30%). Sediment D had no significant effect on pelagic and benthic organisms. Finally, sediment E, the most toxic, limited duckweed growth (inhibition of 82%) and impaired daphnid survival (0% of survival). Amphipods were impaired dramatically by this sediment (0% of survival), in contrast with chironomids, for which no toxic effect was measured. The 2-L, indoor microcosm assay successfully was applied to the assessment of those five contaminated sediments. Sediments A, B, C, and E should not be deposited in gravel quarries, and new, more sensitive endpoint measurements should be developed.

  8. Nondestructive assay methods for irradiated nuclear fuels

    Energy Technology Data Exchange (ETDEWEB)

    Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

    1978-01-01

    This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed.

  9. Quantum dot-based cell motility assay.

    Science.gov (United States)

    Pellegrino, Teresa; Parak, Wolfgang J; Boudreau, Rosanne; Le Gros, Mark A; Gerion, Daniele; Alivisatos, A Paul; Larabell, Carolyn A

    2003-12-01

    Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.

  10. Thread as a matrix for biomedical assays.

    Science.gov (United States)

    Reches, Meital; Mirica, Katherine A; Dasgupta, Rohit; Dickey, Michael D; Butte, Manish J; Whitesides, George M

    2010-06-01

    This paper describes the use of thread as a matrix for the fabrication of diagnostic assay systems. The kinds of thread used for this study are inexpensive, broadly available, and lightweight; some of them are already familiar materials in healthcare. Fluids wick along these threads by capillary action; no external power source is necessary for pumping. This paper demonstrates three designs for diagnostic assays that use different characteristics of the thread. The first two designs-the "woven array" and the "branching design"-take advantage of the ease with which thread can be woven on a loom to generate fluidic pathways that enable multiple assays to be performed in parallel. The third design-the "sewn array"-takes advantage of the ease with which thread can be sewn through a hydrophobic polymer sheet to incorporate assays into bandages, diapers and similar systems. These designs lead to microfluidic devices that may be useful in performing simple colorimetric assays that require qualitative results. We demonstrate the function of thread-based microfluidic devices in the context of five different colorimetric assays: detection of ketones, nitrite, protein, and glucose in artificial urine, and detection of alkaline phosphatase in artificial plasma.

  11. Application of frog embryo teratogenesis assay-Xenopus to ecological risk assessment.

    Science.gov (United States)

    Hoke, Robert A; Ankley, Gerald T

    2005-10-01

    An expert workshop recently was convened to consider the frog embryo teratogenesis assay-Xenopus (FETAX) as a screening method for identifying the potential developmental toxicity of single chemicals and chemical mixtures. One recommendation from the workshop was that, in order to determine the utility of FETAX for ecological risk assessments, additional consideration of how the assay is conducted is necessary. In addition, a comparative evaluation would be useful of FETAX endpoints (i.e., survival, malformations, growth) versus each other, endpoints from aquatic toxicity tests using more commonly tested species of cladocerans and fish, and tests with other amphibian species. This review provides an evaluation and critique of the current FETAX protocol from two perspectives: Practical considerations relative to conducting the test and sensitivity of the assay (and associated endpoints) compared to tests with other species. Several aspects of the current standard protocol, including test temperature, diet, loading rates, and chemical exposure options, need to be modified to ensure that the assay is robust technically. Evaluation of FETAX data from the open literature indicates that growth is the most sensitive endpoint in the assay, followed by malformations and then survival; unfortunately, the growth endpoint often is not considered or reported in the assay. Comparison of FETAX data with acute toxicity data from tests with other amphibians or traditional aquatic test species indicates FETAX is relatively insensitive. This suggests that environmental risk assessments using acute hazard data from tests with traditional aquatic test species usually would be more protective of native amphibian species than risk assessments that use hazard data from FETAX.

  12. A multi-centre evaluation of the intra-assay and inter-assay variation of commercial and in-house anti-cardiolipin antibody assays.

    Science.gov (United States)

    Wong, Richard; Favaloro, Emmanuel; Pollock, Wendy; Wilson, Robert; Hendle, Michelle; Adelstein, Stephen; Baumgart, Karl; Homes, Paul; Smith, Stuart; Steele, Richard; Sturgess, Allan; Gillis, David

    2004-04-01

    To assess the intra-assay (intra-run) and inter-assay (inter-run) variation of commercial and in-house IgG and IgM anti-cardiolipin antibody (aCL) assays/kits, and to determine an appropriate maximum value for inclusion in consensus guidelines. Frozen aliquots of two patient specimens and one commercial control were sent to nine laboratories for the evaluation of eight commercial kits and one in-house assay. Intra-assay and inter-assay evaluations were performed with all three samples for IgG aCL, and one patient specimen for IgM aCL. The IgG and IgM aCL values varied considerably between the nine assays/kits. The majority of assays/kits demonstrated less than 20% intra-assay and inter-assay variation, with lower intra-assay and inter-assay variation observed with the commercial control. Single calibrator assays were not consistently associated with higher inter-assay variation than multi-point calibrator assays. An inter-assay coefficient of variation of 20% was determined to be an appropriate maximum value for inclusion in the Australasian aCL Working Party consensus guidelines. Improved standardisation between different assay/kits is still required.

  13. Diurnal cortisol and survival in epithelial ovarian cancer.

    Science.gov (United States)

    Schrepf, Andrew; Thaker, Premal H; Goodheart, Michael J; Bender, David; Slavich, George M; Dahmoush, Laila; Penedo, Frank; DeGeest, Koen; Mendez, Luis; Lubaroff, David M; Cole, Steven W; Sood, Anil K; Lutgendorf, Susan K

    2015-03-01

    Hypothalamic-pituitary-adrenal (HPA) deregulation is commonly observed in cancer patients, but its clinical significance is not well understood. We prospectively examined the association between HPA activity, tumor-associated inflammation, and survival in ovarian cancer patients prior to treatment. Participants were 113 women with ovarian cancer who provided salivary cortisol for three days prior to treatment for calculation of cortisol slope, variability, and night cortisol. Cox proportional hazard regression analyses were used to examine associations between cortisol and survival in models adjusting for disease stage, tumor grade, cytoreduction and age. On a subsample of 41 patients with advanced disease ascites fluid was assayed for levels of interleukin-6 (IL-6) and correlated with cortisol variables. Each cortisol measure was associated with decreased survival time, adjusting for covariates (all pcortisol was associated with a 46% greater likelihood of death. Patients in the high night cortisol group survived an estimated average of 3.3 years compared to 7.3 years for those in the low night cortisol group. Elevated ascites IL-6 was associated with each cortisol measure (all r>36, all pcortisol rhythms assessed prior to treatment are associated with decreased survival in ovarian cancer and increased inflammation in the vicinity of the tumor. HPA abnormalities may reflect poor endogenous control of inflammation, dysregulation caused by tumor-associated inflammation, broad circadian disruption, or some combination of these factors. Nocturnal cortisol may have utility as a non-invasive measure of HPA function and/or disease severity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Individual social capital and survival

    DEFF Research Database (Denmark)

    Ejlskov, Linda; Mortensen, Rikke N; Overgaard, Charlotte

    2014-01-01

    BACKGROUND: The concept of social capital has received increasing attention as a determinant of population survival, but its significance is uncertain. We examined the importance of social capital on survival in a population study while focusing on gender differences. METHODS: We used data from...... a Danish regional health survey with a five-year follow-up period, 2007-2012 (n = 9288, 53.5% men, 46.5% women). We investigated the association between social capital and all-cause mortality, performing separate analyses on a composite measure as well as four specific dimensions of social capital while...... controlling for covariates. Analyses were performed with Cox proportional hazard models by which hazard ratios and 95% confidence intervals were calculated. RESULTS: For women, higher levels of social capital were associated with lower all-cause mortality regardless of age, socioeconomic status, health...

  15. Campylobacter virulence and survival factors.

    Science.gov (United States)

    Bolton, Declan J

    2015-06-01

    Despite over 30 years of research, campylobacteriosis is the most prevalent foodborne bacterial infection in many countries including in the European Union and the United States of America. However, relatively little is known about the virulence factors in Campylobacter or how an apparently fragile organism can survive in the food chain, often with enhanced pathogenicity. This review collates information on the virulence and survival determinants including motility, chemotaxis, adhesion, invasion, multidrug resistance, bile resistance and stress response factors. It discusses their function in transition through the food processing environment and human infection. In doing so it provides a fundamental understanding of Campylobacter, critical for improved diagnosis, surveillance and control. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Does Intelligence Provide Survival Value?

    OpenAIRE

    Cairns, John

    2009-01-01

    Intelligence is defined as a general mental capacity to reason, plan, solve problems, think abstractly, comprehend ideas, and learn. Intelligence can also be defined as the ability to acquire and apply information gathered from the environment to modify its behavior. It is this intelligence that has allowed the genus Homo to survive for 2 million years. However, recently the global financial meltdown and the deleterious effects of climate change raise the question of whether intelligence has ...

  17. Survival on Land and Sea

    Science.gov (United States)

    1943-01-01

    flower, leaf steams, and young leaves may be eaten cooked as greens provided you cook them in several changes of water to remove the bitter taste and...and become a weed almost throughout the tropics. Its bladdery pods contain a single retl tomato-like fruit that is edible. Raspberries, blackberries ...plant that 108 SURVIVAL OK LAND AND SEA is about one foot In height and has blue flowers and inflated leaf stems. The young leaves, leafy stalks

  18. Columbia Crew Survival Investigation Report

    Science.gov (United States)

    2009-01-01

    NASA commissioned the Columbia Accident Investigation Board (CAIB) to conduct a thorough review of both the technical and the organizational causes of the loss of the Space Shuttle Columbia and her crew on February 1, 2003. The accident investigation that followed determined that a large piece of insulating foam from Columbia s external tank (ET) had come off during ascent and struck the leading edge of the left wing, causing critical damage. The damage was undetected during the mission. The CAIB's findings and recommendations were published in 2003 and are available on the web at http://caib.nasa.gov/. NASA responded to the CAIB findings and recommendations with the Space Shuttle Return to Flight Implementation Plan. Significant enhancements were made to NASA's organizational structure, technical rigor, and understanding of the flight environment. The ET was redesigned to reduce foam shedding and eliminate critical debris. In 2005, NASA succeeded in returning the space shuttle to flight. In 2010, the space shuttle will complete its mission of assembling the International Space Station and will be retired to make way for the next generation of human space flight vehicles: the Constellation Program. The Space Shuttle Program recognized the importance of capturing the lessons learned from the loss of Columbia and her crew to benefit future human exploration, particularly future vehicle design. The program commissioned the Spacecraft Crew Survival Integrated Investigation Team (SCSIIT). The SCSIIT was asked to perform a comprehensive analysis of the accident, focusing on factors and events affecting crew survival, and to develop recommendations for improving crew survival for all future human space flight vehicles. To do this, the SCSIIT investigated all elements of crew survival, including the design features, equipment, training, and procedures intended to protect the crew. This report documents the SCSIIT findings, conclusions, and recommendations.

  19. Statistical analysis of survival data.

    Science.gov (United States)

    Crowley, J; Breslow, N

    1984-01-01

    A general review of the statistical techniques that the authors feel are most important in the analysis of survival data is presented. The emphasis is on the study of the duration of time between any two events as applied to people and on the nonparametric and semiparametric models most often used in these settings. The unifying concept is the hazard function, variously known as the risk, the force of mortality, or the force of transition.

  20. LATERAL SURVIVAL: AN OT ACCOUNT

    Directory of Open Access Journals (Sweden)

    Moira Yip

    2004-12-01

    Full Text Available When laterals are the targets of phonological processes, laterality may or may not survive. In a fixed feature geometry, [lateral] should be lost if its superordinate node is eliminated by either the spreading of a neighbouring node, or by coda neutralization. So if [lateral] is under Coronal (Blevins 1994, it should be lost under Place assimilation, and if [lateral] is under Sonorant Voicing (Rice & Avery 1991 it should be lost by rules that spread voicing. Yet in some languages lateral survives such spreading intact. Facts like these argue against a universal attachment of [lateral] under either Coronal or Sonorant Voicing, and in favour of an account in terms of markedness constraints on feature-co-occurrence (Padgett 2000. The core of an OT account is that IFIDENTLAT is ranked above whatever causes neutralization, such as SHARE-F or *CODAF. laterality will survive. If these rankings are reversed, we derive languages in which laterality is lost. The other significant factor is markedness. High-ranked feature co-occurrence constraints like *LATDORSAL can block spreading from affecting laterals at all.

  1. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  2. The comet assay in male reproductive toxicology.

    Science.gov (United States)

    Baumgartner, A; Cemeli, E; Anderson, D

    2009-02-01

    Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.

  3. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins

    2014-10-01

    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  4. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Directory of Open Access Journals (Sweden)

    Bas-jan Van Der Leede

    2015-08-01

    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  5. Comparison of Elecsys Anti-HCV II Assay With Other HCV Screening Assays.

    Science.gov (United States)

    Li, Dongdong; Zhu, Siyuan; Wang, Tingting; An, Jingna; Wang, Lanlan; Tao, Chuanmin

    2016-09-01

    Early detection of hepatitis C virus (HCV) is an important step in preventing progression to cirrhosis and hepatocellular carcinoma. Serologic assays for anti-HCV antibody are valuable first-line tests in the screening and diagnosis of HCV infection. This study's aim was to evaluate the sensitivity and specificity of Elecsys Anti-HCV II assay for HCV screening. A total of 1,044 routine sera, 20 known HCV-positive samples, plus 54 preselected weakly positive samples were tested for anti-HCV with Elecsys Anti-HCV II assay, Elecsys Anti-HCV assays, InTec HCV enzymoimmunoassay (EIA), and Livzon Anti-HCV EIA. Interference test was assessed with additional 423 specimens without clinical evidence of HCV infection: preselected HCV weak reactive samples; dialysis samples; anti-HBc (antibody to HBV core antigen) (+), anti-Treponema pallidum (+), and anti-HIV (+) sera; and samples form autoimmune/alcoholic hepatitis or systemic Lupus erythematosus (SLE). Discrepant results were evaluated with recombinant immunoblot assay. The seroconversion panels were evaluated to assess how early each assay could detect HCV infection. The specificity (99.81%) of the Elecsys Anti-HCV II assay was less than that with the two EIA comparison methods. However, false-negative results were easily seen in the EIA assays. When serial bleeds of HCV panels were compared with the above-mentioned methods, the assay detected acute HCV infection only 3.5 days after a positive HCV-RNA nucleic acid test and earlier than the comparator assays. Sensitivities and specificities of the anti-HCV assays were sufficiently high for use in this study. The Elecsys Anti-HCV II assay is suitable for screening and reliable early detection of HCV infection. © 2015 Wiley Periodicals, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-12

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

  7. 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

  8. Use of 5-cyano-2,3-ditolyl-tetrazolium chloride staining as an indicator of biocidal activity in a rapid assay for anti-Acanthamoeba agents.

    Science.gov (United States)

    Kobayashi, Takeshi; Mito, Tsuyoshi; Watanabe, Narumi; Suzuki, Takashi; Shiraishi, Atsushi; Ohashi, Yuichi

    2012-05-01

    The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r = 0.83 and P activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r = 0.70 and P biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.

  9. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  10. Comet assay in genetic ecotoxicology: a review.

    Science.gov (United States)

    Cotelle, S; Férard, J F

    1999-01-01

    The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells. Since the protocol was published by Singh et al. [1988], its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology, and genetic ecotoxicology. This study is a review of the investigations that have involved the alkaline version of the Comet assay in genetic ecotoxicology. It focuses mainly on the type of organisms (plants, worms, molluscs, fish, amphibians, and mammalians) but also on the type of cells that have been used for ecotoxicological studies. In the 23 papers published since 1993 and presented here, the original test procedure may have been slightly modified according to the cell type. In vitro and in vivo experiments as well as in situ studies have been carried out in various environments (water, soil, and air). Although the Comet assay is able to detect genotoxic effects of chemical and physical agents, only chemical substances and environmental complex mixtures will be considered in this review. Copyright 1999 Wiley-Liss, Inc.

  11. Assay Portal | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

  12. Assay Characterization Guidance Documents | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    CPTAC characterized assays are defined as those that meet the criteria described in the Assay Characterization Guidance Document. This guidance document aligns with recommendations by the research community as “fit-for-purpose” validation requirements of targeted proteomics assays.

  13. Competitive protein binding assay for piritrexim

    Energy Technology Data Exchange (ETDEWEB)

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. (Wellcome Research Laboratories, Research Triangle Park, NC (USA))

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  14. Nuclear Resonance Fluorescence for Materials Assay

    Science.gov (United States)

    Quiter, Brian J.; Ludewigt, Bernhard A.; Mozin, Vladimir V.; Prussin, Stanley G.

    2011-04-01

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  15. Stage at diagnosis and ovarian cancer survival

    DEFF Research Database (Denmark)

    Maringe, Camille; Walters, Sarah; Butler, John

    2012-01-01

    We investigate what role stage at diagnosis bears in international differences in ovarian cancer survival.......We investigate what role stage at diagnosis bears in international differences in ovarian cancer survival....

  16. Goal-directed Hemostatic Resuscitation of Trauma-induced Coagulopathy: A Pragmatic Randomized Clinical Trial Comparing a Viscoelastic Assay to Conventional Coagulation Assays.

    Science.gov (United States)

    Gonzalez, Eduardo; Moore, Ernest E; Moore, Hunter B; Chapman, Michael P; Chin, Theresa L; Ghasabyan, Arsen; Wohlauer, Max V; Barnett, Carlton C; Bensard, Denis D; Biffl, Walter L; Burlew, Clay C; Johnson, Jeffrey L; Pieracci, Fredric M; Jurkovich, Gregory J; Banerjee, Anirban; Silliman, Christopher C; Sauaia, Angela

    2016-06-01

    Massive transfusion protocols (MTPs) have become standard of care in the management of bleeding injured patients, yet strategies to guide them vary widely. We conducted a pragmatic, randomized clinical trial (RCT) to test the hypothesis that an MTP goal directed by the viscoelastic assay thrombelastography (TEG) improves survival compared with an MTP guided by conventional coagulation assays (CCA). This RCT enrolled injured patients from an academic level-1 trauma center meeting criteria for MTP activation. Upon MTP activation, patients were randomized to be managed either by an MTP goal directed by TEG or by CCA (ie, international normalized ratio, fibrinogen, platelet count). Primary outcome was 28-day survival. One hundred eleven patients were included in an intent-to-treat analysis (TEG = 56, CCA = 55). Survival in the TEG group was significantly higher than the CCA group (log-rank P = 0.032, Wilcoxon P = 0.027); 20 deaths in the CCA group (36.4%) compared with 11 in the TEG group (19.6%) (P = 0.049). Most deaths occurred within the first 6 hours from arrival (21.8% CCA group vs 7.1% TEG group) (P = 0.032). CCA patients required similar number of red blood cell units as the TEG patients [CCA: 5.0 (2-11), TEG: 4.5 (2-8)] (P = 0.317), but more plasma units [CCA: 2.0 (0-4), TEG: 0.0 (0-3)] (P = 0.022), and more platelets units [CCA: 0.0 (0-1), TEG: 0.0 (0-0)] (P = 0.041) in the first 2 hours of resuscitation. Utilization of a goal-directed, TEG-guided MTP to resuscitate severely injured patients improves survival compared with an MTP guided by CCA and utilizes less plasma and platelet transfusions during the early phase of resuscitation.

  17. Mathematical Model of Serodiagnostic Immunochromatographic Assay.

    Science.gov (United States)

    Sotnikov, Dmitriy V; Zherdev, Anatoly V; Dzantiev, Boris B

    2017-04-18

    This article describes the mathematical model for an immunochromatographic assay for the detection of specific immunoglobulins against a target antigen (antibodies) in blood/serum (serodiagnosis). The model utilizes an analytical (non-numerical) approach and allows the calculation of the kinetics of immune complexes' formation in a continuous-flow system using commonly available software, such as Microsoft Excel. The developed model could identify the nature of the influence of immunochemical interaction constants and reagent concentrations on the kinetics of the formation of the detected target complex. On the basis of the model, recommendations are developed to decrease the detection limit for an immunochromatographic assay of specific immunoglobulins.

  18. A new fluorescent assay for enalapril maleate.

    Science.gov (United States)

    de los A Oliva, María; Sombra, Lorena L; Olsina, Roberto A; Masi, Adriana N

    2005-09-01

    A new spectrofluorimetric method for the enalapril maleate monitoring was studied. Enalapril maleate was found to be highly photolabile. This drug was evaluated according to photodegradation assay at pH 2.5 and 6. Enalapril maleate was exposed to UVA-UVB radiations. Under these specific conditions was found as degradation product, the diketopiperazine. The modification of the fluorescent properties of enalapril maleate in solution after exposure UV-radiation and the degradation mechanisms were studied. The photodegradation was followed by the developed spectrofluorimetric assay.

  19. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  20. Starvation-Survival in Haloarchaea

    Science.gov (United States)

    Winters, Yaicha D.; Lowenstein, Tim K.; Timofeeff, Michael N.

    2015-01-01

    Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea—microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1

  1. Starvation-Survival in Haloarchaea

    Directory of Open Access Journals (Sweden)

    Yaicha D. Winters

    2015-11-01

    Full Text Available Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea—microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media from cells trapped without nutrients (represented here by experimental starvation in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum

  2. Survival of adult martens in Northern Wisconsin

    Science.gov (United States)

    Nicholas P. McCann; Patrick A. Zollner; Jonathan H. Gilbert

    2010-01-01

    Low adult marten (Martes americana) survival may be one factor limiting their population growth >30 yr after their reintroduction in Wisconsin, USA. We estimated annual adult marten survival at 0.81 in northern Wisconsin, with lower survival during winter (0.87) than summer-fall (1.00). Fisher (Martes pennanti) and raptor kills...

  3. Long-term survival after perforated diverticulitis

    NARCIS (Netherlands)

    J. Vermeulen (Jan); M.P. Gosselink (Martijn Pieter); W.C.J. Hop (Wim); E. van der Harst (Erwin); B.E. Hansen (Bettina); G.H.H. Mannaerts (Guido); P-P. Coene (Peter Paul); W.F. Weidema (Wibo); J.F. Lange (Johan)

    2011-01-01

    textabstractAim: Short-term survival after emergency surgery for perforated diverticulitis is poor. Less is known about long-term survival. The aims of this study were to evaluate long-term survival after discharge from hospital and to identify factors associated with prognosis. Method: All patients

  4. Clustered survival data with left-truncation

    DEFF Research Database (Denmark)

    Eriksson, Frank; Martinussen, Torben; Scheike, Thomas H.

