Sample records for two-hybrid assays showed

  1. Estrogenic/Antiestrogenic Activities of Polycyclic Aromatic Hydrocarbons and Their Monohydroxylated Derivatives by Yeast Two-Hybrid Assay


    Hayakawa, Kazuichi; Onoda, Yu; Tachikawa, Chihiro; Hosoi, Shinzo; Yoshita, Morio; Chung, Sang Woon; Kizu, Ryoichi; Toriba, Akira; Kameda, Takayuki; Tang, Ning


    Estrogenic/antiestrogenic activities of 14 polycyclic aromatic hydrocarbons (PAHs) and 63 monohydroxylated PAHs (OHPAHs) having 2 to 6 rings were evaluated by yeast two-hybrid assay expressing human estrogen receptor α. Relative effective potencies of estrogenic and antiestrogenic activities were calculated as the inverse values of the relative concentration of the test compound that gave the same activities of E2 and 4-hydroxytamoxifen, respectively. PAHs did not show any estrogenic/antiestr...

  2. Determination of estrogen receptor {beta}-mediated estrogenic potencies of hydroxylated PCBS by a yeast two-hybrid assay

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    Kuroki, H.; Kumate, M.; Nakaoka, H.; Yonekura, S. [Daiichi Coll. of Pharmaceutical Sciences, Fukuoka (Japan); Nishikawa, J.; Nishihara, T. [Osaka Univ., Osaka (Japan)


    Several environmental phenolic chemicals such as Nonylphenol and Bisphenol A (BPA) have been previously shown to possess estrogenic properties. In the previous paper, we have investigated the estrogenic activity of a series of hydroxylated PCBs (OH-PCBs) by a yeast two-hybrid assay (estrogen receptor{alpha} (ER{alpha}) -TIF2), in which the expression of estrogenic activity is based on the interaction of chemicals with ER{alpha}, and demonstrated that 4'-OH-CB30 and 4'-OH-CB61 are more estrogenic than BPA, one of the environmental estrogens. We have showed that one chlorine substitution adjacent to 4-OH at 3- or 5-position significantly reduces the ER{alpha}-mediated estrogenic activity of 4-OH-PCBs. Thus, 4'-OH-CB25 and 4-OH-CB56 showed a very weak estrogenicity. We have also showed that 4-OH-PCBs with two chlorine substitutions adjacent to 4-OH at 3- and 5-position such as 4'-OH-CB79 (hydroxylated metabolite of CB77) and persistent 4-OH-PCBs retained in human blood (4-OH-CB107, 4-OH-CB146 and 4-OH-CB187) have no ER{alpha}-mediated estrogenic activity. ER is known to have two subtypes, namely ER{alpha} and ER{beta} and it is reported that ligand, some agonist and antagonist have a different binding affinity for ER{alpha} and ER{beta}. However, there is limited information on ER{beta}-mediated endocrine disrupting potency. In this study, we examined the ER{beta}-mediated estrogenic activity of a series of OH-PCBs, including environmentally relevant 4-OH-PCBs by a yeast two-hybrid assay (ER{beta}-TIF2).

  3. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

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    Bloch, Donald B., E-mail:; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong


    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  4. Screening of agonistic activities against four nuclear receptors in wastewater treatment plants in Japan using a yeast two-hybrid assay

    Institute of Scientific and Technical Information of China (English)

    Daisuke Inoue; Koki Nakama; Kazuko Sawada; Taro Watanabe; Hisae Matsui; Kazunari Sei; Tsuyoshi Nakanishi; Michihiko Ike


    To assess the potential endocrine disruptive effects through multiple nuclear receptors (NRs), especially non-steroidal NRs, in municipal wastewater, we examined the agonistic activities on four NRs (estrogen receptor c, thyroid hormone receptor α, retinoic acid receptor α and retinoid X receptor α) of untreated and treated wastewater from municipal wastewater treatment plants (WWTPs) in Japan using a yeast two-hybrid assay.Investigation of the infiuent and effluent of seven WWTPs revealed that agonistic activities against steroidal and non-steroidal NRs were always detected in the infiuents and partially remained in the effluents.Further investigation of four WWTPs employing conventional activated sludge, pseudo-anoxic-oxic, anoxic-oxic and anaerobic-anoxic-oxic processes revealed that the ability to reduce the agonistic activity against each of the four NRs varies depending on the treatment process.These results indicated that municipal wastewater in Japan commonly contains endocrine disrupting chemicals that exert agonistic activities on steroidal and non-steroidal NRs, and that some of these chemicals are released into the natural aquatic environment.Although the results obtained in yeast assays suggested that measured levels of non-steroidal NR agonists in the effluent of WWTPs were not likely to cause any biological effect, further study is required to assess their possible risks in detail.

  5. Yeast two-hybrid screen. (United States)

    Makuch, Lauren


    Yeast two-hybrid is a method for screening large numbers of gene products (encoded by cDNA libraries) for their ability to interact with a protein of interest. This system can also be used for characterizing and manipulating candidate protein: protein interactions. Interactions between proteins are monitored by the growth of yeast plated on selective media.