    2015-01-01

    Left-truncation occurs frequently in survival studies, and it is well known how to deal with this for univariate survival times. However, there are few results on how to estimate dependence parameters and regression effects in semiparametric models for clustered survival data with delayed entry...

  5. 46 CFR 133.105 - Survival craft.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Survival craft. 133.105 Section 133.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS LIFESAVING SYSTEMS Requirements for All OSVs § 133.105 Survival craft. (a) Each survival craft must be approved and equipped as...

  6. 46 CFR 199.201 - Survival craft.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Survival craft. 199.201 Section 199.201 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.201 Survival craft. (a) Each survival craft must be approved and equipped as follows: (1) Each lifeboat must be...

  7. 46 CFR 199.261 - Survival craft.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Survival craft. 199.261 Section 199.261 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.261 Survival craft. (a) Each survival craft must be approved and equipped as follows: (1) Each lifeboat must be a totally...

  8. 46 CFR 28.120 - Survival craft.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Survival craft. 28.120 Section 28.120 Shipping COAST... VESSELS Requirements for All Vessels § 28.120 Survival craft. (a) Except as provided in paragraphs (b) through (h) of this section and 28.305, each vessel must carry the survival craft specified in Table 28...

  9. 46 CFR 174.320 - Damage survival.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Damage survival. 174.320 Section 174.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... survival. A hopper dredge survives assumed damage if it meets the following conditions: (a) The maximum...

  10. Surviving the crash: T-cell homeostasis

    Indian Academy of Sciences (India)

    TOSHIBA

    Spatial and temporal elements. – Cellular sites for the integration of cell death and survival cues. – Spatial regulation of Notch activity for cell survival. Page 4. Cell survival is determined by the availability and uptake of nutrients live dead. Activated T-cells. T-cells. Page 5. dead wildtype. Bax active -6A7. Nucleus – H33342.

  11. Multiple HPV genotype infection impact on invasive cervical cancer presentation and survival.

    Science.gov (United States)

    Nogueira Dias Genta, Maria Luiza; Martins, Toni Ricardo; Mendoza Lopez, Rossana V; Sadalla, José Carlos; de Carvalho, João Paulo Mancusi; Baracat, Edmund Chada; Levi, José Eduardo; Carvalho, Jesus Paula

    2017-01-01

    Invasive cervical cancer (ICC) is the third most common malignant neoplasm affecting Brazilian women. Little is known about the impact of specific HPV genotypes in the prognosis of ICC. We hypothesized that HPV genotype would impact ICC clinical presentation and survival. Women diagnosed with ICC at the Instituto do Câncer do Estado de São Paulo (ICESP) between May 2008 and June 2012 were included in the study and were followed until December 2015. HPV genotype was detected from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples using Onclarity™ system (BD Viper™ LT automated system). 292 patients aged 50±14 years were analyzed. HPVDNA was detected in 84% of patients. The HPV genotypes studied were: HPV16 (64%), HPV18 (10%), HPV33-58 (7%), HPV45 (5%), HPV31 (4%) and other high-risk HPV genotypes (11%). HPV genotypes showed different distributions regarding histological type and clinical stage. Patients were followed for 35±21 months. The overall survival at 5 years after diagnosis of cervical cancer was 54%. Age, clinical staging, histological type and multiple HPV genotypes infection detected in the same tumor specimen were associated with poorer overall survival on multivariate Cox proportional hazard analysis (pgenotype affected survival. Multiple HPV genotype infection was associated with poorer ICC survival in our study, compared with single genotype infection. HPV genotyping from FFPE tumor tissue using an automated assay such as the Onclarity BD™ assay provides a simpler alternative for routine clinical use. This is the largest study employing an automated HPV genotyping assay using FFPE of ICC. Multiple HPV genotype infection adversely influenced survival.

  12. Increased sensitivity of SPR assays in plasma through efficient parallel assay optimization.

    Science.gov (United States)

    Moberg, Anna; Lager, Anna; Hämäläinen, Markku D; Jarhede, Tanja

    2013-05-05

    The sensitivity of biosensor assays in complex media such as plasma or serum is often limited by non-specific binding. The degree of binding often varies between individuals and therefore a large number of different plasma samples have to be used during assay development. Some surface plasmon resonance (SPR) biosensors allow for parallel screening of several running buffer compositions, with a number of different immobilization levels for each buffer. These technical possibilities combined with statistical design of experiments (DoE) enable efficient parallel optimization of multiple assay conditions. In this paper we outline how to increase the sensitivity in SPR-based assays by minimizing background binding and variability from negative control plasma while retaining high signals from positive samples. To mimic immunogenicity studies of biotherapeutics we have used a model assay with anti-rituximab as an anti-drug antibody to be detected in plasma by binding to immobilized rituximab. Immobilization level and sodium chloride concentration were found to be the most important factors to optimize. There were also a number of significant interaction effects and strong non-linearites between the buffer composition/immobilization level and the assay performance, which necessitated DoE based optimization strategies. The applicability of the optimized conditions was verified with several assays (anti-erythropoietin, omalizumab, anti-IgE and anti-myoglobin) in spiked plasma samples resulting in detection levels in the range of 80-170 ng ml(-1). The buffer conditions presented in this paper can be used for other immunogenicity assays on biosensor platforms or as a good starting point for further assay development for new immunogenicity assays. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Insights on the antitumor effects of kahweol on human breast cancer: Decreased survival and increased production of reactive oxygen species and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Cárdenas, Casimiro [Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, E-29071 Málaga (Spain); IBIMA (Biomedical Research Institute of Málaga), E-29071 Málaga (Spain); Research Support Central Services (SCAI) of the University of Málaga, E-29071 Málaga (Spain); Quesada, Ana R. [Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, E-29071 Málaga (Spain); IBIMA (Biomedical Research Institute of Málaga), E-29071 Málaga (Spain); CIBER de Enfermedades Raras (CIBERER), E-29071 Málaga (Spain); Medina, Miguel Ángel, E-mail: medina@uma.es [Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, E-29071 Málaga (Spain); IBIMA (Biomedical Research Institute of Málaga), E-29071 Málaga (Spain); CIBER de Enfermedades Raras (CIBERER), E-29071 Málaga (Spain)

    2014-05-09

    Highlights: • Kahweol inhibits growth and attachment-independent proliferation of tumor cells. • Kahweol induces apoptosis in MDA-MB231 human breast cancer cells. • Kahweol-induced apoptosis involves caspase activation and cytochrome c release. • Kahweol does not protect against hydrogen peroxide cytotoxicity. • Kahweol increases hydrogen peroxide production by human breast cancer cells. - Abstract: The present study aims to identify the modulatory effects of kahweol, an antioxidant diterpene present in coffee beans, on a panel of human tumor cell lines. Kahweol inhibits tumor cell proliferation and clonogenicity and induces apoptosis in several kinds of human tumor cells. In the estrogen receptor-negative MDA-MB231 human breast cancer, the mentioned effects are accompanied by caspases 3/7 and 9 activation and cytochrome c release. On the other hand, kahweol increases the production of reactive oxygen species and their cytotoxicity in human breast cancer cells but not in normal cells. Taken together, our data suggest that kahweol is an antitumor compound with inhibitory effects on tumor cell growth and survival, especially against MDA-MB231 breast cancer cells.

  14. Survival strategies in arctic ungulates

    Directory of Open Access Journals (Sweden)

    N. J. C. Tyler

    1990-09-01

    Full Text Available Arctic ungulates usually neither freeze nor starve to death despite the rigours of winter. Physiological adaptations enable them to survive and reproduce despite long periods of intense cold and potential undernutrition. Heat conservation is achieved by excellent insulation combined with nasal heat exchange. Seasonal variation in fasting metabolic rate has been reported in several temperate and sub-arctic species of ungulates and seems to occur in muskoxen. Surprisingly, there is no evidence for this in reindeer. Both reindeer and caribou normally maintain low levels of locomotor activity in winter. Light foot loads are important for reducing energy expenditure while walking over snow. The significance and control of selective cooling of the brain during hard exercise (e.g. escape from predators is discussed. Like other cervids, reindeer and caribou display a pronounced seasonal cycle of appetite and growth which seems to have an intrinsic basis. This has two consequences. First, the animals evidently survive perfectly well despite enduring negative energy balance for long periods. Second, loss of weight in winter is not necessarily evidence of undernutrition. The main role of fat reserves, especially in males, may be to enhance reproductive success. The principal role of fat reserves in winter appears to be to provide a supplement to, rather than a substitute for, poor quality winter forage. Fat also provides an insurance against death during periods of acute starvation.

  15. Pre-transplant levels of ficolin-3 are associated with kidney graft survival

    DEFF Research Database (Denmark)

    Bay, Jakob T; Hein, Estrid; Sørensen, Søren S

    2013-01-01

    . 97 blood donors served as controls. Ficolin-3, C4 and C3 were measured in pre-transplant as well as in control serum samples. In controls, deposition of ficolin-3, C4, C3 and the terminal complement complex (TCC) was measured in an assay based on acetylated albumin as matrix. The ficolin-3 levels...... correlated with the serum levels of C4 and C3. The serum levels of ficolin-3 correlated with the deposition of ficolin-3, C4, C3 and TCC. Survival analyses showed that high pre-transplant serum levels of ficolin-3 were associated with decreased graft survival. These results suggest an important role...

  16. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Science.gov (United States)

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  17. Development and validation of ultraviolet spectrophotometric assay ...

    African Journals Online (AJOL)

    In order to validate the method, the timeabsorbance relationship, optical characteristics (limit of detection, limit of quantitation, wavelength of maximum absorption, slope, linearity, intercept and Beer's law limits), accuracy, inter-day precision and intra-day precision of the assay method were studied. The absorbance of ...

  18. A novel fluorescent assay for sucrose transporters

    Directory of Open Access Journals (Sweden)

    Gora Peter J

    2012-04-01

    Full Text Available Abstract Background We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae is routinely used for the heterologous expression of SUTs and does not take up esculin. Results When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS. Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant and BY4742. Conclusions The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

  19. Assay for Angiotensin-Converting Enzyme.

    Science.gov (United States)

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  20. Optimization of the sulforhodamine B colorimetric assay.

    Science.gov (United States)

    Papazisis, K T; Geromichalos, G D; Dimitriadis, K A; Kortsaris, A H

    1997-10-27

    Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.

  1. Relationships between ytterbium precipitation assay, colorimetric ...

    African Journals Online (AJOL)

    vitro biological activities of tannins and the commonly used, rapid colorimetric and gravimetric assays of phenolic concentration was of interest. An in vitro gas production bioassay was used to assess the potential biological effect of tannins by incubating tree fruits with and without tannin-binding PEG. Material and Methods.

  2. Wafer-scale Mitochondrial Membrane Potential Assays

    Science.gov (United States)

    Lim, Tae-Sun; Davila, Antonio; Zand, Katayoun; Douglas, Wallace C.; Burke, Peter J.

    2012-01-01

    It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4” standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems. PMID:22627274

  3. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero

    2013-12-01

    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  4. Protein biomarker validation via proximity ligation assays.

    Science.gov (United States)

    Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

    2014-05-01

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  5. Foreign Ownership and Long-term Survival

    DEFF Research Database (Denmark)

    Kronborg, Dorte; Thomsen, Steen

    2006-01-01

    Does foreign ownership enhance or decrease a firm's chances of survival? Over the 100 year period 1895-2001 this paper compares the survival of foreign subsidiaries in Denmark to a control sample matched by industry and firm size. We find that foreign-owned companies have higher survival...... probability. On average exit risk for domestic companies is 2.3 times higher than for foreign companies. First movers like Siemens, Philips, Kodak, Ford, GM or Goodyear have been active in the country for almost a century. Relative foreign survival increases with company age. However, the foreign survival...

  6. The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

    Science.gov (United States)

    O'Dowd, Colm; Mothersill, Carmel E; Cairns, Michael T; Austin, Brian; McClean, Brendan; Lyng, Fiona M; Murphy, James E J

    2006-10-01

    The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

  7. Paraprotein interference with turbidimetric gentamicin assay.

    Science.gov (United States)

    Dimeski, Goce; Bassett, Kendra; Brown, Nigel

    2015-01-01

    Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays. A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser. The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery. IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.

  8. Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients.

    Science.gov (United States)

    Katarkar, Atul; Mukherjee, Sanjit; Khan, Masood H; Ray, Jay G; Chaudhuri, Keya

    2014-09-01

    Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in management of the disease and improve survival rates. The comet assay has been successfully utilised to detect DNA damage in oral premalignant or malignancy. However, due to the invasive nature of collecting blood, it may be painful for many unwilling patients. This study compares the micronucleus (MN) assay in oral buccal mucosa cells with the comet assay in peripheral blood cells in a subset of oral habit-induced precancer and cancer patients. For this, MN assay of exfoliated epithelial cells was compared with comet assay of peripheral blood leucocytes among 260 participants, including those with oral lichen planus (OLP; n = 52), leukoplakia (LPK; n = 51), oral submucous fibrosis (OSF; n = 51), oral squamous cell carcinoma (OSCC; n = 54) and normal volunteers (n = 52). Among the precancer groups, LPK patients showed significantly higher levels of DNA damage as reflected by both comet tail length (P cancer patients was OLP oral habits, it was multiple habits > cigarette + khaini > cigarette smokers > areca + khaini > areca. There was no significant difference in the comet length and MNi frequency between males and females who had oral chewing habits. An overall significant correlation was observed between MNi frequency and comet tail length with r = 0.844 and P oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  10. 21 CFR 866.3305 - Herpes simplex virus serological assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices... herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus antisera...

  11. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

  12. Survival paths through the forest

    DEFF Research Database (Denmark)

    Mogensen, Ulla Brasch

    when the information is high-dimensional e.g. when there are many thousands of genes or markers. In these situations machine learning methods such as the random forest can still be applied and provide reasonable prediction accuracy. The main focus in this talk is the performance of random forest...... in particular when the response is three-dimensional. In a diagnostic study of inflammatory bowel disease three classes of patients have to be diagnosed based on microarray gene-expression data. The performance of random forest is compared on a probability scale and on a classification scale to elastic net....... In survival analysis with competing risks I present an extension of random forest using time-dependent pseudo-values to build event risk prediction models. This approach is evaluated with data from Copenhagen stroke study. Further, I will explain how to use the R-package "pec" to evaluate random forests using...

  13. MRI survival guide; MRT Basiskurs

    Energy Technology Data Exchange (ETDEWEB)

    Cardoza, J. [Alta Imaging Medical Group and Magnetic Imaging Affiliates, Berkeley, CA (United States); Herfkens, R.J. [eds.] [Stanford Univ., CA (United States). Medical Center

    1999-07-01

    The book is a German translation of an American textbook with the original title ''MRI Survival Guide'' and is intended to serve as an introductory guide for beginners or a reference book for quick information. Readers will find information on the fundamentals of the technology and methodology of MRI as well as all details of relevance to practice in a precise and easy-to-grasp arrangement, covering all anatomic areas of interest, illustrations and descriptions of characteristic signs of pathologic processes, high-quality and unusually large-sized diagnostic pictures, a modern didactic concept for quick orientiation, including surveys, tables, and reproductions for visualisation of contents. (orig./CB) [German] Praktisch jeder Mediziner wird im Laufe seiner Berufstaetigkeit mit der MRT konfrontiert - unabhaengig davon ob in der Radiologie, Orthopaedie, Gynaekologie, Chirurgie, Neurologie oder sonstigen Fachrichtung. Das Buch ist eine Uebersetzung eines amerikanischen Lehrbuches mit dem Originaltitel ''MRI Survival Guide'' und will eine wesentliche Erleichterung als 'Ueberlebenshandbuch fuer die MRT' bieten: - Darstellung aller relevanten Grundlagen zu Technik, verschiedenen Sequenzen und Methodik der MRT - knapp und mit maximalem Praxisbezug - Beruecksichtigung aller moeglichen Untersuchungsregionen und strukturierte Orientierung daran (Gehirn, Wirbelsaeule, Kopf/Nacken, Brustkorb, Bauch, Becken, Muskel-Skelett-Bereich) - Illustration und Beschreibung der charakteristischen Erscheinungsmerkmale aller haeufigen pathologischen Prozesse im MRT, inklusive direkt umsetzbarer differentialdiagnostischer Abgrenzungskriterien - hochwertiges und aussergewoehnlich gross dimensioniertes Bildmaterial - modernes, didaktisches Konzept fuer die rasche Orientierung mit vielen Uebersichten, Tabellen und Abbildungen zur Visualisierung der Inhalte, Praxistips und Aufzeigen von Fehlermoeglichkeiten - zum Einstieg, zur Rekapitulation

  14. A novel assay reveals hygrotactic behavior in Drosophila.

    Directory of Open Access Journals (Sweden)

    Feiteng Ji

    Full Text Available Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. Here, we established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, we found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while arista was required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in arista which is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, we demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.

  15. The citotoxicity of calcium hydroxide intracanal dressing by MTT assay

    Directory of Open Access Journals (Sweden)

    Nanik Zubaidah

    2007-12-01

    Full Text Available Calcium hydroxide had been used as the intracanal dressing in endodontic treatment due to its high alkaline and high antimicrobial capacity. It also be able to dissolve the necrotic tissue, prevent the root resorbtion and regenerate a new hard tissue. The aim of this study is to identify the concentration of calcium hydroxide that has the lowest citotoxicity. There are 5 groups, each group had 8 samples with different concentration of calcium hydroxide. Group I: 50%, Group II: 55%, Group III: 60%, Group IV: 65% and Group V: 70%. The citotoxicity test by using enzymatic assay of MTT [3-(4.5- dimethylthiazol-2yl ]-2.5 diphenyl tetrazolium bromide, against fibroblast cell (BHK-21. The result of susceptibility test was showed by the citotoxicity detection of the survive cell of fibroblast that was measured spectrophotometrically using 595 nm beam. The data was analyzed using One-Way ANOVA test with significant difference α = 0.05 and subsequently LSD test. The result showed that in concentration 50%, 55%, 60%, 65%, and 70% calcium hydroxide had low toxicity, but calcium hydroxide 60%, had the lowest toxicity.

  16. Oxygen Control For Bioreactors And In-vitro Cell Assays

    Science.gov (United States)

    Nock, V.; Blaikie, R. J.; David, T.

    2009-07-01

    Dissolved oxygen (DO) is an important parameter in biomedical and cell-culture applications. Several studies have found cell survival and function to be intimately linked to oxygen concentration. Laminar flow, as observed in microfluidic devices, provides an ideal environment to manipulate and control concentration gradients. In this paper we demonstrate the first characterization of integrated fluorescence-based oxygen sensors for DO measurement within a cell-culture bioreactor device. Solid-state PtOEPK/PS sensor patterns were integrated into the PDMS-based bioreactor and calibrated for detection of DO concentration with a superimposed layer of collagen and Ishikawa human endometrial cancer cells. The sensor signal of the layer subjacent to the cells was found to follow a Stern-Volmer model and the intensity ratio was measured to I0/I100 = 3.9 after 3 days in culture. The device provides a novel tool for the control and spatially-resolved measurement of oxygen levels in cellular assays and cell-culture applications.

  17. Adult survival of Delphastus catalinae (Coleoptera: Coccinellidae), a predator of whiteflies (Hemiptera: Aleyrodidae), on diets of whiteflies, honeydew and honey

    Science.gov (United States)

    Delphastus catalinae (Horn) (Coleoptera: Coccinellidae) is a predator that is commercially sold for the management of whiteflies. A study was conducted to assay the effect of selected diets on the survival of adult D. catlinae. Treatments of water (as a control), 10% honey, honeydew, and whiteflie...

  18. The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration

    DEFF Research Database (Denmark)

    Mogensen, Helle Smidt; Hack, N; Balázs, R

    1994-01-01

    The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from d...

  19. Identification of irradiated pepper with comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail: efprieto@ceaden.edu.cu; damaris@ceaden.edu.cu; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)

    2007-07-01

    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  20. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  1. Delayed gamma technique for fissile material assay

    Energy Technology Data Exchange (ETDEWEB)

    Mozin, Vladimir [Los Alamos National Laboratory; Tobin, Stephen [Los Alamos National Laboratory; Vujie, Jasmina [UC BERKELEY; Hunt, Alan [IDAHO ACCELERATOR CENTER

    2010-01-01

    Research sponsored by the Next Generation Safeguards Initiative are investigating several non-destructive assay techniques for the quantification of fissile plutonium mass in spent nuclear fuel assemblies. AppHcation of the delayed gamma signatures is investigated in this context. The objective of the research is to assess whether the delayed gamma assay instrument can provide sufficient sensitivity, isotope specificity and accuracy as required in nuclear material safeguards. This effort includes theoretical and experimental components for the optimal combination of interrogation parameters. A new modeling algorithm offering a high level of detail was developed specifically for this purpose and was validated in series of benchmark experiments. Preliminary modeling of the delayed gamma response from spent fuel assemblies was accomplished offering a future direction for the design process.

  2. DNA Origami Seesaws as Comparative Binding Assay.

    Science.gov (United States)

    Nickels, Philipp C; Høiberg, Hans C; Simmel, Stephanie S; Holzmeister, Phil; Tinnefeld, Philip; Liedl, Tim

    2016-06-16

    The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami-based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw-like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  3. Quantifiable Lateral Flow Assay Test Strips

    Science.gov (United States)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  4. Carbohydrate microarrays for assaying galactosyltransferase activity.

    Science.gov (United States)

    Park, Sungjin; Shin, Injae

    2007-04-26

    [reaction: see text] Carbohydrate microarrays have been used recently for the rapid analysis of glycan-protein or glycan-cell interactions and for the detection of pathogens. As a demonstration of its significance and versatility, the microarray technology has been applied in this effort to assay glycosyltransferase activities. In addition, carbohydrate microarray based methods have been employed to quantitatively determine binding affinities between lectins and carbohydrates.

  5. Clinical mutation assay of tumors: new developments.

    Science.gov (United States)

    Starostik, Petr

    2017-01-01

    Mutation detection in tumors started with classical cytogenetics as the method of choice more than 50 years ago. Karyotyping proved to be sensitive enough to detect deletions or duplications of large chromosome segments, and translocations. Over time, new techniques were developed to detect mutations that are much smaller in scope. The availability of Sanger sequencing and the invention of the PCR improved the discriminatory power of mutation detection to just one base change in the genomic DNA sequence. Techniques derived from PCR (allele-specific PCR, qPCR) and improved or modified sequencing methods (capillary electrophoresis, pyrosequencing) considerably increased the efficiency and sample throughput of mutation detection assays. With the advent of massive parallel sequencing [also called next-generation sequencing (NGS)] in the past decade, a major shift to even higher sample throughput and a significant decrease in cost per sequenced base occurred. The application of the new technology provided a whole slew of novel biomarkers and potential therapy targets to improve diagnosis and treatment. It even led to changes in cancer classification as new information on the mutation profile of tumors became available that characterizes some disease entities better than morphology. NGS, which usually interrogates multiple genes at once and is a prime example of a multianalyte assay, started to replace older single analyte assays focused on analysis of one target, one gene. However, the transition to these extremely complex NGS-based assays is associated with multiple challenges. There are issues with adequate tissue source of nucleic acids, sequencing library preparation, bioinformatics, government regulations and oversight, reimbursement, and electronic medical records that need to be resolved to successfully implement the new technology in a clinical laboratory.

  6. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  7. Radon assay and radioactivity database in Kamioka

    Science.gov (United States)

    Takeuchi, Yasuo; Tasaka, Shigeki; Nakano, Yuuki

    2017-09-01

    A new cooperative program among underground experiments, theorists, and low-background researchers was started in 2014 in Japan. The purpose of this program is to achieve technical and scientific synergies among underground researchers. In this report, the R&D of radon assay technique as an activity in this program is shown. The current status of the radioactivity database system in Kamioka is also shown.

  8. Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice.

    NARCIS (Netherlands)

    J. Groen (Jan); A.D.M.E. Osterhaus (Albert); H.W.J. Broeders; H.E.M. Spijkers (Ine)

    1989-01-01

    textabstractThe sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks

  9. Probiotic bacteria survive in Cheddar cheese and modify populations of other lactic acid bacteria.

    Science.gov (United States)

    Ganesan, B; Weimer, B C; Pinzon, J; Dao Kong, N; Rompato, G; Brothersen, C; McMahon, D J

    2014-06-01

    Starter lactic acid bacteria in Cheddar cheese face physico-chemical stresses during manufacture and ageing that alter their abilities to survive and to interact with other bacterial populations. Nonstarter bacteria are derived from milk handling, cheese equipment and human contact during manufacture. Probiotic bacteria are added to foods for human health benefits that also encounter physiological stresses and microbial competition that may mitigate their survival during ageing. We added probiotic Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium animalis subsp. lactis to full-fat, reduced-fat and low-fat Cheddar cheeses, aiming to study their survival over 270 days of ageing and to determine the role of the cheese matrix in their survival. Probiotic and other lactic acid bacterial populations were enumerated by quantitative PCR using primers specifically targeting the different bacterial genera or species of interest. Bifidobacteria were initially added at 10(6) CFU g(-1) cheese and survived variably in the different cheeses over the 270-day ageing process. Probiotic lactobacilli that were added at 10(7) CFU g(-1) cheese and incident nonstarter lactobacilli (initially at 10(8) CFU g(-1) cheese) increased by 10- to 100-fold over 270 days. Viable bacterial populations were differentiated using propidium monoazide followed by species-specific qPCR assays, which demonstrated that the starter and probiotic microbes survived over ageing, independent of cheese type. Addition of probiotic bacteria, at levels 100-fold below that of starter bacteria, modified starter and nonstarter bacterial levels. We demonstrated that starter lactococci, nonstarter lactobacilli and probiotic bacteria are capable of surviving throughout the cheesemaking and ageing process, indicating that delivery via hard cheeses is possible. Probiotic addition at lower levels may also alter starter and nonstarter bacterial survival. We applied qPCR to study

  10. Circulating 25-hydroxyvitamin D and survival in women with ovarian cancer.

    Science.gov (United States)

    Webb, Penelope M; de Fazio, Anna; Protani, Melinda M; Ibiebele, Torukiri I; Nagle, Christina M; Brand, Alison H; Blomfield, Penelope I; Grant, Peter; Perrin, Lewis C; Neale, Rachel E

    2015-07-01

    Vitamin D status might be associated with cancer survival. Survival after ovarian cancer is poor, but the association with vitamin D has rarely been examined. We evaluated the association between serum 25-hydroxyvitamin D [25(OH)D], a marker of vitamin D status, and ovarian cancer survival. Participants were women with invasive ovarian cancer diagnosed between 2002 and 2005 who participated in the Australian Ovarian Cancer Study. Serum samples, collected at diagnosis (n = 670) or after completion of primary treatment and before recurrence (n = 336), were assayed for 25(OH)D. Sociodemographic, dietary, and lifestyle data came from questionnaires self-completed at recruitment, and clinical and survival data were from medical records, supplemented by linkage to the Australian National Death Index (October 2011). Cox proportional hazards regression was used to estimate HRs and 95% CIs for the association between circulating 25(OH)D and survival. Overall, 59% of the women died during follow-up, with 95% of deaths resulting from ovarian cancer. Circulating 25(OH)D concentrations (mean: 44 nmol/L) were significantly associated with age, state of residence, season of blood collection, and body mass index but not with tumor histology, stage or grade, or comorbidities. Higher 25(OH)D concentrations at diagnosis were significantly associated with longer survival (adjusted HR: 0.93; 95% CI: 0.88, 0.99 per 10 nmol/L), but there was no significant association with progression-free survival or for 25(OH)D measured after primary treatment. In our cohort, higher serum 25(OH)D concentrations at diagnosis were associated with longer survival among women with ovarian cancer. If confirmed in other studies, this suggests that vitamin D status at diagnosis may be an independent predictor of prognosis. Furthermore, if the association is found to be causal, improving vitamin D status may improve ovarian cancer survival rates. © 2015 American Society for Nutrition.

  11. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  12. The Engineered SVA Trans-mobilization Assay.

    Science.gov (United States)

    Bock, Anja; Schumann, Gerald G

    2016-01-01

    Mammalian genomes harbor autonomous retrotransposons coding for the proteins required for their own mobilization, and nonautonomous retrotransposons, such as the human SVA element, which are transcribed but do not have any coding capacity. Mobilization of nonautonomous retrotransposons depends on the recruitment of the protein machinery encoded by autonomous retrotransposons. Here, we summarize the experimental details of SVA trans-mobilization assays which address multiple questions regarding the biology of both nonautonomous SVA elements and autonomous LINE-1 (L1) retrotransposons. The assay evaluates if and to what extent a noncoding SVA element is mobilized in trans by the L1-encoded protein machinery, the structural organization of the resulting marked de novo insertions, if they mimic endogenous SVA insertions and what the roles of individual domains of the nonautonomous retrotransposon for SVA mobilization are. Furthermore, the highly sensitive trans-mobilization assay can be used to verify the presence of otherwise barely detectable endogenously expressed functional L1 proteins via their marked SVA trans-mobilizing activity.

  13. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  14. Screening of fullerene toxicity by hemolysis assay.

    Science.gov (United States)

    Tramer, Federica; Da Ros, Tatiana; Passamonti, Sabina

    2012-01-01

    Fullerene is a compound formed during carbon burst that has been produced synthetically starting from the 1990s. The spherical shape and the characteristic carbon bonds of this allotrope (C(60)) have made it a suitable molecule for many applications. During the last decade, the low aqueous solubility of this molecule has been improved by chemical functionalization allowing the use of fullerene derivatives in biological fluids. The characterization of the toxicity potential of fullerenes is therefore of growing interest for any biomedical application. Intravenous injection is one of the possible routes of their administrations and therefore red blood cells are among the first targets of fullerene cytotoxicity. Human red blood cells are easily available and separated from plasma. Membrane disruption by toxic compounds is easily detected in red blood cells as release of hemoglobin in the cell medium, which can be assayed spectrophotometrically at λ = 415 nm. Due to the high molar extinction coefficient of hemoglobin, the assay can be performed on a small amount of both red blood cells and the test compounds, which might be available only in small quantities. So, the hemolysis assay is a simple screening test, whose results can guide further investigations on cytotoxicity in more complex experimental models.

  15. Variant Interpretation: Functional Assays to the Rescue.

    Science.gov (United States)

    Starita, Lea M; Ahituv, Nadav; Dunham, Maitreya J; Kitzman, Jacob O; Roth, Frederick P; Seelig, Georg; Shendure, Jay; Fowler, Douglas M

    2017-09-07

    Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  16. In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells

    OpenAIRE

    Jiang, Ling; Xiong, Xiao-Peng; Hu, Chao-Su; Ou, Zhou-Luo; Zhu, Guo-Pei; Ying

    2012-01-01

    Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at on...

  17. Assessment of PARP protein expression in epithelial ovarian cancer by ELISA pharmacodynamic assay and immunohistochemistry.

    Science.gov (United States)

    Veskimäe, K; Staff, S; Grönholm, A; Pesu, M; Laaksonen, M; Nykter, M; Isola, J; Mäenpää, J

    2016-09-01

    Targeting Poly (ADP-ribose) polymerase 1 (PARP-1) involved in base excision repair (BER) has been shown to be a clinically effective treatment strategy in epithelial ovarian cancer (EOC) defective in homologous recombination (HR). The aim of this study was to evaluate fresh EOC tumor tissue in regard to PAR (Poly (ADP-ribose)) concentration as a surrogate marker for PARP activity and PARP protein expression in archival samples by immunohistochemistry (IHC). The prospective study cohort consisted of 57 fresh tumor samples derived from patients undergoing primary (n = 38) or interval debulking surgery (n = 19) for EOC and parallel archival paraffin-embedded tumor samples. PARP activity in fresh frozen tumor tissue was assessed by an enzymatic chemiluminescence assay and PARP protein expression in paraffin-embedded tumor tissue by IHC. No correlation was detected between PARP enzyme activity and PARP staining by IHC (p = 0.82). High PARP activity was associated with platinum sensitivity both in the entire study cohort (p = 0.022) and in the high-grade subgroup (p = 0.017). High PARP activity was also associated with improved progression-free survival (PFS) (32 vs 14 months, log-rank p = 0.009). However, PARP immunostaining pattern was not predictive of patient survival. In conclusion, we present a novel finding of high PARP activity associated with platinum sensitivity and improved PFS in EOC. There was no association between PARP IHC and pharmacodynamic assay, and the correlation of PARP IHC with clinico-pathological characteristics and patient survival was poor. Pharmacodynamic assay rather than IHC seems to reflect better biologically significant PARP.

  18. Skp2 expression unfavorably impacts survival in resectable esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Liang Yi

    2012-05-01

    Full Text Available Abstract Background The correlation of S-phase kinase–associated protein 2 (Skp2 with metastasis and prognosis in esophageal squamous cell carcinoma (ESCC is controversial. The purpose of this study was to explore whether there was a correlation between the expression of Skp2 evaluated by immunohistochemistry and the clinical outcome of patients with operable ESCC, and to further determine the possible mechanism of the impact of Skp2 on survival. Methods Tissue microarrays that included 157 surgically resected ESCC specimens was successfully generated for immunohistochemical evaluation. The clinical/prognostic significance of Skp2 expression was analyzed. Kaplan-Meier analysis was used to compare the postoperative survival between groups. The prognostic impact of clinicopathologic variables and Skp2 expression was evaluated using a Cox proportional hazards model. A cell proliferation assay and a colony formation assay were performed in ESCC cell lines to determine the function of Skp2 on the progression of ESCC in vitro. Results Skp2 expression correlated closely with the T category (p = 0.035 and the pathological tumor-node-metastasis (TNM stage (p = 0.027. High expression of Skp2 was associated with poor overall survival in resectable ESCC (p = 0.01. The multivariate Cox regression analysis demonstrated that pathological T category, pathological N category, cell differentiation, and negative Skp2 expression were independent factors for better overall survival. In vitro assays of ESCC cell lines demonstrated that Skp2 promoted the proliferative and colony-forming capacity of ESCCs. Conclusions Negative Skp2 expression in primary resected ESCC is an independent factor for better survival. Skp2 may play a pro-proliferative role in ESCC cells.

  19. Rapid and sensitive detection of Sclerotium rolfsii associated with collar rot disease of Amorphophallus paeoniifolius by species-specific polymerase chain reaction assay.

    Science.gov (United States)

    Pravi, V; Jeeva, M L; Archana, P V

    2014-09-01

    Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.

  20. Survival of pathogenic and lactobacilli species of fermented olives during simulated human digestion.

    Science.gov (United States)

    Arroyo-López, Francisco N; Blanquet-Diot, Stéphanie; Denis, Sylvain; Thévenot, Jonathan; Chalancon, Sandrine; Alric, Monique; Rodríguez-Gómez, Francisco; Romero-Gil, Verónica; Jiménez-Díaz, Rufino; Garrido-Fernández, Antonio

    2014-01-01

    The present survey uses a dynamic gastric and small intestinal model to assess the survival of one pathogenic (Escherichia coli O157:H7 EDL 933) and three lactobacilli bacteria with probiotic potential (Lactobacillus rhamnosus GG, L. pentosus TOMC-LAB2, and L. pentosus TOMC-LAB4) during their passage through the human gastrointestinal tract using fermented olives as the food matrix. The data showed that the survival of the E. coli strain in the stomach and duodenum was very low, while its transit through the distal parts (jejunum and ileum) resulted in an increase in the pathogen population. The production of Shiga toxins by this enterohemorrhagic microorganism in the ileal effluents of the in vitro system was too low to be detected by ELISA assays. On the contrary, the three lactobacilli species assayed showed a considerable resistance to the gastric digestion, but not to the intestinal one, which affected their survival, and was especially evident in the case of both L. pentosus strains. In spite of this, high population levels for all assayed microorganisms were recovered at the end of the gastrointestinal passage. The results obtained in the present study show the potential use of table olives as a vehicle of beneficial microorganisms to the human body, as well as the need for good hygienic practices on the part of olive manufacturers in order to avoid the possibility of contamination by food-borne pathogens.

  1. Survival of pathogenic and lactobacilli species of fermented olives during simulated human digestion

    Directory of Open Access Journals (Sweden)

    Francisco Noé eArroyo López

    2014-10-01

    Full Text Available The present survey uses a dynamic gastric and small intestinal model to assess the survival of one pathogenic (Escherichia coli O157:H7 EDL 933 and three lactobacilli bacteria with probiotic potential (Lactobacillus rhamnosus GG, Lactobacillus pentosus TOMC-LAB2 and Lactobacillus pentosus TOMC-LAB4 during their passage through the human gastrointestinal tract using fermented olives as the food matrix. The data showed that the survival of the E. coli strain in the stomach and duodenum was very low, while its transit through the distal parts (jejunum and ileum resulted in an increase in the pathogen population. The production of Shiga toxins by this enterohemorrhagic microorganism in the ileal effluents of the in vitro system was too low to be detected by ELISA assays. On the contrary, the three lactobacilli species assayed showed a considerable resistance to the gastric digestion, but not to the intestinal one, which affected their survival, and was especially evident in the case of both L. pentosus strains. In spite of this, high population levels for all assayed microorganisms were recovered at the end of the gastrointestinal passage. The results obtained in the present study show the potential use of table olives as a vehicle of beneficial microorganisms to the human body, as well as the need for good hygienic practices on the part of olive manufacturers in order to avoid the possibility of contamination by food-borne pathogens.

  2. Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients

    Directory of Open Access Journals (Sweden)

    Alicia D. Powers

    2012-01-01

    Full Text Available Cancer treatment is often hindered by inadequate methods for diagnosing the disease or insufficient predictive capacity regarding therapeutic efficacy. Targeted cancer treatments, including Bcr-Abl and EGFR kinase inhibitors, have increased survival for some cancer patients but are ineffective in other patients. In addition, many patients who initially respond to targeted inhibitor therapy develop resistance during the course of treatment. Molecular analysis of cancer cells has emerged as a means to tailor treatment to particular patients. While DNA analysis can provide important diagnostic information, protein analysis is particularly valuable because proteins are more direct mediators of normal and diseased cellular processes. In this review article, we discuss current and emerging protein assays for improving cancer treatment, including trends toward assay miniaturization and measurement of protein activity.

  3. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.

    Science.gov (United States)

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S; Yamamoto, Kyoko; Zervantonakis, Ioannis K; Sudo, Ryo; Kamm, Roger D; Chung, Seok

    2012-06-07

    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

  4. Comet assay in evaluating deoxyribonucleic acid damage after out-of-hospital cardiac arrest

    Science.gov (United States)

    Hazuková, Radka; Řezáčová, Martina; Köhlerová, Renata; Tomek, Tomáš; Čermáková, Eva; Kočí, Jaromír; Pleskot, Miloslav

    2017-01-01

    Objective: This study aimed to investigate whether out-of-hospital cardiac arrest (OHCA) may induce severe DNA damage measured using comet assay in successfully resuscitated humans and to evaluate a short-term prognostic role. Methods: In this prospective, controlled, blinded study (1/2013–1/2014), 41 patients (age, 63±14 years) successfully resuscitated from non-traumatic OHCA and 10 healthy controls (age, 53±17 years) were enrolled. DNA damage [double-strand breaks (DSBs) and single-strand breaks (SSBs)] was measured using comet assay in peripheral lymphocytes sampled at admission. Clinical data were recorded (according to Utstein style). A good short-term prognosis was defined as survival for 30 days. Results: Among the patients, there were 71% (29/41) short-term survivors. After OHCA, DNA damage (DSBs and SSBs) was higher (11.0±7.6% and 0.79±2.41% in tail) among patients than among controls (1.96±1.63% and 0.02±0.03% in tail), and it was more apparent for DSBs (pcomet assay in patients successfully resuscitated from OHCA. Whether DNA damage measured using comet assay may be a prognostic marker remains unknown, although our data may encourage some suggestions. PMID:28639949

  5. Shared Frailty Model for Left-Truncated Multivariate Survival Data

    DEFF Research Database (Denmark)

    Jensen, Henrik; Brookmeyer, Ron; Aaby, Peter

    multivariate survival data, left truncation, multiplicative hazard model, shared gamma frailty, conditional model, piecewise exponential model, childhood survival......multivariate survival data, left truncation, multiplicative hazard model, shared gamma frailty, conditional model, piecewise exponential model, childhood survival...

  6. The Survival and Birth of Firms

    OpenAIRE

    Leon Shilton; Craig Stanley

    1999-01-01

    Using a modified form of the location quotient, a "growth quotient," this study traces the survival and growth for the headquarters of publicly listed firms in the United States. At the county level, the spatial concentrations of headquarters listed in 1997 are correlated with the spatial concentrations of corporate headquarters that survived from 1986 though 1996. Counties that house the headquarters of many different survival firms continue to spawn new headquarters. Counties with headquart...

  7. Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay

    Directory of Open Access Journals (Sweden)

    Dilek Özyurt

    2007-07-01

    Full Text Available It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET− and hydrogen atom transfer (HAT−based assays. The majority of HAT assays are kinetics-based, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET−based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II-neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity method. This method offers distinct advantages over other ET−based assays, namely the selection of working pH at physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively, applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH, completion of the redox reactions for most common flavonoids (unlike FRAP, selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay –SH bearing antioxidants (unlike FRAP. Other similar ET–based antioxidant assays that we have developed or modified for phenolics are the Fe

  8. Reflexive Aero Structures for Enhanced Survivability Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Cornerstone Research Group Inc. (CRG) proposes to develop an advanced reflexive structure system to increase the survivability of aerostructures. This reflexive...

  9. Surviving severe traumatic brain injury in Denmark

    DEFF Research Database (Denmark)

    Odgaard, Lene; Poulsen, Ingrid; Kammersgaard, Lars Peter

    2015-01-01

    PURPOSE: To identify all hospitalized patients surviving severe traumatic brain injury (TBI) in Denmark and to compare these patients to TBI patients admitted to highly specialized rehabilitation (HS-rehabilitation). PATIENTS AND METHODS: Patients surviving severe TBI were identified from...... severe TBI were admitted to HS-rehabilitation. Female sex, older age, and non-working status pre-injury were independent predictors of no HS-rehabilitation among patients surviving severe TBI. CONCLUSION: The incidence rate of hospitalized patients surviving severe TBI was stable in Denmark...

  10. Reflexive Aero Structures for Enhanced Survivability Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Cornerstone Research Group Inc. (CRG) will develop an advanced reflexive structure technology system to increase the survivability of future systems constructed of...

  11. In vitro phagocytosis and intracellular survival of Campylobacter jejuni with phagocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kiehlbauch, J.A.

    1986-01-01

    In vitro phagocytosis and intracellular survival of Campylobacter jejuni was studied using three types of mononuclear phagocytes: a J774G8 peritoneal macrophage line, resident BABL/c peritoneal macrophages and human peripheral blood monocytes. In phagocytosis assays using CFU determinations, phagocytosis increased steadily over an 8 hr time period. Results obtained using a /sup 51/Cr assay indicated no consistent significant difference between phagocytosis of C. jejuni between the three mononuclear phagocytes or PMN's and that maximum infection occurred prior to 0.5 hr and maintained throughout the 4 hr assay. Further investigation of the mechanism of attachment and entry of C. jejuni revealed this process required the expenditure of energy by the phagocyte, but was not inhibited by inhibitors of microfilament functions. In addition, phagocytosis was enhanced by the presence of 20% FCS,

  12. The in vitro and in vivo comet assays.

    Science.gov (United States)

    Burlinson, Brian

    2012-01-01

    The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromosome aberration or breakage (clastogenicity), and chromosome loss or gain (aneuploidy). The current generalized strategy consists of assays capable of detecting all of these endpoints using in vitro assays such as the Ames test for detecting gene mutations in bacteria, the human peripheral lymphocyte chromosome aberration (CA) test for detecting clastogenicity, and the in vitro micronucleus test for clastogenicity and aneuploidy. The primary in vivo assay, and generally the only in vivo assay required, is the in vivo rodent bone marrow micronucleus assay. However, there are instances when these assays alone are inadequate and further testing is required, especially in vivo. Historically, the preferred second assay has been the rodent liver unscheduled DNA synthesis assay but recently this has been superseded by the rodent single cell gel electrophoresis or Comet assay. This assay has numerous advantages especially in vivo, where virtually any tissue can be examined. The status of the in vitro comet assay in regulatory testing is much less clear although a preliminary review of data from the assay has shown it to be more specific than other in vitro genotoxicity tests and less prone to false positives.Detailed here are general protocols for both the in vitro and in vivo comet assays which will form the basis of the pending OECD guideline for the assay.

  13. Improving Survival in Decompensated Cirrhosis

    Directory of Open Access Journals (Sweden)

    Amar Nath Mukerji

    2012-01-01

    Full Text Available Mortality in cirrhosis is consequent of decompensation, only treatment being timely liver transplantation. Organ allocation is prioritized for the sickest patients based on Model for End Stage Liver Disease (MELD score. In order to improve survival in patients with high MELD score it is imperative to preserve them in suitable condition till transplantation. Here we examine means to prolong life in high MELD score patients till a suitable liver is available. We specially emphasize protection of airways by avoidance of sedatives, avoidance of Bilevel Positive Airway Pressure, elective intubation in grade III or higher encephalopathy, maintaining a low threshold for intubation with lesser grades of encephalopathy when undergoing upper endoscopy or colonoscopy as pre transplant evaluation or transferring patient to a transplant center. Consider post-pyloric tube feeding in encephalopathy to maintain muscle mass and minimize risk of aspiration. In non intubated and well controlled encephalopathy, frequent physical mobility by active and passive exercises are recommended. When renal replacement therapy is needed, night-time Continuous Veno-Venous Hemodialysis may be useful in keeping the daytime free for mobility. Sparing and judicious use of steroids needs to be borne in mind in treatment of ARDS and acute hepatitis from alcohol or autoimmune process.