  6. LA PROTEÍNA PTHB DE Xanthomonas axonopodis pv. Manihotis ES AUTOACTIVA EN ENSAYOS DE DOBLE HÍBRIDO The PthB Protein from Xanthomonas axonopodis pv. Manihotis is an Autoactive in Yeast Two-Hybrid Assays

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    the yeast two hybrid expression vector pLAW10, generating a fusion protein with the Binding Domain (BD of the transcription factor GAL4. In this work, PthB was cloned in a translational fusion with Gal4-BD (DNA Binding Domain. After transforming this construct into a yeast strain, autoactivation of the reporter genes was observed, even at the highest concentrations of 3-AT. The deletion of the first, second or both NLS and the AAD did not eliminate the ability of autoactivation of PthB. These results show the impossibility of using PthB to screen a cassava cDNA library to identify the proteins interacting with PthB.

  7. Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

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    González-Méndez Ricardo


    Full Text Available Abstract Background Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins. Results Using the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD, a glyceraldehyde-3-P dehydrogenase (GAPDH and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment. Conclusions This study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association

  8. Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease (United States)

    Li, Quan; Henry, Eric R.; Hofrichter, James; Smith, Jeffrey F.; Cellmer, Troy; Dunkelberger, Emily B.; Metaferia, Belhu B.; Jones-Straehle, Stacy; Boutom, Sarah; Christoph, Garrott W.; Wakefield, Terri H.; Link, Mary E.; Staton, Dwayne; Vass, Erica R.; Miller, Jeffery L.; Hsieh, Matthew M.; Tisdale, John F.; Eaton, William A.


    Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort (“sickle”) the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms. PMID:28096387

  9. [Identification of C(2)M interacting proteins by yeast two-hybrid screening]. (United States)

    Shanshan, Yue; Laixin, Xia


    The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

  10. Novel Luminex Assay for Telomere Repeat Mass Does Not Show Well Position Effects Like qPCR.

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    Muhammad G Kibriya

    Full Text Available Telomere length is a potential biomarker of aging and risk for age-related diseases. For measurement of relative telomere repeat mass (TRM, qPCR is typically used primarily due to its low cost and low DNA input. But the position of the sample on a plate often impacts the qPCR-based TRM measurement. Recently we developed a novel, probe-based Luminex assay for TRM that requires ~50ng DNA and involves no DNA amplification. Here we report, for the first time, a comparison among TRM measurements obtained from (a two singleplex qPCR assays (using two different primer sets, (b a multiplex qPCR assay, and (c our novel Luminex assay. Our comparison is focused on characterizing the effects of sample positioning on TRM measurement. For qPCR, DNA samples from two individuals (K and F were placed in 48 wells of a 96-well plate. For each singleplex qPCR assay, we used two plates (one for Telomere and one for Reference gene. For the multiplex qPCR and the Luminex assay, the telomere and the reference genes were assayed from the same well. The coefficient of variation (CV of the TRM for Luminex (7.2 to 8.4% was consistently lower than singleplex qPCR (11.4 to 14.9% and multiplex qPCR (19.7 to 24.3%. In all three qPCR assays the DNA samples in the left- and right-most columns showed significantly lower TRM than the samples towards the center, which was not the case for the Luminex assay (p = 0.83. For singleplex qPCR, 30.5% of the variation in TL was explained by column-to-column variation and 0.82 to 27.9% was explained by sample-to-sample variation. In contrast, only 5.8% of the variation in TRM for the Luminex assay was explained by column-to column variation and 50.4% was explained by sample-to-sample variation. Our novel Luminex assay for TRM had good precision and did not show the well position effects of the sample that were seen in all three of the qPCR assays that were tested.

  11. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. (United States)

    James, P; Halladay, J; Craig, E A


    The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

  12. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast. (United States)

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing


    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  13. Cytochrome C oxidase Ⅲ interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Dan Li; Xiao-Zhong Wang; Jie-Ping Yu; Zhi-Xin Chen; Yue-Hong Huang; Qi-Min Tao


    AIM: To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC).METHODS: With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with β-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified,sequenced and analyzed with bioinformatics. Mating experiment was peformed to confirm the binding of putative proteins to X protein in the yeast cells.RESULTS: Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through β-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase Ⅲ(COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific.CONCLUSION: COXIII protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system,and may contribute to the development of HCC through the interaction with X protein.

  14. Yeast Two-Hybrid: State of the Art

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    Van Criekinge Wim


    Full Text Available Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination.

  15. Media composition influences yeast one- and two-hybrid results

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    Gonzalez Kim L


    Full Text Available Abstract Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.

  16. 与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析%Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library

    Institute of Scientific and Technical Information of China (English)

    任高伟; 崔鑫; 马艳萍; 齐莹; 阮强; 孙峥嵘


    Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.%目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1

  17. Using the Two-Hybrid Screen in the Classroom Laboratory


    Odom, Daniel P.; Grossel, Martha J


    The National Science Foundation and others have made compelling arguments that research be incorporated into the learning of undergraduates. In response to these arguments, a two-hybrid research project was incorporated into a molecular biology course that contained both a lecture section and a laboratory section. The course was designed around specific goals for educational outcomes, including introducing research to a wide range of students, teaching students experimental design and data an...