  14. Survival of Viral Biowarfare Agents in Disinfected Waters

    Directory of Open Access Journals (Sweden)

    Mary Margaret Wade

    2010-01-01

    Full Text Available Protecting civilian and military water supplies has received more attention since the United States began its war on terror in 2001. Both chlorine and bromine are used by branches of the U.S. military for disinfecting water supplies; however, limited data exists as to the effectiveness of these additives when used against viral biowarfare agents. The present study sought to evaluate the survival of selected viral biothreat agents in disinfected water. Disinfected water samples were spiked with vaccinia virus strain WR and Venezuelan equine encephalitis (VEE virus strain TC-83 each separately to a final concentration of approximately 1×106 PFU/mL, and survival was assessed by plaque assay. Both viruses were inactivated by 1 mg/L free available chlorine (FAC and 2mg/L total bromine within one hour. In conclusion, these results demonstrate that both chlorine and bromine are effective disinfectants against vaccinia virus and VEE strain TC-83 at the concentrations tested.

  15. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  16. Steroid hormone control of cell death and cell survival: molecular insights using RNAi.

    Directory of Open Access Journals (Sweden)

    Suganthi Chittaranjan

    2009-02-01

    Full Text Available The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087, was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-alpha3 and Smr with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2mbn cells (p<0.05 following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.

  17. Using a Candidate Gene-Based Genetic Linkage Map to Identify QTL for Winter Survival in Perennial Ryegrass.

    Directory of Open Access Journals (Sweden)

    Cristiana Paina

    Full Text Available Important agronomical traits in perennial ryegrass (Lolium perenne breeding programs such as winter survival and heading date, are quantitative traits that are generally controlled by multiple loci. Individually, these loci have relatively small effects. The aim of this study was to develop a candidate gene based Illumina GoldenGate 1,536-plex assay, containing single nucleotide polymorphism markers designed from transcripts involved in response to cold acclimation, vernalization, and induction of flowering. The assay was used to genotype a mapping population that we have also phenotyped for winter survival to complement the heading date trait previously mapped in this population. A positive correlation was observed between strong vernalization requirement and winter survival, and some QTL for winter survival and heading date overlapped on the genetic map. Candidate genes were located in clusters along the genetic map, some of which co-localized with QTL for winter survival and heading date. These clusters of candidate genes may be used in candidate gene based association studies to identify alleles associated with winter survival and heading date.

  18. Assaying the Potency of Influenza Vaccines

    Directory of Open Access Journals (Sweden)

    Philip D. Minor

    2015-02-01

    Full Text Available The potency of vaccines must be determined to ensure that the appropriate dose is given. The manufacture and assessment of influenza vaccines are complicated by the continuously changing nature of the pathogen, which makes efficacy estimates difficult but also confounds attempts to produce a well-validated, consistent potency assay. Single radial diffusion has been used for decades and provides a relatively simple way to measure the amount of biologically active materials present in the vaccine. It requires reagents, which are updated on a regular, frequently yearly, basis and alternative methods continue to be sought.

  19. Bicarbonate alters cellular responses in respiration assays.

    Science.gov (United States)

    Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J; Muoio, Deborah M; James, David E

    2017-08-05

    Metabolic assay buffers often omit bicarbonate, which is susceptible to alkalinisation in an open environment. Here, we assessed the effect of including bicarbonate in respirometry experiments. By supplementing HEPES-buffered media with low concentrations of bicarbonate, we found increased respiration in adipocytes and hepatocytes, but not myotubes. This was observed across multiple respirometry platforms and was independent of effects on enhanced insulin sensitivity, pH drift, or mitochondrial function. Permeabilised cell experiments suggest that bicarbonate increases substrate availability, likely by acting as a cofactor for carboxylase enzymes. This emphasises the importance of buffer choice in experimental biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Clostridium difficile spore-macrophage interactions: spore survival.

    Directory of Open Access Journals (Sweden)

    Daniel Paredes-Sabja

    Full Text Available BACKGROUND: Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI, C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s to efficiently persist in the host colonic environment. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we provide evidence that C. difficile spores are well suited to survive the host's innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells' ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.

  1. Survival From Childhood Hematological Malignancies in Denmark

    DEFF Research Database (Denmark)

    Erdmann, Friederike; Winther, Jeanette Falck; Dalton, Susanne Oksbjerg

    2016-01-01

    .76 (CI 2.01; 16.51) were observed for the fourth or later born children with ALL (N = 41) and AML (N = 9), respectively. Children with older parents showed a tendency toward inferior ALL survival, while for AML young maternal age was related to poorer survival. Based on small numbers, a trend toward...

  2. Zimbabwe's Exodus: Crisis, Migration, Survival | IDRC - International ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    2010-07-01

    Zimbabwe's Exodus: Crisis, Migration, Survival. Book cover Zimbabwe's Exodus: Crisis, Migration, Survival. Editor(s):. Jonathan Crush and Daniel Tevera. Publisher(s):. Southern African Migration Programme, IDRC. July 1, 2010. ISBN: 9781920409227. 420 pages. e-ISBN: 9781552504994. Download PDF · Read the e- ...

  3. Domesticating Ugandan local earthworms: Survival of African ...

    African Journals Online (AJOL)

    3.5; E, 90±2.8 and E. eugeniae There was significant effect (P<0.05) of feeding rate on the survival of both species and the Fisher's LSD multiple comparison test also showed significant different (P<0.05) in the survival among the two species.

  4. Cancer rehabilitation: a barometer for survival?

    Science.gov (United States)

    Saotome, Takako; Klein, Linda; Faux, Steven

    2015-10-01

    This pilot study was conducted to describe the clinical features and functional outcomes of patients attending inpatient rehabilitation for cancer-related deconditioning and neurological deficits and to explore factors associated with improved survival. Using a retrospective audit, demographic characteristics, discharge outcomes, survival time, and functional status as measured by Functional Independence Measure (FIM) were recorded for 73 patients. Clinical status was estimated by Karnofsky Performance Status Scale (KPS). Cox regression was used to assess factors associated with improved survival following discharge from rehabilitation. Significant functional gains following rehabilitation were observed in total FIM (p = 0.02), motor FIM (p = 0.001), and KPS (p = 0.003). Length of survival ranged from 9.0 to 25.0 months, with 26 cases surviving to the end of study (censored). Patients scoring a total FIM of ≥80 survived significantly longer than patients scoring <80 (p = 0.002). At discharge, motor FIM scores (p = 0.004), FIM Efficiency (p = 0.001), KPS scores (p = 0.022), ambulation ability (p = 0.026), return to home (p = 0.009), and receipt of in-home services (p = 0.045) were significantly associated with improved survival. Functional improvement achieved through inpatient rehabilitation was associated with prolonged survival among cancer patients. Rehabilitation leading to improved independence among cancer patients may act as a marker of those with greater likelihood of better prognosis.

  5. Nematode survival in relation to soil moisture

    NARCIS (Netherlands)

    Simons, W.R.

    1973-01-01

    Established nematode populations are very persistent in the soil. It is known that they need sufficient soil moisture for movement, feeding and reproduction (fig. 5), and that there are adverse soil moisture conditions which they cannot survive. The influence of soil moisture on survival

  6. Socioeconomic position and survival after cervical cancer

    DEFF Research Database (Denmark)

    Ibfelt, E H; Kjær, S K; Høgdall, C

    2013-01-01

    In an attempt to decrease social disparities in cancer survival, it is important to consider the mechanisms by which socioeconomic position influences cancer prognosis. We aimed to investigate whether any associations between socioeconomic factors and survival after cervical cancer could...... be explained by socioeconomic differences in cancer stage, comorbidity, lifestyle factors or treatment....

  7. MECHANICAL TRANSMISSION AND SURVIVAL OF BACTERIAL ...

    African Journals Online (AJOL)

    jen

    The transmission of enset bacterial wilt with contaminated knives and the survival of the causal agent in soil and enset plant debris was studied ... Xanthomonas campestris pv. musacearum (Xcm) isolates were observed to survive in the soil up to. 9 days. Thereafter the .... and needle, while pathogenicity tests were carried ...

  8. Long-term survival in Patau syndrome.

    Science.gov (United States)

    Tunca, Y; Kadandale, J S; Pivnick, E K

    2001-04-01

    A female patient with an extra chromosome 13 (Patau syndrome) is described. There are only five previous reports of patients with trisomy 13 who have survived past the first decade. It is concluded that non-lethal congenital anomalies and aggressive medical care play an important role in the survival of patients with trisomy 13.

  9. EFFECT OF SALINITY ON SURVIVAL AND LARVAL ...

    African Journals Online (AJOL)

    Laboratory investigations was conducted to gain a better insight into the effect of changing salinity regime on the development and survival of Macrobrachium vollenhoveli larvae. At water temperature of 28 ± 2oC, larvae reared in the salinity range of 0 to 10 ppt showed low survival (<48%), whereas those reared at 12 ...

  10. Escherichia coli survival in waters: Temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  11. Relief for surviving relatives following a suicide.

    NARCIS (Netherlands)

    Oud, MJT; de Groot, MH

    2006-01-01

    Relief for surviving relatives following a suicide. - After the suicide of a 43-year-old woman with known depression, a 41-year-old paraplegic man who recently developed diarrhoea and a 41-year-old woman with probable depression with symptoms of psychosis, the general practitioners of the surviving

  12. Survival after in-hospital Cardiopulmonary Resuscitation

    Directory of Open Access Journals (Sweden)

    M Adib Hajbaghery

    2005-05-01

    Full Text Available Background: During recent years, cardiopulmonary resuscitation (CPR in hospital has received much attention. However, the survival rate of CPR in Iran’s hospitals is unknown. This study was designed to evaluate outcome of in-hospital CPR in Kashan. Methods: A longitudinal case registry study was conducted on all cases of in-hospital CPR during 6 months at 2002. Necessary data including; age, sex, underlying disease, working shift, time from cardiac arrest until initiating of CPR and until defibrillation, duration and result of CPR, frequency of tracheal intubations and time served for it were collected in a checklist. Results: In six months study, 206 cases of cardiopulmonary resuscitation attempted. The survival rate was similar for both sexes. Short-term survival observed in19.9% of cases and only 5.3% survived to discharge. Conclusions: Duration of CPR, time of the first defibrillation, response time and the location of cardiac arrest are the key predictors of survival to hospital discharge and in-hospital CPR strategies require improvement. This study promotes a national study on post CPR survival for accurate data on our performance in attention to chain of survival. KeyWords: Cardiopulmonary Resuscitation (CPR, Survival rate, Iran

  13. Racial differences in survival from gynecologic cancer.

    Science.gov (United States)

    Morgan, M A; Behbakht, K; Benjamin, I; Berlin, M; King, S A; Rubin, S C

    1996-12-01

    To determine whether survival from gynecologic cancer is different between African-American and white patients at an inner-city hospital with both a large clinic and a private service. We studied 538 patients (89 African American, 449 white) diagnosed with cervical, uterine, or ovarian cancer at a single institution from January 1, 1989 through December 31, 1993. Information was obtained on age, stage, site of disease, histology, and type of health insurance (public or commercial). Insurance coverage was used as a proxy for socioeconomic status. Overall survival was estimated by the method of Kaplan and Meier and compared by the log-rank test. Cox proportional hazard modeling was used to evaluate the effects of multiple factors on survival. African-American patients were significantly older and were more likely to have cervical cancer and public insurance than white patients. Overall survival was worse for African-American patients than for white patients (P whites, and African-American patients older than 65 years had a worse survival than whites of similar age. On multivariate analysis, only stage and insurance coverage were significant predictors of survival. African-American patients with gynecologic cancer at our institution have worse overall survival than white patients. The survival difference seems to be due predominantly to differences in socioeconomic status and stage at diagnosis.

  14. Sensitive Troponin Assay and the Classification of Myocardial Infarction

    Science.gov (United States)

    Shah, Anoop S.V.; McAllister, David A.; Mills, Rosamund; Lee, Kuan Ken; Churchhouse, Antonia M.D.; Fleming, Kathryn M.; Layden, Elizabeth; Anand, Atul; Fersia, Omar; Joshi, Nikhil V.; Walker, Simon; Jaffe, Allan S.; Fox, Keith A.A.; Newby, David E.; Mills, Nicholas L.

    2015-01-01

    Background Lowering the diagnostic threshold for troponin is controversial because it may disproportionately increase the diagnosis of myocardial infarction in patients without acute coronary syndrome. We assessed the impact of lowering the diagnostic threshold of troponin on the incidence, management, and outcome of patients with type 2 myocardial infarction or myocardial injury. Methods Consecutive patients with elevated plasma troponin I concentrations (≥50 ng/L; n = 2929) were classified with type 1 (50%) myocardial infarction, type 2 myocardial infarction or myocardial injury (48%), and type 3 to 5 myocardial infarction (2%) before and after lowering the diagnostic threshold from 200 to 50 ng/L with a sensitive assay. Event-free survival from death and recurrent myocardial infarction was recorded at 1 year. Results Lowering the threshold increased the diagnosis of type 2 myocardial infarction or myocardial injury more than type 1 myocardial infarction (672 vs 257 additional patients, P infarction were at higher risk of death compared with those with type 1 myocardial infarction (37% vs 16%; relative risk [RR], 2.31; 95% confidence interval [CI], 1.98-2.69) but had fewer recurrent myocardial infarctions (4% vs 12%; RR, 0.35; 95% CI, 0.26-0.49). In patients with troponin concentrations 50 to 199 ng/L, lowering the diagnostic threshold was associated with increased healthcare resource use (P infarction and death for patients with type 1 myocardial infarction (31% vs 20%; RR, 0.64; 95% CI, 0.41-0.99), but not type 2 myocardial infarction or myocardial injury (36% vs 33%; RR, 0.93; 95% CI, 0.75-1.15). Conclusions After implementation of a sensitive troponin assay, the incidence of type 2 myocardial infarction or myocardial injury disproportionately increased and is now as frequent as type 1 myocardial infarction. Outcomes of patients with type 2 myocardial infarction or myocardial injury are poor and do not seem to be modifiable after reclassification despite

  15. Microbial survival and odor in laundry

    DEFF Research Database (Denmark)

    Jepsen, Signe Munk; Johansen, Charlotte; Stahnke, Louise Heller

    2001-01-01

    , hydrophobic odorants [(Z)-4- heptenal, (E)-2-nonenal, and guaiacol] adhered more strongly to polyester than the acids. The odor formed by surviving skin microflora attached to textiles soiled with human sebum and sweat after laundering at 30 degreesC was studied by sensory evaluation and aroma extract......-were evaluated on cotton textile. A significant survival and transfer between textiles were found for all four test strains washed in E.U. and U.S. color detergents (without bleach), whereas no survival was observed in bleach-containing detergents. Gram-negative strains generally survived in greater numbers than...... Gram-positive strains. A greater survival was observed in U.S. detergents at U.S. conditions (30 degreesC, 12 min) than in E.U. detergents at E.U. conditions (40 degreesC, 30 min). The adhesion of odorants to cotton and polyester textiles during washing and drying was studied using six previously...

  16. [Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

    Science.gov (United States)

    Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

    2014-03-01

    The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

  17. Sperm DNA damage measured by comet assay.

    Science.gov (United States)

    Simon, Luke; Carrell, Douglas T

    2013-01-01

    Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidation-mediated DNA damage. The Comet assay or single-cell gel electrophoresis is a relatively simple and sensitive method for measuring strand breaks in DNA in individual sperm. During this procedure, sperm cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent under high salt conditions. This process removes protamines and histones allowing the nucleus to form a nucleoid-like structure containing supercoiled loops of DNA. Alkaline pH conditions result in unwinding of double-stranded DNA, and subsequent electrophoresis results in the migration of broken strands towards the anode, forming a comet tail, when observed under fluorescence microscope. The amount of DNA in the head and tail is reflected by its fluorescent intensity. The relative fluorescence in the tail compared with its head serves as a measure of the level of DNA damage. In this chapter, we describe the alkaline version of the Comet assay, which is highly sensitive for measuring single- and double-strand DNA breaks.

  18. Sulforhodamine B colorimetric assay for cytotoxicity screening.

    Science.gov (United States)

    Vichai, Vanicha; Kirtikara, Kanyawim

    2006-01-01

    The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

  19. Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay.

    Science.gov (United States)

    Lah, B; Vidic, T; Glasencnik, E; Cepeljnik, T; Gorjanc, G; Marinsek-Logar, Romana

    2008-04-01

    Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO(2)) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.

  20. Seasonal survival of adult female mottled ducks

    Science.gov (United States)

    Moon, Jena A.; Haukos, David A.; Conway, Warren C.

    2017-01-01

    The mottled duck (Anas fulgivula) is a non-migratory duck dependent on coastal habitats to meet all of its life cycle requirements in the Western Gulf Coast (WGC) of Texas and Louisiana, USA. This population of mottled ducks has experienced a moderate decline during the past 2 decades. Adult survival has been identified as an important factor influencing population demography. Previous work based on band-recovery data has provided only annual estimates of survival. We assessed seasonal patterns of female mottled duck survival from 2009 to 2012 using individuals marked with satellite platform transmitter terminals (PTTs). We used temperature and movement sensors within each PTT to indicate potential mortality events. We estimated cumulative weekly survival and ranked factors influential in patterns of mortality using known-fate modeling in Program MARK. Models included 4 predictors: week; hunting and non-hunting periods; biological periods defined as breeding, brooding, molt, and pairing; and mass at time of capture. Models containing hunt periods, during and outside the mottled duck season, comprised essentially 100% of model weights where both legal and illegal harvest had a negative influence on mottled duck survival. Survival rates were low during 2009–2011 (12–38% annual rate of survival), when compared with the long-term banding average of 53% annual survival. During 2011, survival of female mottled ducks was the lowest annual rate (12%) ever documented and coincided with extreme drought. Management actions maximizing the availability of wetlands and associated upland habitats during hunting seasons and drought conditions may increase adult female mottled duck survival.

  1. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    Science.gov (United States)

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  2. Evaluation of an immunoradiometric assay protocol for determining ...

    African Journals Online (AJOL)

    BDL) for prostate-specific antigen (PSA) by an assay system are essential for successful prediction of recurrent disease after radical prostatectomy. A study was conducted to evaluate an Immunoradiometric Assay (IRMA) system to establish the ...

  3. The Statistics of wood assays for preservative retention

    Science.gov (United States)

    Patricia K. Lebow; Scott W. Conklin

    2011-01-01

    This paper covers general statistical concepts that apply to interpreting wood assay retention values. In particular, since wood assays are typically obtained from a single composited sample, the statistical aspects, including advantages and disadvantages, of simple compositing are covered.

  4. a positive control plasmid for reporter gene assay

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-04

    903 for ... qualification as a positive control for luciferase reporter gene assays. Key words: Reporter gene plasmid, luciferase assay, .... tissue-specific promoters by gene gun. Br. J. Dermatol. 144: 34-39. Naylor LH (1999).

  5. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    Science.gov (United States)

    Kawaguchi, Satomi; Nakamura, Takanori; Yamamoto, Ayumi; Honda, Gisho; Sasaki, Yu F.

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  6. Analytical evaluation of nine serological assays for diagnosis of syphilis.

    Science.gov (United States)

    Malm, K; Andersson, S; Fredlund, H; Norrgren, H; Biague, A; Månsson, F; Ballard, R; Unemo, M

    2015-12-01

    The diagnosis of syphilis is most frequently dependent on antibody detection with serological assays. Assays for both treponemal and non-treponemal antibodies are needed to provide a sensitive and specific diagnosis. For decades, a first screening has been done with non-treponemal assays, followed by treponemal. However, in recent years, following laboratory automation, the reverse sequence screening algorithms have been developed, using a treponemal assay as the initial screening test. To evaluate serological assays for treponemal and non-treponemal antibodies, to use in reverse algorithm screening of syphilis. Six treponemal assays (one IgM-specific assay), two non-treponemal assays and one novel dual point-of-care (POC) assay for serological diagnosis of syphilis were evaluated. Serum samples from Guinea-Bissau and Sweden were examined, as well as two performance panels and samples from blood donors. Sensitivity and specificity were calculated for each assay, using different assays as gold standard test. The Macro-Vue RPR Card test was the most sensitive non-treponemal test and the TrepSure Anti-Treponema EIA Screen and the SeroDia TP-PA were the most sensitive and specific treponemal assays. Among the automated assays, both the Liaison Treponema Screen and Architect Syphilis TP showed high sensitivity, however, the former had clearly higher specificity. In resourced settings, where the reverse sequence algorithm is preferred for screening, an automated treponemal immunoassay for initial screening subsequently followed by the TrepSure test or TP-PA assay as a second treponemal assay appear highly effective. Finally, a quantitative highly sensitive non-treponemal assay, e.g. the Macro-Vue RPR Card test, could then be used as a supplementary test to evaluate activity of the syphilis infection. © 2015 European Academy of Dermatology and Venereology.

  7. Development of cross-priming amplification assays for rapid and sensitive detection of Aeromonas hydrophila.

    Science.gov (United States)

    Meng, S; Wang, Y; Wang, Y; Liu, D; Ye, C

    2015-08-01

    Aeromonas hydrophila has been increasingly implicated as the aetiologic agent of various human diseases. Therefore, reliable laboratory detection and identification of this bacterium has become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method for rapid detection of Aer. hydrophila using a cross-priming amplification (CPA) assay with the desA gene as the target. The desA gene is crucial for the survival and growth of Aer. hydrophila under iron starvation. The results can be visualized as colour changes without opening the reaction tubes. No false-positive results were observed for the 33 non-Aer. hydrophila strains tested to evaluate assay specificity. The limit of detection for Aer. hydrophila was approximately 200 copies of desA per reaction (on reference plasmids) and 5 × 10(3)  CFU g(-1) Aer. hydrophila in simulated human stool, which is the same sensitivity as a qPCR assay. The performance of the CPA assay was also evaluated with 100 stool specimens from diarrhoea patients and 40 environmental water samples. In conclusion, the simplicity, cost-effectiveness and nearly instrument-free platform of the CPA assay make it practical for use in primary care facilities and smaller clinical laboratories. Aeromonas hydrophila is a human pathogen that infects via exposed wounds or ingestion of contaminated water and food. In this study, a CPA-based PCR method was developed for specific, rapid, cost-effective detection of Aer. hydrophila, and the test results could be visualized without opening the reaction tubes. This is the first report on the application of the CPA method for the detection of Aer. hydrophila. This novel method could be practical for use in primary care facilities and smaller clinical laboratories. © 2015 The Society for Applied Microbiology.

  8. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  9. 21 CFR 864.7415 - Abnormal hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864... hemoglobin assay. (a) Identification. An abnormal hemoglobin assay is a device consisting of the reagents... hemoglobin types. (b) Classification. Class II (performance standards). [45 FR 60618, Sept. 12, 1980] ...

  10. 21 CFR 864.7470 - Glycosylated hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section... Glycosylated hemoglobin assay. (a) Identification. A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a, A1b, and A1c) in a patient's blood by a column chromatographic...

  11. Systems, devices, and methods for agglutination assays using sedimentation

    Energy Technology Data Exchange (ETDEWEB)

    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

    2016-01-26

    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  12. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation...

  13. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, Thomas R. (Somerville, MA); Liber, Howard L. (Brookline, MA); Penman, Bruce W. (Cambridge, MA); Thilly, William G. (Cambridge, MA); Hoppe, IV, Henry (Arlington, MA)

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  14. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, T.R.; Liber, H.L.; Penman, B.W.; Thilly, W.G.; Hoppe, H. IV.

    1981-11-24

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay. 7 figs.

  15. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  16. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866... Plasmodium species antigen detection assays. (a) Identification. A Plasmodium species antigen detection assay... malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum, Plasmodium...

  17. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the...

  18. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT PCR assays and two new assays that we have developed. To determine the analytical sensitivity of

  19. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

    Science.gov (United States)

    Maslanka, S E; Gheesling, L L; Libutti, D E; Donaldson, K B; Harakeh, H S; Dykes, J K; Arhin, F F; Devi, S J; Frasch, C E; Huang, J C; Kriz-Kuzemenska, P; Lemmon, R D; Lorange, M; Peeters, C C; Quataert, S; Tai, J Y; Carlone, G M

    1997-03-01

    A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

  20. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  1. Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays.

    Science.gov (United States)

    Singer, Timothy M; Lambert, Iain B; Williams, Andrew; Douglas, George R; Yauk, Carole L

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently--the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  2. Cancer survival in Cuba, 1994-1995.

    Science.gov (United States)

    Garrote, L F; Alvarez, Y G; Babie, P T; Yi, M G; Alvarez, M G; Cicili, M L

    2011-01-01

    The population-based cancer registry in Cuba is a national cancer registry established in 1964; cancer registration is entirely done by passive methods. Data on survival from 13 cancer sites or types registered during 1994-1995 are reported. Follow-up has been carried out predominantly by passive methods, with median follow-up ranging from 13-54 months. The proportion with histologically verified diagnosis for various cancers ranged between 34-100%; death certificates only (DCOs) comprised 8-50%; 50-89% of total registered cases were included for the survival analysis. The 5-year age-standardized relative survival for selected cancers were breast (69%), colon (41%), cervix (56%), urinary bladder (64%), rectum (48%) and non-Hodgkin lymphoma (49%). The 5-year relative survival by age group showed no distinct pattern or trend, and was fluctuating. A decreasing survival with increasing clinical extent of disease was noted for all cancers studied. The data on survival trend revealed that the 5-year relative survival of most cancers diagnosed in 1994-1995 was greater than that in 1988-1989.

  3. Linking age, survival, and transit time distributions

    Science.gov (United States)

    Calabrese, Salvatore; Porporato, Amilcare

    2015-10-01

    Although the concepts of age, survival, and transit time have been widely used in many fields, including population dynamics, chemical engineering, and hydrology, a comprehensive mathematical framework is still missing. Here we discuss several relationships among these quantities by starting from the evolution equation for the joint distribution of age and survival, from which the equations for age and survival time readily follow. It also becomes apparent how the statistical dependence between age and survival is directly related to either the age dependence of the loss function or the survival-time dependence of the input function. The solution of the joint distribution equation also allows us to obtain the relationships between the age at exit (or death) and the survival time at input (or birth), as well as to stress the symmetries of the various distributions under time reversal. The transit time is then obtained as a sum of the age and survival time, and its properties are discussed along with the general relationships between their mean values. The special case of steady state case is analyzed in detail. Some examples, inspired by hydrologic applications, are presented to illustrate the theory with the specific results. This article was corrected on 11 Nov 2015. See the end of the full text for details.

  4. Survival analysis of orthodontic mini-implants.

    Science.gov (United States)

    Lee, Shin-Jae; Ahn, Sug-Joon; Lee, Jae Won; Kim, Seong-Hun; Kim, Tae-Woo

    2010-02-01

    Survival analysis is useful in clinical research because it focuses on comparing the survival distributions and the identification of risk factors. Our aim in this study was to investigate the survival characteristics and risk factors of orthodontic mini-implants with survival analyses. One hundred forty-one orthodontic patients (treated from October 1, 2000, to November 29, 2007) were included in this survival study. A total of 260 orthodontic mini-implants that had sandblasted (large grit) and acid-etched screw parts were placed between the maxillary second premolar and the first molar. Failures of the implants were recorded as event data, whereas implants that were removed because treatment ended and those that were not removed during the study period were recorded as censored data. A nonparametric life table method was used to visualize the hazard function, and Kaplan-Meier survival curves were generated to identify the variables associated with implant failure. Prognostic variables associated with implant failure were identified with the Cox proportional hazard model. Of the 260 implants, 22 failed. The hazard function for implant failure showed that the risk is highest immediately after placement. The survival function showed that the median survival time of orthodontic mini-implants is sufficient for relatively long orthodontic treatments. The Cox proportional hazard model identified that increasing age is a decisive factor for implant survival. The decreasing pattern of the hazard function suggested gradual osseointegration of orthodontic mini-implants. When implants are placed in a young patient, special caution is needed to lessen the increased probability of failure, especially immediately after placement.

  5. Abbott ARCHITECT iPhenytoin assay versus similar assays for measuring free phenytoin concentrations.

    Science.gov (United States)

    Tacker, Danyel Hermes; Robinson, Randy; Perrotta, Peter L

    2014-01-01

    To measure free phenytoin (FP) concentrations in filtered specimens using the Abbott ARCHITECT iPhenytoin assay and to compare results from this method with results from the Abbott TDx/FLx assays. We verified accuracy, analytic measurement range, and precision for FP measurements. For correlation and therapeutic interval studies, we used filtered calibrators, controls, proficiency-testing materials, and surplus clinical samples. After implementation, we determined proficiency testing results. The analytic measurement range was 2.0 to 25.0 micromol/L. Quality control materials (6.1, 12.6, and 20.1 micromol/L) provided mean (SD) recoveries of 96.1 (5.0%), 99.2 (5.0%), and 99.3 (5.7%), respectively, and coefficients of variation of 5.2%, 5.0%, and 5.8%, respectively. Clinical specimens produced mean (SD) FP recovery levels of 103.7 (10.6%) (bias, 0.1 [0.3] micromol/L). Altering the FP therapeutic range (4.0-8.0 micromol/L) was unnecessary. Proficiency testing yielded consistently acceptable results. Our accuracy, precision, and correlation results were similar for the TDx/FLx and ARCHITECT assays, which demonstrates that the ARCHITECT iPhenytoin assay is acceptable for clinical FP measurements.

  6. Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

    Science.gov (United States)

    2017-07-27

    The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

  7. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

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

  8. Lab-on-a-Chip Multiplex Assays.

    Science.gov (United States)

    Peter, Harald; Wienke, Julia; Bier, Frank F

    2017-01-01

    Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial species and detection of antibiotic resistance. The use of DNA microarrays allows a fast adaptation of new biomarkers enabling the identification of different genes as well as single-nucleotide-polymorphisms (SNPs) within these genes. In this protocol we describe a DNA microarray developed for identification of Staphylococcus aureus and the mecA resistance gene.

  9. Stable isotope dilution assays in mycotoxin analysis.

    Science.gov (United States)

    Rychlik, Michael; Asam, Stefan

    2008-01-01

    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.

  10. COMPARATIVE STUDY ON MILK CASEIN ASSAY METHODS

    Directory of Open Access Journals (Sweden)

    RODICA CĂPRIłĂ

    2008-05-01

    Full Text Available Casein, the main milk protein was determined by different assay methods: the gravimetric method, the method based on the neutralization of the NaOH excess used for the casein precipitate solving and the method based on the titration of the acetic acid used for the casein precipitation. The last method is the simplest one, with the fewer steps, and also with the lowest error degree. The results of the experiment revealed that the percentage of casein from the whole milk protein represents between 72.6–81.3% in experiment 1, between 73.6–81.3% in experiment 2 and between 74.3–81% in experiment 3.

  11. Rapid Automated Sample Preparation for Biological Assays

    Energy Technology Data Exchange (ETDEWEB)

    Shusteff, M

    2011-03-04

    Our technology utilizes acoustic, thermal, and electric fields to separate out contaminants such as debris or pollen from environmental samples, lyse open cells, and extract the DNA from the lysate. The objective of the project is to optimize the system described for a forensic sample, and demonstrate its performance for integration with downstream assay platforms (e.g. MIT-LL's ANDE). We intend to increase the quantity of DNA recovered from the sample beyond the current {approx}80% achieved using solid phase extraction methods. Task 1: Develop and test an acoustic filter for cell extraction. Task 2: Develop and test lysis chip. Task 3: Develop and test DNA extraction chip. All chips have been fabricated based on the designs laid out in last month's report.

  12. Nondestructive Assay Options for Spent Fuel Encapsulation

    Energy Technology Data Exchange (ETDEWEB)

    Tobin, Stephen J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Jansson, Peter [Uppsala Univ. (Sweden)

    2014-10-02

    This report describes the role that nondestructive assay (NDA) techniques and systems of NDA techniques may have in the context of an encapsulation and deep geological repository. The potential NDA needs of an encapsulation and repository facility include safeguards, heat content, and criticality. Some discussion of the facility needs is given, with the majority of the report concentrating on the capability and characteristics of individual NDA instruments and techniques currently available or under development. Particular emphasis is given to how the NDA techniques can be used to determine the heat production of an assembly, as well as meet the dual safeguards needs of 1) determining the declared parameters of initial enrichment, burn-up, and cooling time and 2) detecting defects (total, partial, and bias). The report concludes with the recommendation of three integrated systems that might meet the combined NDA needs of the encapsulation/repository facility.

  13. Stable isotope dilution assays in mycotoxin analysis

    Energy Technology Data Exchange (ETDEWEB)

    Rychlik, Michael; Asam, Stefan [Universitaet Muenchen, Lehrstuhl fuer Lebensmittelchemie der Technischen, Garching (Germany)

    2008-01-15

    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis. (orig.)

  14. Development of a Radioactive Waste Assay System

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-12-31

    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  15. Universal fieldable assay with unassisted visual detection

    Science.gov (United States)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

  16. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...... limit was 3 nmol/l. Within run and between-run precision was 7.4% and 13.5% for control 10 nmol/l and 1.2% and 3.2% for control 50 nmol/l. Recovery was 85-115% of MTX added to haemolysed erythrocytes. We found the method useful for pharmacokinetic studies of MTX in erythrocytes in MTX-treated patients...

  17. Die Sulforhodamien B Seltellingsmetode in vergelyking met drie algemene seltellingsmetodes vir sitotoksisiteitstoetse/The Sulforhodamine B Assay in comparison with three commonly used cytotoxicity assays

    National Research Council Canada - National Science Library

    A van Tonder; A Joubert; A D Cromarty

    2014-01-01

      The sulforhodamine B (SRB) assay was compared to the MTT assay, the neutral red uptake assay and the resazurin reduction assay with regards to sensitivity, interference with glycolysis inhibitors, reproducibility and cost-effectiveness...

  18. Foreign Ownership and long-term Survival

    OpenAIRE

    Kronborg, Dorte; Thomsen, Steen

    2006-01-01

    Does foreign ownership enhance or decrease a firm’s chances of survival? Over the 100 year period 1895-2001 this paper compares the survival of foreign subsidiaries in Denmark to a control sample matched by industry and firm size. We find that foreign-owned companies have higher survival probability. On average exit risk for domestic companies is 2.3 times higher than for foreign companies. First movers like Siemens, Philips, Kodak, Ford, GM or Goodyear have been active in the country for alm...

  19. Controlling chaotic transients: Yorke's game of survival

    DEFF Research Database (Denmark)

    Aguirre, Jacobo; D'ovidio, Francesco; Sanjuán, Miguel A. F.

    2004-01-01

    . This problem is focused as a two-person, mathematical game between two players called "the protagonist" and "the adversary." The protagonist's goal is to survive. He can lose but cannot win; the best he can do is survive to play another round, struggling ad infinitum. In the absence of actions by either player...... knows the action of the adversary in choosing his response and is permitted to choose the initial point x(0) of the game. We use the "slope 3" tent map in an example of this problem. We show that it is possible for the protagonist to survive....

  20. Empirical likelihood method in survival analysis

    CERN Document Server

    Zhou, Mai

    2015-01-01

    Add the Empirical Likelihood to Your Nonparametric ToolboxEmpirical Likelihood Method in Survival Analysis explains how to use the empirical likelihood method for right censored survival data. The author uses R for calculating empirical likelihood and includes many worked out examples with the associated R code. The datasets and code are available for download on his website and CRAN.The book focuses on all the standard survival analysis topics treated with empirical likelihood, including hazard functions, cumulative distribution functions, analysis of the Cox model, and computation of empiric

  1. A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

    Science.gov (United States)

    Faulkner, Ashton; Purcell, Robert; Hibbert, Andrew; Latham, Sally; Thomson, Scott; Hall, Wendy L; Wheeler-Jones, Caroline; Bishop-Bailey, David

    2014-12-05

    Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

  2. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    Pas, S D; Fries, E; De Man, R A; Osterhaus, A D; Niesters, H G

    A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV

  3. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    S.D. Pas (Suzan); E. Fries; H.G.M. Niesters (Bert); R.A. de Man (Robert); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractA highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel,

  4. 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.

  5. Circulating free light chain measurement in the diagnosis, prognostic assessment and evaluation of response of AL amyloidosis: comparison of Freelite and N latex FLC assays.

    Science.gov (United States)

    Palladini, Giovanni; Jaccard, Arnaud; Milani, Paolo; Lavergne, David; Foli, Andrea; Bender, Sebastien; Lavatelli, Francesca; Bosoni, Tiziana; Valentini, Veronica; Pirolini, Laura; Ferraro, Giovanni; Basset, Marco; Russo, Francesca; Nuvolone, Mario; Albertini, Riccardo; Cogne, Michel; Merlini, Giampaolo

    2017-10-26

    The measurement of circulating free light chain (FLC) is essential in the diagnosis, prognostic stratification and evaluation of response to therapy in light chain (AL) amyloidosis. For more than 10 years, this has been done with an immunonephelometric assay based on polyclonal antibodies (Freelite), and cutoffs for staging and response assessment have been validated with this method. Recently, a new assay based on monoclonal antibodies (N latex FLC) has been marketed in Europe. We evaluated and compared the clinical performance of the two assays in 426 patients with newly diagnosed AL amyloidosis. We found suboptimal agreement between the two methods, with differences between values obtained with the Freelite and N latex FLC assays increasing with the concentration of clonal FLC. The diagnostic sensitivity of the Freelite (82%) and N latex FLC (84%) assays was similar, and both improved to 98% in combination with serum and urine immunofixation. The concentration of FLC measured with both methods had prognostic significance. Less pronounced decreases in FLC best predicted improved survival with the N latex FLC assay (33% vs. 50%), and there was poor concordance (84%) in discrimination of responders. The two assays have similar diagnostic and prognostic performance. However, they are not interchangeable, and follow-up should be done with either one. New response criteria are needed for the N latex FLC assay.

  6. Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina

    Science.gov (United States)

    Neves, Raquel A. F.; Fernandes, Tainá; dos Santos, Luciano Neves; Nascimento, Silvia M.

    2017-01-01

    Harmful algae may differently affect their primary grazers, causing sub-lethal effects and/or leading to their death. The present study aim to compare the effects of three toxic benthic dinoflagellates on clearance and grazing rates, behavioral changes, and survival of Artemia salina. Feeding assays consisted in 1-h incubations of brine shrimps with the toxic Prorocentrum lima, Gambierdiscus excentricus and Ostreopsis cf. ovata and the non-toxic Tetraselmis sp. Brine shrimps fed unselectively on all toxic and non-toxic algal preys, without significant differences in clearance and ingestion rates. Acute toxicity assays were performed with dinoflagellate cells in two growth phases during 7-h to assess differences in cell toxicity to A. salina. Additionally, exposure to cell-free medium was performed to evaluate its effects on A. salina survival. The behavior of brine shrimps significantly changed during exposure to the toxic dinoflagellates, becoming immobile at the bottom by the end of the trials. Dinoflagellates significantly affected A. salina survival with 100% mortality after 7-h exposure to cells in exponential phase (all treatments) and to P. lima in stationary phase. Mortality rates of brine shrimps exposed to O. cf. ovata and G. excentricus in stationary phase were 91% and 75%, respectively. However, incubations of the brine shrimps with cell-free medium did not affect A. salina survivorship. Significant differences in toxic effects between cell growth phases were only found in the survival rates of A. salina exposed to G. excentricus. Acute exposure to benthic toxic dinoflagellates induced harmful effects on behavior and survival of A. salina. Negative effects related to the toxicity of benthic dinoflagellates are thus expected on their primary grazers making them more vulnerable to predation and vectors of toxins through the marine food webs. PMID:28388672

  7. Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina.

    Directory of Open Access Journals (Sweden)

    Raquel A F Neves

    Full Text Available Harmful algae may differently affect their primary grazers, causing sub-lethal effects and/or leading to their death. The present study aim to compare the effects of three toxic benthic dinoflagellates on clearance and grazing rates, behavioral changes, and survival of Artemia salina. Feeding assays consisted in 1-h incubations of brine shrimps with the toxic Prorocentrum lima, Gambierdiscus excentricus and Ostreopsis cf. ovata and the non-toxic Tetraselmis sp. Brine shrimps fed unselectively on all toxic and non-toxic algal preys, without significant differences in clearance and ingestion rates. Acute toxicity assays were performed with dinoflagellate cells in two growth phases during 7-h to assess differences in cell toxicity to A. salina. Additionally, exposure to cell-free medium was performed to evaluate its effects on A. salina survival. The behavior of brine shrimps significantly changed during exposure to the toxic dinoflagellates, becoming immobile at the bottom by the end of the trials. Dinoflagellates significantly affected A. salina survival with 100% mortality after 7-h exposure to cells in exponential phase (all treatments and to P. lima in stationary phase. Mortality rates of brine shrimps exposed to O. cf. ovata and G. excentricus in stationary phase were 91% and 75%, respectively. However, incubations of the brine shrimps with cell-free medium did not affect A. salina survivorship. Significant differences in toxic effects between cell growth phases were only found in the survival rates of A. salina exposed to G. excentricus. Acute exposure to benthic toxic dinoflagellates induced harmful effects on behavior and survival of A. salina. Negative effects related to the toxicity of benthic dinoflagellates are thus expected on their primary grazers making them more vulnerable to predation and vectors of toxins through the marine food webs.

  8. Ministerial Importance and Survival in Government

    DEFF Research Database (Denmark)

    Bright, Jonathan; Döring, Holger; Little, Conor

    2015-01-01

    Are holders of important ministerial positions more likely to survive in cabinet than their colleagues who hold less important positions? This study examines the relationship between the importance of a ministerial position and the length of time ministers are able to survive in government....... It is based on an original dataset of cabinet ministers in seven West European countries from 1945 to 2011. Employing a little-used measure of ministerial survival based on overall time in government, it is found that holders of important ministerial positions are more durable than their colleagues who hold...... less important ministerial positions. Age, prior government experience and the size of the party to which the minister belongs are also associated with consistently significant effects. Further, the study explores the determinants of survival for two types of risk – exiting government with one’s party...

  9. SURVIVAL OF SALMONELLA SPECIES IN RIVER WATER.

    Science.gov (United States)

    The survival of four Salmonella strains in river water microcosms was monitored using culturing techniques, direct counts, whole cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytrometry. Plate counts of...

  10. Surviving Sepsis: Taming a Deadly Immune Response

    Science.gov (United States)

    ... Issues Subscribe August 2014 Print this issue Surviving Sepsis Taming a Deadly Immune Response En español Send ... Mouth? Looking at Lupus Wise Choices Signs of Sepsis Sepsis can be hard to spot, because its ...

  11. Cognitive function in families with exceptional survival

    DEFF Research Database (Denmark)

    Barral, Sandra; Cosentino, Stephanie; Costa, Rosann

    2012-01-01

    members in the offspring generation demonstrate significantly better performance on multiple tasks requiring attention, working memory, and semantic processing when compared with individuals without a family history of exceptional survival, suggesting that cognitive performance may serve as an important...

  12. Bay Scallop Spawning, Survival, Growth Records

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Bay Scallops are selected and cultured according to criteria of growth and survival. Morphological attributes have also been selected to assess heretibility....

  13. Zooplankton Data - Ocean Survival of Salmonids

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A study to evaluate the role of changing ocean conditions on growth and survival of juvenile salmon from the Columbia River basin as they enter the Columbia River...

  14. Juvenile Salmonid Metrics - Ocean Survival of Salmonids

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A study to evaluate the role of changing ocean conditions on growth and survival of juvenile salmon from the Columbia River basin as they enter the Columbia River...

  15. Oceanographic Trawl Data - Ocean Survival of Salmonids

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A study to evaluate the role of changing ocean conditions on growth and survival of juvenile salmon from the Columbia River basin as they enter the Columbia River...

  16. CTD Oceanographic Data - Ocean Survival of Salmonids

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A study to evaluate the role of changing ocean conditions on growth and survival of juvenile salmon from the Columbia River basin as they enter the Columbia River...

  17. Abiraterone Improves Survival in Metastatic Prostate Cancer

    Science.gov (United States)

    A multinational phase III trial found that the drug abiraterone acetate prolonged the median survival of patients with metastatic castration-resistant prostate cancer by 4 months compared with patients who received a placebo.

  18. New Firm Survival: Industry versus Firm Effects

    NARCIS (Netherlands)

    D.B. Audretsch (David); P. Houweling (Patrick); A.R. Thurik (Roy)

    1997-01-01

    textabstractRecent studies show that the likelihood of survival differs significantly across firms. Both firm and industry characteristics are hypothesized to account for this heterogenity. Using a longitudinal database of manufacturing firms we investigate whether firm or industry characteristics

  19. FCS Vehicle Transportability, Survivability, and Reliability Analysis

    National Research Council Canada - National Science Library

    Dion-Schwarz, Cynthia; Hirsch, Leon; Koehn, Phillip; Macheret, Jenya; Sparrow, Dave

    2005-01-01

    .... The investigation into metrics for transportability revealed that the C130 Transportability requirement for FCS vehicles is a constraint that leads to a less survivable platform but without improving Unit of Action (UA) transportability...