  18. Precision and recall estimates for two-hybrid screens. (United States)

    Huang, Hailiang; Bader, Joel S


    Yeast two-hybrid screens are an important method to map pairwise protein interactions. This method can generate spurious interactions (false discoveries), and true interactions can be missed (false negatives). Previously, we reported a capture-recapture estimator for bait-specific precision and recall. Here, we present an improved method that better accounts for heterogeneity in bait-specific error rates. For yeast, worm and fly screens, we estimate the overall false discovery rates (FDRs) to be 9.9%, 13.2% and 17.0% and the false negative rates (FNRs) to be 51%, 42% and 28%. Bait-specific FDRs and the estimated protein degrees are then used to identify protein categories that yield more (or fewer) false positive interactions and more (or fewer) interaction partners. While membrane proteins have been suggested to have elevated FDRs, the current analysis suggests that intrinsic membrane proteins may actually have reduced FDRs. Hydrophobicity is positively correlated with decreased error rates and fewer interaction partners. These methods will be useful for future two-hybrid screens, which could use ultra-high-throughput sequencing for deeper sampling of interacting bait-prey pairs. All software (C source) and datasets are available as supplemental files and at under the Lesser GPL v. 3 license.

  19. Yeast Two-Hybrid, a Powerful Tool for Systems Biology

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    Uwe Schlattner


    Full Text Available A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

  20. Tuberculin skin test and interferon-γ release assay show better correlation after the tuberculin 'window period' in tuberculosis contacts. (United States)

    Anibarro, Luis; Trigo, Matilde; Villaverde, Carlos; Pena, Alberto; González-Fernández, Africa


    T-cell interferon-gamma release assays (IGRAs) have been shown to be effective tools for the detection of Mycobacterium tuberculosis infection, offering an enhanced specificity compared to the tuberculin skin test (TST). Most tuberculosis (TB) contact studies have shown a better correlation of IGRA with the intensity of M. tuberculosis exposure than that obtained using the TST. However, the correlation between tests performed before and after the tuberculin 'window period' (time between infection and when the immunological response becomes measurable) remains to be studied. A longitudinal prospective analysis was performed in TB contacts. We analyzed the correlation between a commercially available IGRA (QuantiFERON®-TB Gold in-Tube, QFT) and the TST before and after the tuberculin window period (2 months). Concordance between both tests was assessed using the Kappa coefficient (κ). Correlation of both tests with the degree of TB exposure was also analyzed. One hundred and fifty-two TB contacts were included in the study. Agreement between the TST and IGRA was better after the window period (κ = 0.60 at the first visit and κ = 0.73 after 2 months), especially for non-BCG vaccinated subjects (κ = 0.81). Both a positive TST and QFT were correlated, after the window period, with the size of place of contact (the smaller the place of contact, the higher the probability of having a positive test) (p = 0.022 and p = 0.02, respectively) and with the total numbers of hours spent with the index case (p = 0.006 for TST and p = 0.007 for QFT). IGRAs are a good alternative to the TST in contact tracing studies, especially after the tuberculin window period.

  1. A non-invasive stress assay shows that tadpole populations infected with Batrachochytrium dendrobatidis have elevated corticosterone levels.

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    Caitlin R Gabor

    Full Text Available Batrachochytrium dendrobatidis (Bd is a fungus that causes the disease chytridiomycosis and is associated with widespread amphibian declines. Populations vary in their susceptibility to Bd infections, and the virulence of the infecting lineage can also vary. Both of these factors may manifest as a differential physiological stress response. In addition, variation in disease susceptibility across amphibian populations may be influenced by immunosuppression caused by chronic stress imposed by environmental factors. Here, we use a non-invasive water-borne hormone technique to assess stress levels (corticosterone of free-living tadpole populations that are infected by Bd. We found that corticosterone release rates were higher in infected populations of two species of tadpoles (Alytes obstetricans and A. muletensis than in an uninfected population for both species. The relationship between corticosterone and the intensity of infection differed between species, with only the infected A. obstetricans population showing a significant positive correlation. The higher corticosterone release rates found in A. obstetricans may be an outcome of infection by a highly virulent lineage of Bd (BdGPL, whereas A. muletensis is infected with a less virulent lineage (BdCAPE. These results suggest that different lineages of Bd impose different levels of stress on the infected animals, and that this may influence survival. The next step is to determine whether higher corticosterone levels make individuals more susceptible to Bd or if Bd infections drive the higher corticosterone levels.

  2. Characterization of single chain antibody targets through yeast two hybrid

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    Vielemeyer Ole


    Full Text Available Abstract Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID, efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise

  3. Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

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    Cataldi Angel A


    Full Text Available Abstract Background The exported repetitive protein (erp gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. Results In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC. The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. Conclusion We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.

  4. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐兵; 齐义鹏; Masuo; Yutsudo; 刘青珍


    asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of

  5. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)


    asy gene is a novel apoptosis-inducing gene,but its mechanism is unclear.To investigate the mechanism of asy inducing apoptosis,a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system.The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels.Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end,and share a common open reading frame encoding a polypeptide of 236 amino acids.Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence.Two highly hydrophobic regions encoding potentially two transmembrane domains are present.The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu).Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells.Compared with asy gene,overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

  6. Yeast two-hybrid screening for proteins that interact with α1-adrenergic receptors

    Institute of Scientific and Technical Information of China (English)