  20. HMSRP Hawaiian Monk Seal Survival Factors

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains records of survival factors recorded by PSD personnel and cooperating scientists as part of the ongoing monk seal population assessment...

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

    Science.gov (United States)

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

    2017-09-01

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

  2. Modelling population-based cancer survival trends using join point models for grouped survival data.

    Science.gov (United States)

    Yu, Binbing; Huang, Lan; Tiwari, Ram C; Feuer, Eric J; Johnson, Karen A

    2009-04-01

    In the United States cancer as a whole is the second leading cause of death and a major burden to health care, thus the medical progress against cancer is a major public health goal. There are many individual studies to suggest that cancer treatment breakthroughs and early diagnosis have significantly improved the prognosis of cancer patients. To better understand the relationship between medical improvements and the survival experience for the patient population at large, it is useful to evaluate cancer survival trends on the population level, e.g., to find out when and how much the cancer survival rates changed. In this paper, we analyze the population-based grouped cancer survival data by incorporating joinpoints into the survival models. A joinpoint survival model facilitates the identification of trends with significant change points in cancer survival, when related to cancer treatments or interventions. The Bayesian Information Criterion is used to select the number of joinpoints. The performance of the joinpoint survival models is evaluated with respect to cancer prognosis, joinpoint locations, annual percent changes in death rates by year of diagnosis, and sample sizes through intensive simulation studies. The model is then applied to the grouped relative survival data for several major cancer sites from the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute. The change points in the survival trends for several major cancer sites are identified and the potential driving forces behind such change points are discussed.

  3. Effect of temperature and relative humidity on the survival of foodborne viruses during food storage.

    Science.gov (United States)

    Lee, Su Jin; Si, Jiyeon; Yun, Hyun Sun; Ko, GwangPyo

    2015-03-01

    Millions of people suffer from foodborne diseases throughout the world every year, and the importance of food safety has grown worldwide in recent years. The aim of this study was to investigate the survival of hepatitis A virus (HAV) and viral surrogates of human norovirus (HuNoV) (bacteriophage MS2 and murine norovirus [MNV]) in food over time. HAV, MNV, and MS2 were inoculated onto either the digestive gland of oysters or the surface of fresh peppers, and their survival on these food matrices was measured under various temperature (4°C, 15°C, 25°C, and 40°C) and relative humidity (RH) (50% and 70%) conditions. Inoculated viruses were recovered from food samples and quantified by a plaque assay at predetermined time points over 2 weeks (0, 1, 3, 7, 10, and 14 days). Virus survival was influenced primarily by temperature. On peppers at 40°C and at 50% RH, >4- and 6-log reductions of MNV and HAV, respectively, occurred within 1 day. All three viruses survived better on oysters. In addition, HAV survived better at 70% RH than at 50% RH. The survival data for HAV, MS2, and MNV were fit to three different mathematical models (linear, Weibull, and biphasic models). Among them, the biphasic model was optimum in terms of goodness of fit. The results of this study suggest that major foodborne viruses such as HAV and HuNoV can survive over prolonged periods of time with a limited reduction in numbers. Because a persistence of foodborne virus on contaminated foods was observed, precautionary preventive measures should be performed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Relevance Vector Machine for Survival Analysis.

    Science.gov (United States)

    Kiaee, Farkhondeh; Sheikhzadeh, Hamid; Mahabadi, Samaneh Eftekhari

    2016-03-01

    An accelerated failure time (AFT) model has been widely used for the analysis of censored survival or failure time data. However, the AFT imposes the restrictive log-linear relation between the survival time and the explanatory variables. In this paper, we introduce a relevance vector machine survival (RVMS) model based on Weibull AFT model that enables the use of kernel framework to automatically learn the possible nonlinear effects of the input explanatory variables on target survival times. We take advantage of the Bayesian inference technique in order to estimate the model parameters. We also introduce two approaches to accelerate the RVMS training. In the first approach, an efficient smooth prior is employed that improves the degree of sparsity. In the second approach, a fast marginal likelihood maximization procedure is used for obtaining a sparse solution of survival analysis task by sequential addition and deletion of candidate basis functions. These two approaches, denoted by smooth RVMS and fast RVMS, typically use fewer basis functions than RVMS and improve the RVMS training time; however, they cause a slight degradation in the RVMS performance. We compare the RVMS and the two accelerated approaches with the previous sparse kernel survival analysis method on a synthetic data set as well as six real-world data sets. The proposed kernel survival analysis models have been discovered to be more accurate in prediction, although they benefit from extra sparsity. The main advantages of our proposed models are: 1) extra sparsity that leads to a better generalization and avoids overfitting; 2) automatic relevance sample determination based on data that provide more accuracy, in particular for highly censored survival data; and 3) flexibility to utilize arbitrary number and types of kernel functions (e.g., non-Mercer kernels and multikernel learning).

  5. Minding the body: psychotherapy and cancer survival.

    Science.gov (United States)

    Spiegel, David

    2014-09-01

    This article reviews evidence regarding effects of psychotherapy on overall cancer survival time. Special emphasis is given to research on adverse effects of depression on cancer survival, breast cancer, and mediating psychophysiological pathways linking psychosocial support to longer survival. It reviews all published clinical trials addressing effects of psychotherapy on cancer survival, emphasizing depression, breast cancer, and psychophysiological evidence linking stress, depression, and support to cancer survival. Systematic literature review and synthesis. Eight of 15 published trials indicate that psychotherapy enhances cancer survival time. No studies show an adverse effect of psychotherapy on cancer survival. Potential psychophysiological mechanisms linking stress to shorter survival include dysregulation of diurnal cortisol, increased pro-inflammatory cytokines, reduced natural killer cell activity, shorter telomeres and lower telomerase activity, glucocorticoid-mediated suppression of p53 and BrCA1 gene expression, and sympathetic nervous system activation of vascular endothelial growth factor. Stress and support affect the course of cancer progression. What is known? Stress and support have been thought to be related to cancer risk and progression, but evidence has been mixed. Depression is a natural co-morbid condition with cancer. It has not been clear how stress and support could physiologically affect the rate of cancer progression. Immune function was not thought to have much relevance to cancer progression. Few other physiological mechanisms linking stress to cancer progression were known. What does this paper add? There is evidence from 15 RCTs indicating that effective psychosocial support improves quantity as well as quality of life with cancer. There is evidence that chronic depression predicts poorer prognosis with cancer. Dysregulated circadian cortisol patterns predict more rapid cancer progression. Inflammatory processes affect cancer

  6. Helping mothers survive bleeding after birth

    DEFF Research Database (Denmark)

    Nelissen, Ellen; Ersdal, Hege; Ostergaard, Doris

    2014-01-01

    OBJECTIVE: To evaluate "Helping Mothers Survive Bleeding After Birth" (HMS BAB) simulation-based training in a low-resource setting. DESIGN: Educational intervention study. SETTING: Rural referral hospital in Northern Tanzania. POPULATION: Clinicians, nurse-midwives, medical attendants, and ambul......OBJECTIVE: To evaluate "Helping Mothers Survive Bleeding After Birth" (HMS BAB) simulation-based training in a low-resource setting. DESIGN: Educational intervention study. SETTING: Rural referral hospital in Northern Tanzania. POPULATION: Clinicians, nurse-midwives, medical attendants...

  7. Survival of Burkholderia pseudomallei in Water

    Directory of Open Access Journals (Sweden)

    Woods Donald E

    2008-05-01

    Full Text Available Abstract Background The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB to distilled water. Findings Increased expression of a gene encoding for a putative membrane protein (BPSL0721 was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP, was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC, lipopolysaccharide (LPS oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water. Conclusion The results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

  8. Evolution and Survival of Quantum Entanglement

    Science.gov (United States)

    2015-05-06

    independently for tasks of  quantum  information. These include  quantum  computing,  quantum   cryptography ,  quantum  teleportation and other forms of entanglement...Evolution and Survival of Quantum Entanglement Theoretical foundations for methods to preserve quantum entanglement are explored and explained...Research Triangle Park, NC 27709-2211 quantum entanglement, decoherence, qubit, revival, survival, Jaynes-Cummings, Rabi, rotating wave approximation

  9. Survival function of hypo-exponential distributions

    OpenAIRE

    Lotfy, Mamdouh M.; Abdelsamad, Ali S.

    1985-01-01

    Approved for public release; distribution is unlimited The reliability of a system is the probability that the system will survive or complete an intended mission of certain duration. Describing all possible ways that a system can survive a mission in reliability shorthand gives a simple approach to reliability computations. Reliability computation for a system defined by shorthand notation is greatly dependent upon the convolution problem. Assuming constant component failure rates, this p...

  10. Retrofiting survivability of military vehicles

    Energy Technology Data Exchange (ETDEWEB)

    Canavan, Gregory H [Los Alamos National Laboratory

    2009-01-01

    of 0.5. Over the range from 0.5 to 4.5 cm the shock KE is attenuated by a factor of {approx}70, while its momentum is changed little. The shock and particle velocity falls by a factor of 200 while the mass increases by a factor of 730. In the limit of very porous media u {approx} 1/M, so KE {approx} 1/M, which falls by a factor of {approx}600, while momentum Mu does not change at all. Figure 2 shows the KE, Mu, u, and M for a material with a porosity of 1.05, for which the KE changes little. In the limit of media of very low porosity, u {approx} 1/{radical}M, so KE is constant while Mu {approx} {radical}M, which increases by a factor of 15. Thus, if the goal is to reduce the peak pressure from strong explosions below, very porous materials, which strongly reduce pressure but do not increase momentum, are preferred to non-porous materials, which amplify momentum but do not decrease pressure. These predictions are in qualitative accord with the results of experiments at Los Alamos in which projectiles from high velocity, large caliber cannons were stopped by one to two sandbags. The studies were performed primarily to determine the effectiveness of sand in stopping fragments of various sizes, but could be extended to study sand's effectiveness in attenuating blast pressure. It would also be useful to test the above predictions on the effectiveness of media with higher porosity. Water barriers have been discussed but not deployed in previous retrofit survivability studies for overseas embassies. They would detect the flash from the mine detonation below, trigger a thin layer of explosive above a layer of water, and drive water droplets into the approaching blast wave. The blast loses energy in evaporating the droplets and loses momentum in slowing them. Under favorable conditions that could attenuate the pressure in the blast enough to prevent the penetration or disruption of the vehicle. However, such barriers would depend on prompt and reliable detonation

  11. The survival of Coxiella burnetii in soils

    Science.gov (United States)

    Evstigneeva, A. S.; Ul'Yanova, T. Yu.; Tarasevich, I. V.

    2007-05-01

    Coxiella burnetii is a pathogen of Q-fever—a widespread zoonosis. The effective adaptation of C. burnetii to intracellular existence is in contrast with its ability to survive in the environment outside the host cells and its resistance to chemical and physical agents. Its mechanism of survival remains unknown. However, its survival appears to be related to the developmental cycle of the microorganism itself, i.e., to the formation of its dormant forms. The survival of Coxiella burnetii was studied for the first time. The pathogenic microorganism was inoculated into different types of soil and cultivated under different temperatures. The survival of the pathogen was verified using a model with laboratory animals (mice). Viable C. burnetii were found in the soil even 20 days after their inoculation. The relationship between the organic carbon content in the soils and the survival of C. burnetii was revealed. Thus, the results obtained were the first to demonstrate that the soil may serve as a reservoir for the preservation and further spreading of the Q-fever pathogen in the environment, on the one hand, and reduce the risk of epidemics, on the other.

  12. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Science.gov (United States)

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.

    Science.gov (United States)

    McKenzie, Jennifer Helen; Alwis, K Udeni; Sordillo, Joanne E; Kalluri, Kesava Srinivas; Milton, Donald Kirby

    2011-06-01

    Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.

  14. Post-transplant development of C1q-positive HLA antibodies and kidney graft survival.

    Science.gov (United States)

    Piazza, Antonina; Poggi, Elvira; Ozzella, Giuseppina; Adorno, Domenico

    2013-01-01

    The development of de novo human leukocyte antigen (HLA) donor specific antibodies (DSA), detected by both cytotoxic or solid phase assays, was considered the major risk factor for allograft failure in kidney transplantation. However, it was shown that not all patients with persistent production of DSA suffered loss of their grafts. Modified Luminex-Single Antigen assays, able to identify C1q-fixing antibodies, represent a new strategy in assessing the clinical relevance of detected DSA. This study demonstrated that C1q-fixing capability of de novo DSA is a clinically relevant marker of worse outcome and inferior graft survival in kidney transplantation. In fact, our findings evidenced a very low graft survival only in the patients who developed DSA able to fix C1q during post-transplant course, while patients producing C1q-negative DSA had good graft survival, which was comparable to that found in our previous study for DSA-negative patients. Moreover, anti-HLA class II antibodies had a higher incidence than anti-HLA class I, and the ability to fix C1q was significantly more frequent among anti-DQ DSA than anti-DR DSA. Monitoring of de novo C1q-DSA production represents a useful, non-invasive tool for risk stratification and prediction of graft outcome in kidney transplantation.

  15. Reliability of mycotoxin assays--an update.

    Science.gov (United States)

    Horwitz, W; Albert, R; Nesheim, S

    1993-01-01

    The precision parameters of the method-performance (collaborative) studies for mycotoxins published in the literature through 1991 have been recalculated on a uniform basis by following the International Union of Pure and Applied Chemistry protocol. About 80% of the 793 accepted assays for mycotoxins, almost all of which have been conducted by thin-layer chromatography (TLC), liquid chromatography (LC), and enzyme-linked immunosorbent assays (ELISA), exhibit relative standard deviations among laboratories (RSDR) that are less than 2 times the values predicted from the Horwitz equation: RSDR, % = 2(1-0.5log10C) where C is the concentration expressed as a decimal fraction. The precision of TLC and LC methods is about the same, but that of ELISA is somewhat poorer. For those commodities for which sufficient data exist to provide a meaningful comparison, the methods applied to cottonseed products have the best precision and corn the worst, with peanuts intermediate. Overall, however, the primary factor affecting RSDR is concentration, more or less independent of analyte, method, matrix, and age of the study. If it is assumed that the test results are normally distributed and that an RSDR of 50% is the point where effective control of the results begins to be lost (a value equivalent to the production of 2% false-negative values), then relying on the Horwitz curve, the limit of quantitative measurement is the single digit, i.e., 5, micrograms/kg (10(-9); ppb) concentration for solid food commodities. Such a value must be considered as a limit applicable to a single analyte, aflatoxin B1, and not as a mean, and not applicable to the sum of the individual components, each of whose associated standard deviation would lie in the unacceptable region. Enforcement of a 5 micrograms aflatoxin B1/kg limit, under the assumptions made, requires that a responsible manufacturer and a prudent regulator operate at opposite extremes of tolerance limits: e.g., the producer at 2

  16. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  17. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  18. Tooth survival after root canal treatment.

    Science.gov (United States)

    Balto, Khaled

    2011-01-01

    Medline, the Cochrane Library, hand searches of the International Endodontic Journal, Journal of Endodontics, Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics, Dental Traumatology (& Endodontics) and bibliographies of all relevant articles and review articles. Unpublished studies were identified by searching abstracts and conference proceedings. Personal contacts were used to identify ongoing or unpublished studies. Two reviewers independently assessed and selected the studies with disagreements being resolved by discussion. Clinical studies of RCTx on more than 30 teeth and of at least six-month duration, where the success was based on survival of tooth and the proportion of teeth surviving was given, or could be calculated from the raw data, were included. Data were extracted by two reviewers independently using custom-designed forms. The weighted pooled proportion of teeth surviving after treatment and the combined effects (expressed as odds ratio) of clinical factors on tooth survival were estimated using fixed and random effects meta-analyses using DerSimonean and Laird's methods. The survival data were pooled into three groups based on the duration after treatment: 2 or 3 years; 4 or 5 years; and 8, 9 or 10 years. Statistical heterogeneity amongst the studies was assessed by Cochran's (Q) test. Of the 31 articles identified, 14 studies were included. The majority (10) were retrospective. The reported survival is shown in Table 1. Substantial differences in study characteristics were found to hinder effective direct comparison of findings. Evidence for the effect of prognostic factors on tooth survival was weak. Based on the data available for meta-analysis, four conditions were found to significantly improve tooth survival. In descending order of influence, the conditions increasing observed proportion of survival were as follows: (i) a crown restoration after RCTx; (ii) tooth having both mesial and distal proximal contacts; (iii

  19. Challenges in the estimation of Net SURvival: The CENSUR working survival group.

    Science.gov (United States)

    Giorgi, R

    2016-10-01

    Net survival, the survival probability that would be observed, in a hypothetical world, where the cancer of interest would be the only possible cause of death, is a key indicator in population-based cancer studies. Accounting for mortality due to other causes, it allows cross-country comparisons or trends analysis and provides a useful indicator for public health decision-making. The objective of this study was to show how the creation and formalization of a network comprising established research teams, which already had substantial and complementary experience in both cancer survival analysis and methodological development, make it possible to meet challenges and thus provide more adequate tools, to improve the quality and the comparability of cancer survival data, and to promote methodological transfers in areas of emerging interest. The Challenges in the Estimation of Net SURvival (CENSUR) working survival group is composed of international researchers highly skilled in biostatistics, methodology, and epidemiology, from different research organizations in France, the United Kingdom, Italy, Slovenia, and Canada, and involved in French (FRANCIM) and European (EUROCARE) cancer registry networks. The expected advantages are an interdisciplinary, international, synergistic network capable of addressing problems in public health, for decision-makers at different levels; tools for those in charge of net survival analyses; a common methodology that makes unbiased cross-national comparisons of cancer survival feasible; transfer of methods for net survival estimations to other specific applications (clinical research, occupational epidemiology); and dissemination of results during an international training course. The formalization of the international CENSUR working survival group was motivated by a need felt by scientists conducting population-based cancer research to discuss, develop, and monitor implementation of a common methodology to analyze net survival in order

  20. Mouse lung adhesion assay for Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Burns, K.A.; Freer, J.H. (Department of Microbiology, Alexander Stone Building, Bearsden, Glasgow, Scotland)

    1982-03-01

    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 ..mu..Ci (37 K Bq) of (/sup 14/C)glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation.

  1. Prandiology of Drosophila and the CAFE assay

    Science.gov (United States)

    Ja, William W.; Carvalho, Gil B.; Mak, Elizabeth M.; de la Rosa, Noelle N.; Fang, Annie Y.; Liong, Jonathan C.; Brummel, Ted; Benzer, Seymour

    2007-01-01

    Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7× their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant. PMID:17494737

  2. Fluorometric enzymatic assay of L-arginine

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  3. Virus Characterization by FFF-MALS Assay

    Science.gov (United States)

    Razinkov, Vladimer

    2009-03-01

    Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

  4. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    Science.gov (United States)

    Huang, Kai-Wen; Chieh, Jen-Jie; Shi, Jin-Cheng; Chiang, Ming-Hsien

    2015-07-01

    Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations ( Φ CEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization ( M), remanent magnetization ( M R), saturation magnetization ( M S), and normalized parameters (Δ M R/ M R and Δ M S/ M S) were studied. Here, Δ M R and Δ M s were defined as the difference between any ΦCEA and zero Φ CEA. The parameters M, Δ M R, and Δ M S increased with Φ CEA, and Δ M S showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a Δ M S greater than that of BMNs. Furthermore, the relationship between Δ M S/ M S and Φ CEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic Δ M S/ M S and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials.

  5. Assaying Visual Memory in the Desert Locust

    Directory of Open Access Journals (Sweden)

    Senne Dillen

    2015-04-01

    Full Text Available The involvement of associative learning cues has been demonstrated in several stages of feeding and food selection. Short neuropeptide F (sNPF, an insect neuropeptide whose effects on feeding behavior have previously been well established, may be one of the factors bridging feeding and learning behavior. Recently, it was shown in Drosophila melanogaster that the targeted reduction of Drome-sNPF transcript levels significantly reduced sugar-rewarded olfactory memory. While Drosophila mainly relies on olfactory perception in its food searching behavior, locust foraging behavior is likely to be more visually orientated. Furthermore, a feeding-dependent regulation of Schgr-sNPF transcript levels has previously been observed in the optic lobes of the locust brain, suggesting a possible involvement in visual perception of food and visual associative memory in this insect species. In this study, we describe the development of a robust and reproducible assay allowing visual associative memory to be studied in the desert locust, Schistocerca gregaria. Furthermore, we performed an exploratory series of experiments, studying the role of Schgr-sNPF in this complex process.

  6. Mortality and translocation assay to study the protective capacity of Bifidobacterium lactis INL1 against Salmonella Typhimurium infection in mice.

    Science.gov (United States)

    Zacarías, M F; Reinheimer, J; Forzani, L; Grangette, C; Vinderola, G

    2014-12-01

    The mouse has been largely used for the study of the protective capacity of probiotics against intestinal infections caused by Salmonella. In this work we aimed at comparing the mortality and translocation assay for the study of the protective capacity of the human breast milk-derived strain Bifidobacterium animalis subsp. lactis INL1 on a model of gut infection by Salmonella enterica subsp. enterica serovar Typhimurium. Different doses of S. Typhimurium FUNED and B. animalis subsp. lactis INL 1 were administered to Balb/c mice in a mortality or a translocation assay. The survival of the control group in the mortality assay resulted to be variable along experiments, and then we preferred to use a translocation assay where the preventive administration of 109 cfu of bifidobacteria/mouse for 10 consecutive days significantly reduced the number of infected animals and the levels of translocation to liver and spleen, with enhanced secretory immunoglobulin A and interleukin 10 production in the small and large intestine, respectively. Ten days of B. animalis subsp. lactis strain INL1 administration to mice significantly reduced both the incidence and the severity of Salmonella infection in a mouse model of translocation. This work provided the first evidence that a translocation assay, compared to a mortality assay, could be more useful to study the protective capacity of probiotics against Salmonella infection, as more information can be obtained from mice and less suffering is conferred to animals due to the fact that the mortality assay is shorter than the latter. These facts are in line with the guidelines of animal research recently established by the National Centre for the Replacement, Refinement & Reduction of Animals in Research.

  7. Five-year survival and median survival time of nasopharyngeal carcinoma in Hospital Universiti Sains Malaysia.

    Science.gov (United States)

    Siti-Azrin, Ab Hamid; Norsa'adah, Bachok; Naing, Nyi Nyi

    2014-01-01

    Nasopharyngeal carcinoma (NPC) is the fourth most common cancer in Malaysia. The objective of this study was to determine the five-year survival rate and median survival time of NPC patients in Hospital Universiti Sains Malaysia (USM). One hundred and thirty four NPC cases confirmed by histopathology in Hospital USM between 1st January 1998 and 31st December 2007 that fulfilled the inclusion and exclusion criteria were retrospectively reviewed. Survival time of NPC patients were estimated by Kaplan-Meier survival analysis. Log-rank tests were performed to compare survival of cases among presenting symptoms, WHO type, TNM classification and treatment modalities. The overall five-year survival rate of NPC patients was 38.0% (95% confidence interval (CI): 29.1, 46.9). The overall median survival time of NPC patients was 31.30 months (95%CI: 23.76, 38.84). The significant factors that altered the survival rate and time were age (p=0.041), cranial nerve involvement (p=0.012), stage (p=0.002), metastases (p=0.008) and treatment (p<0.001). The median survival of NPC patients is significantly longer for age≤50 years, no cranial nerve involvement, and early stage and is dependent on treatment modalities.

  8. Inhibition of Prostate Cancer Bone Metastasis by Synthetic TF Antigen Mimic/Galectin-3 Inhibitor Lactulose-l-Leucine

    Directory of Open Access Journals (Sweden)

    Olga V. Glinskii

    2012-01-01

    Full Text Available Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-l-leucine (Lac-l-Leu to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-l-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-l-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-l-Leu resulted in a three-fold (P < .05 decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-l-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

  9. Survivability is more fundamental than evolvability.

    Directory of Open Access Journals (Sweden)

    Michael E Palmer

    Full Text Available For a lineage to survive over long time periods, it must sometimes change. This has given rise to the term evolvability, meaning the tendency to produce adaptive variation. One lineage may be superior to another in terms of its current standing variation, or it may tend to produce more adaptive variation. However, evolutionary outcomes depend on more than standing variation and produced adaptive variation: deleterious variation also matters. Evolvability, as most commonly interpreted, is not predictive of evolutionary outcomes. Here, we define a predictive measure of the evolutionary success of a lineage that we call the k-survivability, defined as the probability that the lineage avoids extinction for k generations. We estimate the k-survivability using multiple experimental replicates. Because we measure evolutionary outcomes, the initial standing variation, the full spectrum of generated variation, and the heritability of that variation are all incorporated. Survivability also accounts for the decreased joint likelihood of extinction of sub-lineages when they 1 disperse in space, or 2 diversify in lifestyle. We illustrate measurement of survivability with in silico models, and suggest that it may also be measured in vivo using multiple longitudinal replicates. The k-survivability is a metric that enables the quantitative study of, for example, the evolution of 1 mutation rates, 2 dispersal mechanisms, 3 the genotype-phenotype map, and 4 sexual reproduction, in temporally and spatially fluctuating environments. Although these disparate phenomena evolve by well-understood microevolutionary rules, they are also subject to the macroevolutionary constraint of long-term survivability.

  10. SURVIVAL ANALYSIS AND LENGTH-BIASED SAMPLING

    Directory of Open Access Journals (Sweden)

    Masoud Asgharian

    2010-12-01

    Full Text Available When survival data are colleted as part of a prevalent cohort study, the recruited cases have already experienced their initiating event. These prevalent cases are then followed for a fixed period of time at the end of which the subjects will either have failed or have been censored. When interests lies in estimating the survival distribution, from onset, of subjects with the disease, one must take into account that the survival times of the cases in a prevalent cohort study are left truncated. When it is possible to assume that there has not been any epidemic of the disease over the past period of time that covers the onset times of the subjects, one may assume that the underlying incidence process that generates the initiating event times is a stationary Poisson process. Under such assumption, the survival times of the recruited subjects are called “lengthbiased”. I discuss the challenges one is faced with in analyzing these type of data. To address the theoretical aspects of the work, I present asymptotic results for the NPMLE of the length-biased as well as the unbiased survival distribution. I also discuss estimating the unbiased survival function using only the follow-up time. This addresses the case that the onset times are either unknown or known with uncertainty. Some of our most recent work and open questions will be presented. These include some aspects of analysis of covariates, strong approximation, functional LIL and density estimation under length-biased sampling with right censoring. The results will be illustrated with survival data from patients with dementia, collected as part of the Canadian Study of Health and Aging (CSHA.

  11. Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.

    Directory of Open Access Journals (Sweden)

    Francesca Necchi

    Full Text Available Serum Bactericidal Activity (SBA assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.

  12. Attenuation caused by infrequently updated covariates in survival analysis

    DEFF Research Database (Denmark)

    Andersen, Per Kragh; Liestøl, Knut

    2003-01-01

    Attenuation; Cox regression model; Measurement errors; Survival analysis; Time-dependent covariates......Attenuation; Cox regression model; Measurement errors; Survival analysis; Time-dependent covariates...

  13. An assay to screen bacterial adhesion to mucus biomolecules.

    Science.gov (United States)

    Tkachenko, A; Da Silva, L; Hearne, J; Parveen, S; Waguespack, Y

    2013-01-01

    To develop an assay for rapid screening of bacterial adhesion to various groups of biomolecules present in fish mucus. A novel assay was developed for investigation of bacterial adhesion to various groups of mucus biomolecules from fish. Lipid-, protein-, carbohydrate- and nucleic acid-rich constituents of mucus were separated using isopycnic density gradient centrifugation techniques. Separated mucus fractions were assayed for bacterial adhesion using a blotting apparatus. The assay was validated using Vibrio vulnificus and skin mucus from hybrid tilapia. A novel assay was developed for the screening of bacterial adhesion to major groups of mucus biomolecules. Adhesion of V. vulnificus MLT403 positively correlated with lipid- and protein-rich mucus constituents and negatively correlated with carbohydrate-rich mucus constituents. The assay can be used as an initial approach in a systematic identification of mucus constituent(s) exhibiting the most favourable adhesion properties for bacteria. © 2012 The Society for Applied Microbiology.

  14. Multiplex cytological profiling assay to measure diverse cellular states.

    Directory of Open Access Journals (Sweden)

    Sigrun M Gustafsdottir

    Full Text Available Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

  15. Comparison of assay formats for drug-tolerant immunogenicity testing.

    Science.gov (United States)

    Butterfield, Anthony M; Chain, Jana S; Ackermann, Bradley L; Konrad, Robert J

    2010-12-01

    Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(®) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.

  16. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    Directory of Open Access Journals (Sweden)

    KHUSNUL YAQIN

    2006-09-01

    Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

  17. Proteomics: from hypothesis to quantitative assay on a single platform. Guidelines for developing MRM assays using ion trap mass spectrometers.

    Science.gov (United States)

    Han, Bomie; Higgs, Richard E

    2008-09-01

    High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

  18. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

    Emilio eRojas; Yolanda eLorenzo; Kristiane eHuag-Berg; Bjørn eNicolaissen; Mahara eValverde

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many org...

  19. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    OpenAIRE

    YAQIN, KHUSNUL

    2006-01-01

    Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The appli...

  20. Enzyme Assay Methods to Validate DIGE Proteomics Data.

    Science.gov (United States)

    Dowd, Andrew

    2018-01-01

    Enzyme activity assay methods can be used to corroborate the results generated by Difference Gel Electrophoresis (DIGE) proteomic experiments. Two assay methods were chosen to demonstrate how this can be achieved. Assays for determining the activity of superoxide dismutase and NADH dehydrogenase are outlined in detail in this paper. These methods were chosen as examples because they are frequently used in conjunction with DIGE proteomics.

  1. Absolute nuclear material assay using count distribution (LAMBDA) space

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Mano K.; Snyderman, Neal J.; Rowland, Mark S.

    2015-12-01

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  2. Evaluation of Resistance to Phytophthora sojae in Soybean Mini Core Collections Using an Improved Assay System.

    Science.gov (United States)

    Jiang, Chang-Jie; Sugano, Shoji; Kaga, Akito; Lee, Sung Shin; Sugimoto, Takuma; Takahashi, Mami; Ishimoto, Masao

    2017-02-01

    Stem and root rot disease caused by Phytophthora sojae is devastating to soybean crops worldwide. Developing host resistance to P. sojae, considered the most effective and stable means to control this disease, is partly hampered by limited germplasm resources. In this study, we first modified conventional methods for a P. sojae resistance assay to a simpler and more cost-effective version, in which the P. sojae inoculum was mixed into the soil and the resistance was evaluated by survival rate (%) of soybean seedlings. This rating had significant correlations (P < 0.01) with the reduction in root fresh weight and the visual root rot severity. Applying this method to evaluate P. sojae resistance in soybean mini core collections comprising either 79 accessions originating from Japan (JMC) or 80 accessions collected around the world (WMC) revealed a wide variation in resistance among the individual varieties. In total, 38 accessions from the JMC and 41 from the WMC exhibited resistance or moderate resistance to P. sojae isolate N1 (with virulence to Rps1b, 3c, 4, 5, and 6), with ≥50% survival. Of these, 26 from the JMC and 29 from the WMC showed at least moderate resistance to P. sojae isolate HR1 (vir Rps1a-c, 1k, 2, 3a-c, 4-6, and 8). Additionally, 24 WCS accessions, in contrast to only 6 from the JMC, exhibited 100% survival after being challenged with both the N1 and HR1 isolates, suggesting a biogeographical difference between the two collections. We further verified two JMC varieties, Daizu and Amagi zairai 90D, for their resistance to an additional four P. sojae isolates (60 to 100% survival), which may provide new and valuable genetic sources for P. sojae resistance breeding in soybean.

  3. Multidimensional Poverty and Child Survival in India

    Science.gov (United States)

    Mohanty, Sanjay K.

    2011-01-01

    Background Though the concept of multidimensional poverty has been acknowledged cutting across the disciplines (among economists, public health professionals, development thinkers, social scientists, policy makers and international organizations) and included in the development agenda, its measurement and application are still limited. Objectives and Methodology Using unit data from the National Family and Health Survey 3, India, this paper measures poverty in multidimensional space and examine the linkages of multidimensional poverty with child survival. The multidimensional poverty is measured in the dimension of knowledge, health and wealth and the child survival is measured with respect to infant mortality and under-five mortality. Descriptive statistics, principal component analyses and the life table methods are used in the analyses. Results The estimates of multidimensional poverty are robust and the inter-state differentials are large. While infant mortality rate and under-five mortality rate are disproportionately higher among the abject poor compared to the non-poor, there are no significant differences in child survival among educationally, economically and health poor at the national level. State pattern in child survival among the education, economical and health poor are mixed. Conclusion Use of multidimensional poverty measures help to identify abject poor who are unlikely to come out of poverty trap. The child survival is significantly lower among abject poor compared to moderate poor and non-poor. We urge to popularize the concept of multiple deprivations in research and program so as to reduce poverty and inequality in the population. PMID:22046384

  4. Survival of pneumococcus on hands and fomites

    Directory of Open Access Journals (Sweden)

    Beissbarth Jemima

    2008-11-01

    Full Text Available Abstract Background Pneumococcal hand contamination in Indigenous children in remote communities is common (37%. It is not clear whether this requires frequent inoculation, or if pneumococci will survive on hands for long periods of time. Thus the aim of this study was to determine the survival time of pneumococci on hands and fomites. Findings The hands of 3 adult volunteers, a glass plate and plastic ball were inoculated with pneumococci suspended in two different media. Survival at specified time intervals was determined by swabbing and re-culture onto horse blood agar. Pneumococci inoculated onto hands of volunteers were recovered after 3 minutes at 4% to 79% of the initial inoculum. Recovery from one individual was consistently higher. By one hour, only a small number of pneumococci were recovered and this was dependent on the suspension medium used. At subsequent intervals and up to 3 hours after inoculation, Conclusion The poor survival of pneumococci on hands suggests that the high prevalence of pneumococcal hand contamination in some populations is related to frequent inoculation rather than long survival. It is plausible that hand contamination plays a (brief role in transmission directly, and indirectly through contamination via fomites. Regular hand washing and timely cleansing or removal of contaminated fomites may aid control of pneumococcal transmission via these routes.

  5. Incidence and overall survival of malignant ameloblastoma.

    Directory of Open Access Journals (Sweden)

    Alexandra Rizzitelli

    Full Text Available Malignant ameloblastoma, comprising metastasizing ameloblastoma and ameloblastic carcinoma, represents 1.6-2.2% of all odontogenic tumors. Due to its rare nature, malignant ameloblastoma has only been reported in the literature in small case series or case reports. Using the Surveillance, Epidemiology and End-Results (SEER database, we have performed a population-based study to determine the incidence rate and the absolute survival of malignant ameloblastoma.Using the International Classification of Diseases for Oncology (ICD-O codes 9310/3 and 9270/3, data from the SEER database were used to calculate the incidence rate and absolute survival rate of population with malignant ameloblastoma.The overall incidence rate of malignant ameloblastoma was 1.79 per 10 million person/year. The incidence rate was higher in males than females and also higher in black versus white population. The median overall survival was 17.6 years from the time of diagnosis and increasing age was associated with a statistically significant poorer survival.To our best knowledge, we report the largest population-based series of malignant ameloblastoma. The incidence rate was 1.79 per 10 million person/year and the overall survival was 17.6 years.

  6. Survivability of a metapopulation under local extinctions

    Science.gov (United States)

    Kundu, Srilena; Majhi, Soumen; Sasmal, Sourav Kumar; Ghosh, Dibakar; Rakshit, Biswambhar

    2017-12-01

    A metapopulation structure in landscape ecology comprises a group of interacting spatially separated subpopulations or patches of the same species that may experience several local extinctions. This makes the investigation of survivability (in the form of global oscillation) of a metapopulation on top of diverse dispersal topologies extremely crucial. However, among various dispersal topologies in ecological networks, which one can provide higher metapopulation survivability under local extinction is still not well explored. In this article, we scrutinize the robustness of an ecological network consisting of prey-predator patches having Holling type I functional response, against progressively extinct population patches. We present a comprehensive study on this while considering global, small-world, and scale-free dispersal of the subpopulations. Furthermore, we extend our work in enhancing survivability in the form of sustained global oscillation by introducing asymmetries in the dispersal rates of the considered species. Our findings affirm that the asynchrony among the patches plays an important role in the survivability of a metapopulation. In order to demonstrate the model independence of the observed phenomenon, we perform a similar analysis for patches exhibiting Holling type II functional response. On the grounds of the obtained results, our work is expected to provide a better perception of the influence of dispersal arrangements on the global survivability of ecological networks.

  7. Multidimensional poverty and child survival in India.

    Science.gov (United States)

    Mohanty, Sanjay K

    2011-01-01

    Though the concept of multidimensional poverty has been acknowledged cutting across the disciplines (among economists, public health professionals, development thinkers, social scientists, policy makers and international organizations) and included in the development agenda, its measurement and application are still limited. OBJECTIVES AND METHODOLOGY: Using unit data from the National Family and Health Survey 3, India, this paper measures poverty in multidimensional space and examine the linkages of multidimensional poverty with child survival. The multidimensional poverty is measured in the dimension of knowledge, health and wealth and the child survival is measured with respect to infant mortality and under-five mortality. Descriptive statistics, principal component analyses and the life table methods are used in the analyses. The estimates of multidimensional poverty are robust and the inter-state differentials are large. While infant mortality rate and under-five mortality rate are disproportionately higher among the abject poor compared to the non-poor, there are no significant differences in child survival among educationally, economically and health poor at the national level. State pattern in child survival among the education, economical and health poor are mixed. Use of multidimensional poverty measures help to identify abject poor who are unlikely to come out of poverty trap. The child survival is significantly lower among abject poor compared to moderate poor and non-poor. We urge to popularize the concept of multiple deprivations in research and program so as to reduce poverty and inequality in the population.

  8. Comparative investigation of various cellulase assay procedures

    Energy Technology Data Exchange (ETDEWEB)

    Canevascini, G.; Gattlen, C.

    1981-07-01

    The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. 1) Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis, analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present. (Refs. 39).

  9. BROMATOMATRIC ASSAY OF GATIFLOXACIN IN PHARMACEUTICALS

    Directory of Open Access Journals (Sweden)

    KALSANG THARPA

    2008-09-01

    Full Text Available Three new, simple, and cost-effective visible spectrophotometric methods are proposed for determination of gatifloxacin (GTF using bromate-bromide mixture, and three dyes, methyl orange, indigocarmine and thymol blue, as reagents.The methods engross the addition of a known excess of bromate-bromide mixture to GTF in hydrochloric acid medium followed by determination of residual bromine by reacting with a fixed amount of either methyl orange andmeasuring the absorbance at 520 nm (method A or indigo carmine and measuring the absorbance at 610 nm (method B or thymol blue and measuring the absorbance at 550 nm (method C. In all the methods, the amount of brominereacted corresponds to the amount of GTF, and the absorbance is found to increase linearly with the concentration of GTF. Under the optimum conditions, GTF could be assayed in the concentration range 0.25-1.5, 0.5-6.0, and 0.5-10μg/mL by method A, method B and method C, respectively. The apparent molar absorptivities are calculated to be 1.6x105, 4.0x104 and 3.2x104 L mol-1 cm-1 for the method A, method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0025, 0.010 and 0.012 μg/cm2. The intra-day and inter-day precision, and the accuracy of the methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the determination of GTF in pharmaceutical preparations without the interference from any of the pharmaceutical adjuvants.

  10. Functional assays for hematopoietic stem cell self-renewal.

    Science.gov (United States)

    Perry, John M; Li, Linheng

    2010-01-01

    Stem cells are defined by the ability to self-renew. Specific functional assays have been developed for the rigorous identification and quantification of hematopoietic stem cells (HSCs), making these cells the benchmark in studies of self-renewal. Here, we review the theory behind these functional stem cell tests and discuss important considerations in choosing and designing these assays. Finally, we provide a basic protocol for the serial-dilution assay, a quantitative assay for HSCs, from which individual researchers can construct their own customized protocols utilizing the guidelines discussed.

  11. Nitrosylcobalamin potentiates the anti-neoplastic effects of chemotherapeutic agents via suppression of survival signaling.

    Directory of Open Access Journals (Sweden)

    Joseph A Bauer

    2007-12-01

    Full Text Available Nitrosylcobalamin (NO-Cbl is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1 measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2 examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA, immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-kappaB activation.Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-kappaB DNA binding activity, inhibition of IkappaB kinase (IKK enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT.

  12. Regression analysis of restricted mean survival time based on pseudo-observations

    DEFF Research Database (Denmark)

    Andersen, Per Kragh; Hansen, Mette Gerster; Klein, John P.

    censoring; hazard function; health economics; regression model; survival analysis; mean survival time; restricted mean survival time; pseudo-observations......censoring; hazard function; health economics; regression model; survival analysis; mean survival time; restricted mean survival time; pseudo-observations...

  13. Regression Analysis of Restricted Mean Survival Time Based on Pseudo-Observations

    DEFF Research Database (Denmark)

    Andersen, Per Kragh; Hansen, Mette Gerster; Klein, John P.

    2004-01-01

    censoring; hazard function; health economics; mean survival time; pseudo-observations; regression model; restricted mean survival time; survival analysis......censoring; hazard function; health economics; mean survival time; pseudo-observations; regression model; restricted mean survival time; survival analysis...

  14. Serial detection of circulating tumour cells by reverse transcriptase-polymerase chain reaction assays is a marker for poor outcome in patients with malignant melanoma

    Directory of Open Access Journals (Sweden)

    Daponte Antonio

    2006-11-01

    Full Text Available Abstract Background Detection of circulating malignant cells (CMCs through a reverse transcriptase-polymerase chain reaction (RT-PCR assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM. Methods One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up (total: 5 samples. Univariate and multivariate analyses were performed after 83 months of median follow-up. Results Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay. A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007. Presence of CMC in a high number (≥2 of analysed blood samples was significantly correlated with a poor clinical outcome (disease-free survival: P = 0.019; overall survival: P = 0.034. Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage. Conclusion Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease.

  15. Physical activity and survival in breast cancer

    DEFF Research Database (Denmark)

    Ammitzbøll, Gunn; Søgaard, Karen; Karlsen, Randi V

    2016-01-01

    the Diet, Cancer, and Health cohort, all enrolled before diagnosis. Self-reported PA was measured as time per activity, and estimated metabolic equivalent task (MET)-hours per week were summed for each activity. We constructed measures for household, exercise, and total PA. The association between......PURPOSE: Knowledge about lifestyle factors possibly influencing survival after breast cancer (BC) is paramount. We examined associations between two types of postdiagnosis physical activity (PA) and overall survival after BC. PATIENTS AND METHODS: We used prospective data on 959 BC survivors from...... from all causes during the study period. In adjusted analyses, exercise PA above eight MET h/week compared to lower levels of activity was significantly associated with improved overall survival (HR, 0.68; confidence interval [CI]: 0.47-0.99). When comparing participation in exercise to non...

  16. Survivability of systems under multiple factor impact

    Energy Technology Data Exchange (ETDEWEB)

    Korczak, Edward [Telecommunications Research Institute, Warsaw (Poland); Levitin, Gregory [Israel Electric Corporation Ltd., Haifa (Israel)]. E-mail: levitin@iec.co.il

    2007-02-15

    The paper considers vulnerable multi-state series-parallel systems operating under influence of external impacts. Both the external impacts and internal failures affect system survivability, which is determined as the probability of meeting a given demand. The external impacts are characterized by several destructive factors affecting the system or its parts simultaneously. In order to increase the system's survivability a multilevel protection against the destructive factors can be applied to its subsystems. In such systems, the protected subsystems can be destroyed only if all of the levels of their protection are destroyed. The paper presents an algorithm for evaluating the survivability of series-parallel systems with arbitrary configuration of multilevel protection against multiple destructive factor impacts. The algorithm is based on a composition of Boolean and the Universal Generating Function techniques. Illustrative examples are presented.

  17. Survival of ovarian cancer patients in Denmark

    DEFF Research Database (Denmark)

    Edwards, Hellen McKinnon; Noer, Mette Calundann; Sperling, Cecilie Dyg

    2016-01-01

    linked via the patients' personal identification number and the analyses included data on cancer stage, age, survival, surgery status and comorbidity. The computed outcome measures were age-adjusted mortality rates and age-adjusted overall and relative survival rates for one and five years. RESULTS: We......BACKGROUND: Ovarian cancer has a high mortality rate, especially in Denmark where mortality rates have been reported higher than in adjacent countries with similar demographics. This study therefore examined recent survival and mortality among Danish ovarian cancer patients over an 18-year study...... period. METHODS: This nationwide registry-based observational study used data from the Danish Gynecology Cancer Database, Danish Pathology Registry, and Danish National Patient Registry. All patients with ovarian cancer diagnosed between 1995 and 2012 were included in the study. The data sources were...