    TanZHANG; QiXU; Feng-rongCHEN; Qi-deHAN; You-yiZHANG


    AIM: To find novel proteins that may bind to α1A-adrenergic receptor (α1A-AR) and investigate their interactions with the other two α1-AR subtypes (α1B-AR and α1D-AR) with an expectation to provide new leads for the function study of the receptors. METHODS: Yeast two-hybrid assay was performed to screen a human brain cDNA library using the C terminus of α1A-AR (α1A-AR-CT) as bait. X-Gal assay and o-nitrophenyl-beta-D-galactopyranoside (ONPG) assay were subsequently conducted to further qualitatively or quantitatively confirm the interactions between receptors and the three identified proteins. RESULTS: (1) Selection medium screening identified segments of bone morphogenetic protein-1 (BMP-1), active Bcr-related protein (Abr), and filamin-C as binding partners of α1A-AR-CT in yeast cells respectively. Besides, protein segments of BMP-1 and Abr could only specifically interact with α1A-AR-CT while filamin-C segment interacted with all three α1-AR subtypes. (2) In X-Gal assay, the cotransformants of α1A-AR-CT and BMP-1 segments turned strong blue at about 30 min while other positive transformants only developed weak blue at about 5-6 h. (3) In ONPG assay, interaction (shown in β-galactosidase activity) between α1A-AR-CT and BMP-1 segments was about 30 times stronger than that of control (P<0.01), while other positive interactions were only about 2-5 times as strong as those of controls (P<0.05). CONCLUSION: In yeast cells BMP-1, Abr and/or filamin-C could interact with three α1-AR subtypes, among which, interaction between BMP-1 and α1A-AR was the strongest while other interactions between proteins and receptors were relatively weak.

  7. Yeast two-hybrid screening for proteins that interact with α1-adrenergic receptors

    Institute of Scientific and Technical Information of China (English)

    Tan ZHANG; Qi XU; Feng-rong CHEN; Qi-de HAN; You-yi ZHANG


    AIM: To find novel proteins that may bind to α1A-adrenergic receptor (α1A-AR) and investigate their interactions with the other two α1-AR subtypes (α1B-AR and α1D-AR) with an expectation to provide new leads for the function study of the receptors. METHODS: Yeast two-hybrid assay was performed to screen a human brain cDNA library using the C terminus of α1A-AR (α1A-AR-CT) as bait. X-Gal assay and o-nitrophenyl-beta-D-galactopyranoside(ONPG) assay were subsequently conducted to further qualitatively or quantitatively confirm the interactions between receptors and the three identified proteins. RESULTS: (1) Selection medium screening identified segments of bone morphogenetic protein-1 (BMP-1), active Bcr-related protein (Abr), and filamin-C as binding partners ofα1A-AR-CT in yeast cells respectively. Besides, protein segments of BMP-1 and Abr could only specifically interact with α1A-AR-CT while filamin-C segment interacted with all three α1-AR subtypes. (2) In X-Gal assay, the cotransformants of α1A-AR-CT and BMP-1 segments turned strong blue at about 30 min while other positive transformants only developed weak blue at about 5-6 h. (3) In ONPG assay, interaction (shown in β-galactosidase activity) between α1A-AR-CT and BMP-1 segments was about 30 times stronger than that of control (P<0.01),while other positive interactions were only about 2-5 times as strong as those of controls (P<0.05). CONCLUSION:In yeast cells BMP-1, Abr and/or filamin-C could interact with three α1-AR subtypes, among which, interaction between BMP-1 and α1A-AR was the strongest while other interactions between proteins and receptors were relatively weak.

  8. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications (United States)

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.


    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  9. An improved yeast two-hybrid approach for detection of interacting proteins

    Institute of Scientific and Technical Information of China (English)

    Wan Bingbing; Shi Yan; Huo Keke


    Yeast two-hybrid approach is popularly used nowadays as an important technical method in the field of studying protein-protein interactions.Although yeast two-hybrid system is obviously advantageous in searching interacting proteins and setting up the network of proteins interaction.not all of proteins can use routine yeast two-hybrid method to search interacting proteins.Many important proteins,such as some nucleoprotein transcriptional factor,carry out the regular method and construct the bait-BD vector to screen the library containing AD vector.However,it usually results in failures because it contains the activate domain and can self-activate the reporter gene.In this study,we changed the research strategy,fused the bait gene(FOXA3)with the AD vector to screen the library containing BD vector,so that we constructed a two-hybrid library containing BD vector and Can bypass the interference of self-activation.And we used this two-hybrid library to screen FOXA3.a hepatocyte nuclear factor,and found out an interacting protein:complement component C3.

  10. Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

    Institute of Scientific and Technical Information of China (English)

    石晓冰; 魏家绵; 沈允钢


    Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast

  11. Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

    Directory of Open Access Journals (Sweden)

    Gonzalez-Mendez Ricardo


    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

  12. Screening of the interacting proteins with NifA in Azospirillum brasilense Sp7 by the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    CHEN Sanfeng; GUAN Yu; TU Ran; SUN Wengai; LI Jilun


    NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in response to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones encode proteins containing PAS domains that play an important role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.

  13. The bacterial two-hybrid system uncovers the involvement of acetylation in regulating of Lrp activity in Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ran Qin


    Full Text Available Nε-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat - or deacetylase CobB-mediated acetylation. Then, the in vitro (deacetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36 in helix-turn-helix (HTH DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

  14. The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium (United States)

    Qin, Ran; Sang, Yu; Ren, Jie; Zhang, Qiufen; Li, Shuxian; Cui, Zhongli; Yao, Yu-Feng


    N𝜀-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat – or deacetylase CobB-mediated acetylation. Then, the in vitro (de)acetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36) in helix-turn-helix (HTH) DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes. PMID:27909434

  15. A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression. (United States)

    Quiñones-Mateu, M E; Ball, S C; Marozsan, A J; Torre, V S; Albright, J L; Vanham, G; van Der Groen, G; Colebunders, R L; Arts, E J


    This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

  16. Interaction of hTCF4 by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)


    To investigate the interaction of hTCF4, the yeast two-hybrid system has been used for testing the interaction of mutants of hTCF4 with themselves. Mutants of hTCF4 (hTCF4Ⅰ) and hTCF4Ⅱ) have been obtained by polymerase chain reaction (PCR). Bait (hTCF4Ⅰ-pDBLeu and hTCF4Ⅱ-pDBLeu) and prey (hTCF4Ⅰ-pPC86 and hTCF4Ⅱ-pPC86) have been constructed by DNA recombination for yeast two-hybrid. Interaction of hTCF4Ⅱ with itself is found in the reverse yeast two-hybrid system (GIBCOBRL Co.). However, no interactions are found in hTCF4Ⅱ with hTCF4Ⅰand in hTCF4Ⅰ with hTCF4Ⅰ. These results suggest that hTCF4 could interact with itself to form homodimer or homocopolymer and perform the transcriptional activating function through LZ or HLH motifs in nucleus of renal cell carcinoma.

  17. Degradation of Acid Orange 7 Dye in Two Hybrid Plasma Discharge Reactors

    Institute of Scientific and Technical Information of China (English)

    SHEN Yongjun; LEI Lecheng; ZHANG Xingwang; DING Jiandong


    To get an optimized pulsed electrical plasma discharge reactor and to increase the energy utilization efficiency in the removal of pollutants,two hybrid plasma discharge reactors were designed and optimized.The reactors were compared via the discharge characteristics,energy transfer efficiency,the yields of the active species and the energy utilization in dye wastewater degradation.The results showed that under the same AC input power,the characteristics of the discharge waveform of the point-to-plate reactor were better.Under the same AC input power,the two reactors both had almost the same peak voltage of 22 kV.The peak current of the point-to-plate reactor was 146 A,while that of the wire-to-cylinder reactor was only 48.8 A.The peak powers of the point-to-plate reactor and the wire-to-cylinder reactor were 1.38 MW and 1.01 MW,respectively.The energy per pulse of the point-to-plate reactor was 0.2221 J,which was about 29.4% higher than that of the wire-to-cylinder reactor (0.1716 J).To remove 50% Acid Orange 7 (AO7),the energy utilizations of the point-to-plate reactor and the wireto-cylinder reactor were 1.02×10-9 mol/L and 0.61×10-9 mol/L,respectively.In the point-to-plate reactor,the concentration of hydrogen peroxide in pure water was 3.6 mmol/L after 40 min of discharge,which was higher than that of the wire-to-cylinder reactor (2.5 mmol/L).The concentration of liquid phase ozone in the point-to-plate reactor (5.7×10-2 mmol/L) was about 26.7% higher than that in the wire-to-cylinder reactor (4.5× 10-2 mmol/L).The analysis results of the variance showed that the type of reactor and reaction time had significant impacts on the yields of the hydrogen peroxide and ozone.The main degradation intermediates of AO7 identified by gas chromatography and mass spectrometry (GCMS) were acetic acid,maleic anhydride,pbenzoquinone,phenol,benzoic acid,phthalic anhydride,coumarin and 2-naphthol.Proposed degradation pathways were elucidated in light of the analyzed

  18. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    Directory of Open Access Journals (Sweden)

    Peter Kusch


    Full Text Available Balanites aegyptiaca (Balanitaceae is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 g/L±3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a of 2.35 g/L and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a of 4.8 g/L. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 M±3 and 47.82 M±2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed.

  19. pISTil: a pipeline for yeast two-hybrid Interaction Sequence Tags identification and analysis

    Directory of Open Access Journals (Sweden)

    de Chassey Benoît


    Full Text Available Abstract Background High-throughput screening of protein-protein interactions opens new systems biology perspectives for the comprehensive understanding of cell physiology in normal and pathological conditions. In this context, yeast two-hybrid system appears as a promising approach to efficiently reconstruct protein interaction networks at the proteome-wide scale. This protein interaction screening method generates a large amount of raw sequence data, i.e. the ISTs (Interaction Sequence Tags, which urgently need appropriate tools for their systematic and standardised analysis. Findings We develop pISTil, a bioinformatics pipeline combined with a user-friendly web-interface: (i to establish a standardised system to analyse and to annotate ISTs generated by two-hybrid technologies with high performance and flexibility and (ii to provide high-quality protein-protein interaction datasets for systems-level approach. This pipeline has been validated on a large dataset comprising more than 11.000 ISTs. As a case study, a detailed analysis of ISTs obtained from yeast two-hybrid screens of Hepatitis C Virus proteins against human cDNA libraries is also provided. Conclusion We have developed pISTil, an open source pipeline made of a collection of several applications governed by a Perl script. The pISTil pipeline is intended to laboratories, with IT-expertise in system administration, scripting and database management, willing to automatically process large amount of ISTs data for accurate reconstruction of protein interaction networks in a systems biology perspective. pISTil is publicly available for download at

  20. Screening of FOXP3-interacted proteins by yeast two-hybrid technique

    Institute of Scientific and Technical Information of China (English)

    Zhou Lina; Wu Jun; Luo Gaoxing; He Weifeng; Chen Xiwei; Bo Ganping; Yuan Shunzong; Zhang Xiaorong; Hu Xiaohong


    Objective: To screen the proteins interacting with the Treg specification factor forkhead box protein P3 (FOXP3) by yeast two-hybrid system. Methods: Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells (PBMC) and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results: The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Conclusion: Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.