  18. Analysis of survival data from telemetry projects

    Science.gov (United States)

    Bunck, C.M.; Winterstein, S.R.; Pollock, K.H.

    1985-01-01

    Telemetry techniques can be used to study the survival rates of animal populations and are particularly suitable for species or settings for which band recovery models are not. Statistical methods for estimating survival rates and parameters of survival distributions from observations of radio-tagged animals will be described. These methods have been applied to medical and engineering studies and to the study of nest success. Estimates and tests based on discrete models, originally introduced by Mayfield, and on continuous models, both parametric and nonparametric, will be described. Generalizations, including staggered entry of subjects into the study and identification of mortality factors will be considered. Additional discussion topics will include sample size considerations, relocation frequency for subjects, and use of covariates.

  19. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L

    2010-01-01

    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

  20. Survival and leaching of Tetracycline resistant bacteria and fecal indicators from manure in field scale experiments

    DEFF Research Database (Denmark)

    Bech, Tina; Amin, Mostofa; Lægdsmand, Mette

    The spreading of manure on agricultural land is an economic and practical solution for improving soil quality; however, animal manure frequently contains zoonotic pathogenic bacteria, such as certain Eschericia coli, Salmonella spp. and Campylobacter spp. The present experiment was conducted...... as a large multidisciplinary project. Pig manure with a natural content of Tetracycline resistant bacteria and fecal indicator organisms was followed in soil columns and a field scale experiment. In the field experiment pig manure was injected into agricultural soil. The distribution and survival of natural...... occurring indicator bacteria around a manure slurry slit in the soil was followed. During a period of two months, sections of soils with different distance to the manure string were assayed to obtain information on survival and spread of bacteriophage, faecal indicators (Enterococci, Bacterioides, E. coli...

  1. Panspermia Survival Scenarios for Organisms that Survive Typical Hypervelocity Solar System Impact Events.

    Science.gov (United States)

    Pasini, D.

    2014-04-01

    Previous experimental studies have demonstrated the survivability of living cells during hypervelocity impact events, testing the panspermia and litho-panspermia hypotheses [1]. It has been demonstrated by the authors that Nannochloropsis Oculata Phytoplankton, a eukaryotic photosynthesizing autotroph found in the 'euphotic zone' (sunlit surface layers of oceans [2]), survive impacts up to 6.93 km s-1 (approx. shock pressure 40 GPa) [3, 4]. Also shown to survive impacts up to 5.49 km s-1 is the tardigrade species Hypsibius dujardini (a complex micro-animal consisting of 40,000 cells) [5, 6]. It has also been shown that they can survive sustained pressures up to 600 MPa using a water filled pressure capsule [7]. Additionally bacteria can survive impacts up to 5.4 km s-1 (~30 GPa) - albeit with a low probability of survival [1], and the survivability of yeast spores in impacts up to 7.4 km s-1 (~30 GPa) has also recently been demonstrated [8]. Other groups have also reported that the lichen Xanthoria elegans is able to survive shocks in similar pressure ranges (~40 GPa) [9]. Here we present various simulated impact regimes to show which scenarios are condusive to the panspermia hypothesis of the natural transfer of life (via an icy body) through space to an extraterrestrial environment.

  2. Survival in common cancers defined by risk and survival of family members

    Directory of Open Access Journals (Sweden)

    Jianguang Ji

    2011-10-01

    Full Text Available Studies on survival between familial and sporadic cancers have been inconclusive and only recent data on a limited number of cancers are available on the concordance of survival between family members. In this review, we address these questions by evaluating the published and unpublished data from the nation-wide Swedish Family-Cancer Database and a total of 13 cancer sites were assessed. Using sporadic cancer as reference, HRs were close to 1.0 for most of the familial cancers in both the offspring and parental generations, which suggested that survival in patients with familial and sporadic cancers was equal, with an exception for ovarian cancer with a worse prognosis. Compared to offspring whose parents had a poor survival, those with a good parental survival had a decreased risk of death for most cancers and HR was significantly decreased for cancers in the breast, prostate, bladder, and kidney. For colorectal and nervous system cancers, favorable survival between the generations showed a borderline significance. These data are consistent in showing that both good and poor survival in certain cancers aggregate in families. Genetic factors are likely to contribute to the results. These observations call for intensified efforts to consider heritability in survival as one mechanism regulating prognosis in cancer patients.

  3. Acute traumatic coagulopathy decreased actual survival rate when compared with predicted survival rate in severe trauma.

    Science.gov (United States)

    Kim, Su Jin; Lee, Sung Woo; Han, Gap Su; Moon, Sung Woo; Choi, Sung Hyuck; Hong, Yun Sik

    2012-11-01

    To determine whether acute traumatic coagulopathy (ATC) should be combined with the trauma and injury severity score (TRISS) to predict outcome in severe trauma patients and investigate effects of the change in coagulation state during early resuscitation on the actual survival rate. This was a retrospective study. Significant variables that affected 28-day mortality were analysed using multivariate logistic regression. Study patients were classified into three groups: no coagulopathy, mild coagulopathy or severe coagulopathy. Concordance between actual and predicted survival rates were compared for each group. The predicted survival rate was calculated using the TRISS method. The study also determined whether changes in the coagulation state during inhospital resuscitation affected the relationship between actual and predicted survival in patients who had rechecked coagulation profile within 12 h after presentation. Data from a total of 336 patients were analysed. At presentation, 20.8% of the study patients had mild coagulopathy, whereas 7.7% had severe coagulopathy. Age, injury severity score, revised trauma score and presence of ATC at presentation were independently associated with 28-day mortality. Actual survival was significantly lower than predicted survival in the mild and severe coagulopathy groups. Aggravation of coagulation state from normal or mild to severe coagulopathy or persistent severe coagulopathy during inhospital resuscitation mainly contributed to the discrepancy between actual and predicted survival. ATC decreased actual survival more than expected. ATC should be combined with TRISS to predict trauma outcome in severely injured patients. Improvement in coagulopathy during resuscitation may reduce the incidence of preventable death after trauma.

  4. Changing Pattern in Malignant Mesothelioma Survival

    Directory of Open Access Journals (Sweden)

    Jennifer Faig

    2015-02-01

    Full Text Available Survival for mesothelioma has been shown to be poor, with marginal improvement over time. Recent advances in the understanding of pathophysiology and treatment of mesothelioma may impact therapy to improve survival that may not be evident from available clinical trials that are often small and not randomized. Therapies may affect survival differently based on mesothelioma location (pleural vs peritoneal. Data are conflicting regarding the effect of asbestos exposure on mesothelioma location. OBJECTIVES: We examined survival in a large cohort of mesothelioma subjects analyzed by tumor location and presence and mode of asbestos exposure. METHODS: Data were analyzed from cases (n = 380 diagnosed with mesothelioma from 1992 to 2012. Cases were either drawn from treatment referrals, independent medical evaluation for medical legal purposes, or volunteers who were diagnosed with mesothelioma. Subjects completed an occupational medical questionnaire, personal interview with the examining physician, and physician review of the medical record. RESULTS: This study reports better survival for mesothelioma than historical reports. Survival for peritoneal mesothelioma was longer than that for pleural mesothelioma (hazard ratio = 0.36, 95% confidence interval = 0.24-0.54, P < .001 after adjusting for gender and age at diagnosis. Non-occupational cases were more likely to be 1 diagnosed with peritoneal mesothelioma, 2 female, 3 exposed, and 4 diagnosed at a younger age and to have a 5 shorter latency compared to occupational cases (P < .001. CONCLUSION: Peritoneal mesothelioma was more likely associated with non-occupational exposure, thus emphasizing the importance of exposure history in enhancing early diagnosis and treatment impact.

  5. Making relative survival analysis relatively easy.

    Science.gov (United States)

    Pohar, Maja; Stare, Janez

    2007-12-01

    In survival analysis we are interested in time from the beginning of an observation until certain event (death, relapse, etc.). We assume that the final event is well defined, so that we are never in doubt whether the final event has occurred or not. In practice this is not always true. If we are interested in cause-specific deaths, then it may sometimes be difficult or even impossible to establish the cause of death, or there may be different causes of death, making it impossible to assign death to just one cause. Suicides of terminal cancer patients are a typical example. In such cases, standard survival techniques cannot be used for estimation of mortality due to a certain cause. The cure to the problem are relative survival techniques which compare the survival experience in a study cohort to the one expected should they follow the background population mortality rates. This enables the estimation of the proportion of deaths due to a certain cause. In this paper, we briefly review some of the techniques to model relative survival, and outline a new fitting method for the additive model, which solves the problem of dependency of the parameter estimation on the assumption about the baseline excess hazard. We then direct the reader's attention to our R package relsurv that provides functions for easy and flexible fitting of all the commonly used relative survival regression models. The basic features of the package have been described in detail elsewhere, but here we additionally explain the usage of the new fitting method and the interface for using population mortality data freely available on the Internet. The combination of the package and the data sets provides a powerful informational tool in the hands of a skilled statistician/informatician.

  6. Targeting A20 decreases glioma stem cell survival and tumor growth.

    Directory of Open Access Journals (Sweden)

    Anita B Hjelmeland

    2010-02-01

    Full Text Available Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs. GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3, a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA. Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

  7. Cell Enumeration Assays: Application of the MTT and Sulforhodamine B Assays to Lipopolysaccharide-Stimulated Neonatal Rodent Microglia.

    Science.gov (United States)

    Facci, Laura; Skaper, Stephen D

    2018-01-01

    Glial cell activation, in particular microglia, plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter compares two established cell enumeration assays, namely, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the protein-binding sulforhodamine B assay for microglia as a function of culture condition and activation state. The pros and cons of each are then described.

  8. Effects of depleted uranium on the health and survival of Ceriodaphnia dubia and Hyalella azteca

    Science.gov (United States)

    Kuhne, W.W.; Caldwell, C.A.; Gould, W.R.; Fresquez, P.R.; Finger, S.

    2002-01-01

    Depleted uranium (DU) has been used as a substitute for the fissionable enriched uranium component of atomic weapons tested at Los Alamos National Laboratory (LANL) (Los Alamos, NM, USA) since the early 1950s, resulting in considerable concentrations of DU in the soils within the test sites. Although the movement of DU into major aquatic systems has been shown to be minimal, there are many small-order ephemeral streams and areas of standing water in canyons throughout LANL that may be affected by inputs of DU via runoff, erosion, and leaching. Ninety-six-hour acute and 7-d chronic toxicity assays were conducted to measure the toxicity of DU on survival and reproduction of Ceriodaphnia dubia. A 14-d water-only assay was conducted to measure survival and growth of Hyalella azteca. The estimated median lethal concentration (LC50) to produce 50% mortality of the test population for the 96-h Ceriodaphnia dubia assay was 10.50 mg/L. Reproductive effects occurred at a lowest-observable-effect concentration ???3.91 mg/L with a no-observable-effect concentration of 1.97 mg/L. The estimated 14-d LC50 for the Hyalella azteca assay was 1.52 mg/L No significant relationship was detected between growth and DU concentrations. Concentrations at which toxicity effects were observed in this study for both invertebrates exceeded concentrations of total uranium observed in runoff from LANL lands. Thus, it is likely that current runoff levels of uranium do not pose a threat to these types of aquatic invertebrates.

  9. Model selection criterion in survival analysis

    Science.gov (United States)

    Karabey, Uǧur; Tutkun, Nihal Ata

    2017-07-01

    Survival analysis deals with time until occurrence of an event of interest such as death, recurrence of an illness, the failure of an equipment or divorce. There are various survival models with semi-parametric or parametric approaches used in medical, natural or social sciences. The decision on the most appropriate model for the data is an important point of the analysis. In literature Akaike information criteria or Bayesian information criteria are used to select among nested models. In this study,the behavior of these information criterion is discussed for a real data set.

  10. Evaluating survival model performance: a graphical approach.

    Science.gov (United States)

    Mandel, M; Galai, N; Simchen, E

    2005-06-30

    In the last decade, many statistics have been suggested to evaluate the performance of survival models. These statistics evaluate the overall performance of a model ignoring possible variability in performance over time. Using an extension of measures used in binary regression, we propose a graphical method to depict the performance of a survival model over time. The method provides estimates of performance at specific time points and can be used as an informal test for detecting time varying effects of covariates in the Cox model framework. The method is illustrated on real and simulated data using Cox proportional hazard model and rank statistics. Copyright 2005 John Wiley & Sons, Ltd.

  11. A stochastic evolutionary model for survival dynamics

    Science.gov (United States)

    Fenner, Trevor; Levene, Mark; Loizou, George

    2014-09-01

    The recent interest in human dynamics has led researchers to investigate the stochastic processes that explain human behaviour in different contexts. Here we propose a generative model to capture the essential dynamics of survival analysis, traditionally employed in clinical trials and reliability analysis in engineering. In our model, the only implicit assumption made is that the longer an actor has been in the system, the more likely it is to have failed. We derive a power-law distribution for the process and provide preliminary empirical evidence for the validity of the model from two well-known survival analysis data sets.

  12. Body mass index and breast cancer survival

    DEFF Research Database (Denmark)

    Guo, Qi; Burgess, Stephen; Turman, Constance

    2017-01-01

    Background: There is increasing evidence that elevated body mass index (BMI) is associated with reduced survival for women with breast cancer. However, the underlying reasons remain unclear. We conducted a Mendelian randomization analysis to investigate a possible causal role of BMI in survival...... from breast cancer. Methods: We used individual-level data from six large breast cancer case-cohorts including a total of 36 210 individuals (2475 events) of European ancestry. We created a BMI genetic risk score (GRS) based on genotypes at 94 known BMI-associated genetic variants. Association between...

  13. Improved survival after rectal cancer in Denmark

    DEFF Research Database (Denmark)

    Bülow, S; Harling, H; Iversen, L H

    2010-01-01

    treated from 1994 to 2006. Method The study was based on the National Rectal Cancer Registry and the National Colorectal Cancer Database, supplemented with data from the Central Population Registry. The analysis included actuarial overall and relative survival. Results A total of 10 632 patients were......Objective In 1995, an analysis showed an inferior prognosis after rectal cancer in Denmark compared with the other Scandinavian countries. The Danish Colorectal Cancer Group (DCCG) was established with the aim of improving the prognosis, and in this study we present a survival analysis of patients...

  14. Graphics and statistics for cardiology: survival analysis.

    Science.gov (United States)

    May, Susanne; McKnight, Barbara

    2017-03-01

    Reports of data in the medical literature frequently lack information needed to assess the validity and generalisability of study results. Some recommendations and standards for reporting have been developed over the last two decades, but few are available specifically for survival data. We provide recommendations for tabular and graphical representations of survival data. We argue that data and analytic software should be made available to promote reproducible research. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  15. Multiple neoplasms, single primaries, and patient survival

    Directory of Open Access Journals (Sweden)

    Amer MH

    2014-03-01

    Full Text Available Magid H Amer Department of Medicine, St Rita's Medical Center, Lima, OH, USA Background: Multiple primary neoplasms in surviving cancer patients are relatively common, with an increasing incidence. Their impact on survival has not been clearly defined. Methods: This was a retrospective review of clinical data for all consecutive patients with histologically confirmed cancer, with emphasis on single versus multiple primary neoplasms. Second primaries discovered at the workup of the index (first primary were termed simultaneous, if discovered within 6 months of the index primary were called synchronous, and if discovered after 6 months were termed metachronous. Results: Between 2005 and 2012, of 1,873 cancer patients, 322 developed second malignancies; these included two primaries (n=284, and three or more primaries (n=38. Forty-seven patients had synchronous primaries and 275 had metachronous primaries. Patients with multiple primaries were predominantly of Caucasian ancestry (91.0%, with a tendency to develop thrombosis (20.2%, had a strong family history of similar cancer (22.3%, and usually presented with earlier stage 0 through stage II disease (78.9%. When compared with 1,551 patients with a single primary, these figures were 8.9%, 15.6%, 18.3%, and 50.9%, respectively (P≤0.001. Five-year survival rates were higher for metachronous cancers (95% than for synchronous primaries (59% and single primaries (59%. The worst survival rate was for simultaneous concomitant multiple primaries, being a median of 1.9 years. The best survival was for patients with three or more primaries (median 10.9 years and was similar to the expected survival for the age-matched and sex-matched general population (P=0.06991. Conclusion: Patients with multiple primaries are usually of Caucasian ancestry, have less aggressive malignancies, present at earlier stages, frequently have a strong family history of similar cancer, and their cancers tend to have indolent

  16. The fitness consequences of honesty: Under-signalers have a survival advantage in song sparrows.

    Science.gov (United States)

    Akçay, Çağlar; Campbell, S Elizabeth; Beecher, Michael D

    2015-12-01

    How honest or reliable signaling can evolve and be maintained has been a major question in evolutionary biology. The question is especially puzzling for a particular class of signals used in aggressive interactions: threat signals. Here, we report a study on song sparrows (Melospiza melodia) in which we assayed males with playbacks on their territories to quantify their aggressiveness (flights and close proximity) and aggressive signaling levels (rates of soft song, a close-range signal reliably predicting attack) and asked whether these traits affect individuals' survival on territory. We found that the effect of aggressive signaling via soft song interacted with aggressive behaviors such that there was a negative correlational selection: among males with low aggression, those males that signaled at higher levels (over-signalers) had higher survival whereas among males with high aggression those that signaled at low levels (under-signalers) survived longer. In other words, males that deviate from reliable signaling have a survival advantage. These results, along with previous research that suggested most of the deviation from reliable signaling in this system is in the form of under-signaling (high-aggression males signaling at low levels) pose a puzzle for future research on how this reliable signaling system is maintained. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

  17. [Evaluation of Abbott Fourth Generation HIV Antigen and Antibody Assays.].

    Science.gov (United States)

    Kang, Hee Jung; Yoo, Kyeong Ha; Kim, Han Sung; Cho, Hyoun Chan

    2006-02-01

    In order to reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and serological diagnosis, new fourth generation screening assays which detect HIV p24 antigen and specific antibody simultaneously have been developed. In this study, we evaluated the performance of a new fourth generation assay. We compared a new fourth generation assay, Architect HIV Ag/Ab combo, with another fourth generation assay AxSYM HIV Ag/Ab combo and a third generation assay, AxSYM HIV 1/2 gO for their performance. The assays were evaluated using 3 HIV seroconversion panels, 305 sera of healthy subjects and 100 sera of patients with HBsAg or anti-HCV antibodies. Within-run and total coefficient variations of the three screening assays were analyzed for the evaluation of precision. Architect HIV Ag/Ab combo shortened the window period by 8.7+/-2.1 days relative to AxSYM HIV 1/2 gO and 2.0+/-2.0 days relative to AxSYM HIV Ag/Ab combo in seroconversion panels. Architect HIV Ag/Ab combo presented the best performance in precision among the three reagents; total CV for positive control was 3.6%, 9.6% and 4.6% for Architect HIV Ag/Ab combo, AxSYM HIV Ag/Ab combo and AxSYM HIV 1/2 gO, respectively. Specificities of three assays were not different in this study. HIV Ag/Ab combined assays reduced the diagnostic window as compared to the third generation screening assays, enabling an earlier diagnosis of HIV infection. A new fourth generation assay, Architect HIV Ag/Ab combo presents a better performance than AxSYM HIV Ag/Ab combo, showing improved seroconversion sensitivity and precision.

  18. Assay optimization for molecular detection of Zika virus

    Science.gov (United States)

    Corman, Victor M; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal BEM; Pas, Suzan D; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M; Koopmans, Marion P; Schmidt-Chanasit, Jonas; Grobusch, Martin P; de Lamballerie, Xavier; Drosten, Christian

    2016-01-01

    Abstract Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. Findings Oligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine. Conclusion We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results. PMID:27994281

  19. Survival during the Breeding Season: Nest Stage, Parental Sex, and Season Advancement Affect Reed Warbler Survival.

    Directory of Open Access Journals (Sweden)

    Kaja Wierucka

    Full Text Available Avian annual survival has received much attention, yet little is known about seasonal patterns in survival, especially of migratory passerines. In order to evaluate survival rates and timing of mortality within the breeding season of adult reed warblers (Acrocephalus scirpaceus, mark-recapture data were collected in southwest Poland, between 2006 and 2012. A total of 612 individuals (304 females and 308 males were monitored throughout the entire breeding season, and their capture-recapture histories were used to model survival rates. Males showed higher survival during the breeding season (0.985, 95% CI: 0.941-0.996 than females (0.869, 95% CI: 0.727-0.937. Survival rates of females declined with the progression of the breeding season (from May to August, while males showed constant survival during this period. We also found a clear pattern within the female (but not male nesting cycle: survival was significantly lower during the laying, incubation, and nestling periods (0.934, 95% CI: 0.898-0.958, when birds spent much time on the nest, compared to the nest building and fledgling periods (1.000, 95% CI: 1.00-1.000, when we did not record any female mortality. These data (coupled with some direct evidence, like bird corpses or blood remains found next to/on the nest may suggest that the main cause of adult mortality was on-nest predation. The calculated survival rates for both sexes during the breeding season were high compared to annual rates reported for this species, suggesting that a majority of mortality occurs at other times of the year, during migration or wintering. These results have implications for understanding survival variation within the reproductive period as well as general trends of avian mortality.

  20. Patulin triggers NRF2-mediated survival mechanisms in kidney cells.

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    Pillay, Y; Phulukdaree, A; Nagiah, S; Chuturgoon, A A

    2015-06-01

    Patulin (PAT), a mycotoxin contaminant of apples and apple products, has been implicated in nephrotoxicity. PAT depletes glutathione (GSH) and elevates reactive oxygen species (ROS). The antioxidant (AO) response is activated by Nuclear erythroid 2-related factor (NRF2) and enhanced by Silent information regulator 3 (SIRT3). The effects of PAT on these molecules have yet to be examined. We investigated the effects of PAT on AO response survival pathways in human embryonic kidney cells (HEK293). PAT cytotoxicity on HEK293 cells was evaluated (MTT assay; 24 h; [0-100 μM]) to determine an IC50. GSH levels were measured using luminometry. Intracellular ROS was evaluated by flow cytometry. Protein expression of Keap1, NRF2, SIRT3 and PGC-1α was quantified by western blotting and gene expression of SOD2, CAT and GPx was evaluated by qPCR. PAT caused a dose dependent decrease in HEK293 cell viability and a significant increase in levels of intracellular ROS (p = 0.0006). A significant increase in protein expression (p = 0.029) was observed. PAT increased gene expression of SOD2 and CAT (p = 0.0043), however, gene expression of GPx was significantly reduced (p = 0.0043). These results show the up-regulation of NRF2 mediated AO mechanisms in response to PAT toxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.