  1. Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

    Institute of Scientific and Technical Information of China (English)

    Qian Yang; Jun Cheng; Jing Dong; Jian Zhang; Shu-Lin Zhang


    AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.

  2. Screening for cardiac HERG potassium channel interacting proteins using the yeast two-hybrid technique. (United States)

    Ma, Qingyan; Yu, Hong; Lin, Jijin; Sun, Yifan; Shen, Xinyuan; Ren, Li


    The human ERG protein (HERG or Kv 11.1) encoded by the human ether-a-go-go-related gene (herg) is the pore-forming subunit of the cardiac delayed rectifier potassium current (IKr) responsible for action potential (AP) repolarization. Mutations in HERG lead to long-QT syndrome, a major cause of arrhythmias. Protein-protein interactions are fundamental for ion channel trafficking, membrane localization, and functional modulation. To identify proteins involved in the regulation of the HERG channel, we conducted a yeast two-hybrid screen of a human heart cDNA library using the C-terminus or N-terminus of HERG as bait. Fifteen proteins were identified as HERG amino terminal (HERG-NT)-interacting proteins, including Caveolin-1 (a membrane scaffold protein with multiple interacting partners, including G-proteins, kinases and NOS), the zinc finger protein, FHL2 and PTPN12 (a non-receptor tyrosine phosphatase). Eight HERG carboxylic terminal (HERG-CT)-interacting proteins were also identified, including the NF-κB-interacting protein myotrophin, We have identified multiple potential interacting proteins that may regulate cardiac IKr through cytoskeletal interactions, G-protein modulation, phosphorylation and downstream second messenger and transcription cascades. These findings provide further insight into dynamic modulation of HERG under physiological conditions and arrhythmogenesis.

  3. New classes of mind bomb-interacting proteins identified from yeast two-hybrid screens. (United States)

    Tseng, Li-Chuan; Zhang, Chengjin; Cheng, Chun-Mei; Xu, Haoying; Hsu, Chia-Hao; Jiang, Yun-Jin


    Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.

  4. Screening of BRD7 interacting proteins by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    余鹰; 朱诗国; 张必成; 周鸣; 李小玲; 李桂源


    BRD7 gene is low or undetectably expressed in nasopharyngeal carcinoma tissue (NPC) and can obviously suppress the growth of NPC cell line HNE1. In this study, the proteins that interacted with BRD7 coding protein were screened by yeast two-hybrid system. The complete reading frame of BRD7 gene was subcloned into pAS2 vector (BRD7-BD). Then the human embryo brain cDNA library was screened by BRD7-BD bait. Eleven positive clones were obtained from 4.8×106 transformed clones. By sequencing directly, 6 interacting proteins of BRD7 coding protein were isolated (Bromodomain-containing 3 protein (BRD3), Bromodomain-containing 2 protein (BRD2), IκB kinase-beta, KIAA1375 protein, interleukin 7 and adaptor-related protein complex 3δ-1 subunit). These results suggest BRD7 protein might form heterogenous dimer or triplex polymer with BRD2 and/or BRD3 to participate in transcriptional regulation.

  5. Homomeric and heteromeric interactions between wild-type and mutant phenylalanine hydroxylase subunits: evaluation of two-hybrid approaches for functional analysis of mutations causing hyperphenylalaninemia. (United States)

    Waters, P J; Scriver, C R; Parniak, M A


    Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH), while mutations in genes encoding the two enzymes (dihydropteridine reductase, DHPR, and pterin-4-alpha-carbinolamine dehydratase, PCD) required for recycling of its cofactor, tetrahydrobiopterin (BH(4)), cause other rarer disease forms of hyperphenylalaninemia. We have applied a yeast two-hybrid method, in which protein--protein interactions are measured by four reporter gene constructs, to the analysis of six PKU-associated PAH missense mutations (F39L, K42I, L48S, I65T, A104D, and R157N). By studying homomeric interactions between mutant PAH subunits, we show that this system is capable of detecting quite subtle aberrations in PAH oligomerization caused by missense mutations and that the observed results generally correlate with the severity of the mutation as determined by other expression systems. The mutant PAH subunits are also shown in this system to be able to interact with wild-type PAH subunits, pointing to an explanation for apparent dominant negative effects previously observed in obligate heterozygotes for PKU mutations. Based on our findings, the applications and limitations of two-hybrid approaches in understanding mechanisms by which PAH missense mutations exert their pathogenic effects are discussed. We have also used this technique to demonstrate homomeric interactions between wild-type DHPR subunits and between wild-type PCD subunits. These data provide a basis for functional studies on HPA-associated mutations affecting these enzymes.

  6. Show Time

    Institute of Scientific and Technical Information of China (English)


    <正> Story: Show Time!The whole class presents the story"Under the Sea".Everyone is so excited and happy.Both Leo and Kathy show their parentsthe characters of the play."Who’s he?"asks Kathy’s mom."He’s the prince."Kathy replies."Who’s she?"asks Leo’s dad."She’s the queen."Leo replieswith a smile.

  7. Snobbish Show

    Institute of Scientific and Technical Information of China (English)



    @@ The State Administration of Radio,Film and Television (SARFT),China's media watchdog,issued a new set of mles on June 9 that strictly regulate TV match-making shows,which have been sweeping the country's primetime programming. "Improper social and love values such as money worship should not be presented in these shows.Humiliation,verbal attacks and sex-implied vulgar content are not allowed" the new roles said.

  8. Yeast Protein–Protein Interaction Assays and Screens

    NARCIS (Netherlands)

    Folter, de S.; Immink, G.H.


    Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein’s function and the molecular mechanism underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and ident


    Institute of Scientific and Technical Information of China (English)


    Visitors look at plane models of the Commercial Aircraft Corp. of China, developer of the count,s first homegrown large passenger jet C919, during the Singapore Airshow on February 16. The biennial event is the largest airshow in Asia and one of the most important aviation and defense shows worldwide. A number of Chinese companies took part in the event during which Okay Airways, the first privately owned aidine in China, signed a deal to acquire 12 Boeing 737 jets.

  10. Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    赵新燕[1; 冯丽冰[2; 周伟国[3; 戴卫列[4; 李昌本[5; 赵寿元[6


    Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3’ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

  11. Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)


    Thrombopoietin(TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl.The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl.First,the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2,and the resulting plasmid is designated as pASMM.Then a human placenta cDNA library was screened using the pASMM as a target plasmid.Seven positive clones were isolated from 150 000 independent transformants.Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3'end and flanking non-translation region was obtained.Therefore,there is an interaction between vimentin and TPO receptor.The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

  12. Identification of the specificity of isolated phage display single-chain antibodies using yeast two-hybrid screens

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik


    A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast ...... two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens....

  13. A split-ubiquitin yeast two-hybrid screen to examine the substrate specificity of atToc159 and atToc132, two Arabidopsis chloroplast preprotein import receptors.

    Directory of Open Access Journals (Sweden)

    Siddhartha Dutta

    Full Text Available Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132, homologous GTPase (G- and Membrane (M- domains, but a highly dissimilar N-terminal acidic (A- domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G, atToc132 G-domain (Toc132G and atToc132 A- plus G-domains (Toc132AG as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.

  14. A yeast two-hybrid screen for SYP-3 interactors identifies SYP-4, a component required for synaptonemal complex assembly and chiasma formation in Caenorhabditis elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Sarit Smolikov


    Full Text Available The proper assembly of the synaptonemal complex (SC between homologs is critical to ensure accurate meiotic chromosome segregation. The SC is a meiotic tripartite structure present from yeast to humans, comprised of proteins assembled along the axes of the chromosomes and central region (CR proteins that bridge the two chromosome axes. Here we identify SYP-4 as a novel structural component of the SC in Caenorhabditis elegans. SYP-4 interacts in a yeast two-hybrid assay with SYP-3, one of components of the CR of the SC, and is localized at the interface between homologs during meiosis. SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes. In the absence of SYP-4, the levels of recombination intermediates, as indicated by RAD-51 foci, are elevated in mid-prophase nuclei, and crossover recombination events are significantly reduced. The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality. Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

  15. The radial immunodiffusion assay for plasma Histidine-rich Glycoprotein (HRG) based on a polyclonal antibody, shows a different specificity towards the two variants of a common amino acid polymorphism

    NARCIS (Netherlands)

    Hennis, B.C.; Hoffmann, J.J.M.L.; Kluft, C.


    Recently, two molecular weight forms of Histidine-rich Glycoprotein have been described which explain 59% of the variation in plasma HRG levels. Here, we demonstrate that this can partly be ascribed to a difference in specificity of the immuno assay for HRG towards the two alleles of the polymorphis

  16. Interaction of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system.

    Directory of Open Access Journals (Sweden)

    Douglas P Gladue

    Full Text Available E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV and bovine viral diarrhea virus (BVDV. E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.

  17. Assessment of two hybrid van der Waals density functionals for covalent and non-covalent binding of molecules (United States)

    Berland, Kristian; Jiao, Yang; Lee, Jung-Hoon; Rangel, Tonatiuh; Neaton, Jeffrey B.; Hyldgaard, Per


    Two hybrid van der Waals density functionals (vdW-DFs) are developed using 25% Fock exchange with (i) the consistent-exchange vdW-DF-cx functional [K. Berland and P. Hyldgaard, Phys. Rev. B 89, 035412 (2014)] and (ii) with the vdW-DF2 functional [K. Lee et al., Phys. Rev. B 82, 081101 (2010)]. The ability to describe covalent and non-covalent binding properties of molecules is assessed. For properties related to covalent binding, atomization energies (G2-1 set), molecular reaction energies (G2RC set), and ionization energies (G21IP set) are benchmarked against experimental reference values. We find that hybrid-vdW-DF-cx yields results that are rather similar to those of the standard non-empirical hybrid PBE0 [C. Adamo and V. Barone, J. Chem. Phys. 110, 6158 (1999)], with mean average deviations (MADs) of 4.9 and 5.0 kcal/mol for the G2-1 set, respectively. In this comparison, experimental reference values are used, back corrected by wavefunction-based quantum-chemistry calculations of zero-point energies. Hybrid vdW-DF2 follows somewhat different trends, showing on average significantly larger deviations from the reference energies, with a MAD of 14.5 kcal/mol for the G2-1 set. Non-covalent binding properties of molecules are assessed using the S22 benchmark set of non-covalently bonded dimers and the X40 set of dimers of small halogenated molecules, using wavefunction-based quantum chemistry results as references. For the S22 set, hybrid-vdW-DF-cx performs better than standard vdW-DF-cx for the mostly hydrogen-bonded systems, with MAD dropping from 0.6 to 0.3 kcal/mol, but worse for purely dispersion-bonded systems, with MAD increasing from 0.2 to 0.6 kcal/mol. Hybrid-vdW-DF2 offers a slight improvement over standard vdW-DF2. Similar trends are found for the X40 set, with hybrid-vdW-DF-cx performing particularly well for binding energies involving the strongly polar hydrogen halides, but poorly for systems with tiny binding energies. Our study of the X40 set

  18. Subunit orientation in the Escherichia coli enterobactin biosynthetic EntA-EntE complex revealed by a two-hybrid approach. (United States)

    Pakarian, Paknoosh; Pawelek, Peter D


    The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the Escherichia coli cytoplasm. We previously reported in vitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E. coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E. coli entE(-) and entA(-) knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entE(-) phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer in vivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cya(-)E. coli BTH101 cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations.

  19. Histograms showing variations in oil yield, water yield, and specific gravity of oil from Fischer assay analyses of oil-shale drill cores and cuttings from the Piceance Basin, northwestern Colorado (United States)

    Dietrich, John D.; Brownfield, Michael E.; Johnson, Ronald C.; Mercier, Tracey J.


    Recent studies indicate that the Piceance Basin in northwestern Colorado contains over 1.5 trillion barrels of oil in place, making the basin the largest known oil-shale deposit in the world. Previously published histograms display oil-yield variations with depth and widely correlate rich and lean oil-shale beds and zones throughout the basin. Histograms in this report display oil-yield data plotted alongside either water-yield or oil specific-gravity data. Fischer assay analyses of core and cutting samples collected from exploration drill holes penetrating the Eocene Green River Formation in the Piceance Basin can aid in determining the origins of those deposits, as well as estimating the amount of organic matter, halite, nahcolite, and water-bearing minerals. This report focuses only on the oil yield plotted against water yield and oil specific gravity.

  20. Effects of in vitro ozone treatment on proteolysis of purified rubisco from two hybrid poplar clones. [Populus maximowizii x trichocarpa

    Energy Technology Data Exchange (ETDEWEB)

    Landry, L.G.; Pell, E.J. (Pennsylvania State Univ., University Park (USA))


    Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH) groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.

  1. Exploring Protein Interactions on a Minimal Type II Polyketide Synthase Using a Yeast Two-Hybrid System

    Directory of Open Access Journals (Sweden)

    Gaetano Castaldo


    Full Text Available Interactions between proteins that form the ’minimal’ type II polyketide synthase in the doxorubicin producing biosynthetic pathway from Streptomyces peucetius were investigated using a yeast two-hybrid system (Y2H. Proteins that function as the so called ’chain length factor’ (DpsB and putative transacylase (DpsD were found to interact with the ketosynthase subunit (DpsA, which can also interact with itself. On the basis of these results we propose a head-to-tail homodimeric structure, which is consistent with previously published in vivo mutagenesis studies. No interactions were found between the acyl-carrier protein (DpsG and any of the other constituents of the complex, however, transient interactions, not detectable using the Y2H system, cannot be discounted and warrant further investigation.

  2. Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein. (United States)

    Yu, You; Li, Yinan; Zhang, Yan


    Alzheimer's disease (AD) is a neurodegenerative disorder in which amyloid β plaques are a pathological characteristic. Little is known about the physiological functions of amyloid β precursor protein (APP). Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1, members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease (PMD) and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

  3. Androgen receptor coregulator ARA267-α interacts with death receptor-6 revealed by the yeast two-hybrid

    Institute of Scientific and Technical Information of China (English)


    ARA267-αis a newly identified androgen receptor coactivator.In order to further elucidate its precise role in cells,using the ARA267- α fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-α-PHD-SET-interacting protein,death receptor-6(DR6),in the yeast two-hybrid screening.DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion(DR6cp)to mediate the cell apoptosis.The interaction between ARA267-α-PHD-SET and DR6cp was confirmed in vitro and in vivo.Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-α and DR6.


    Directory of Open Access Journals (Sweden)

    Ramazan Kurt,


    Full Text Available Experimental parallel strand lumbers (PSLs were manufactured from fast growing rotary peeled I-214 (Populus x euramericana and I-77/51 (Populus deltoides hybrid poplar clones veneer strands with melamine urea formaldehyde (MUF adhesive. The results showed that hybrid poplar clones can be used in PSLs manufacturing. Physical and mechanical properties of PSLs were affected by clone types. The I-77/51 clone had better properties and was found to be more suitable for PSLs manufacturing compared to the I-214 clone. PSLs properties were higher than those of solid woods (SWs and laminated veneer lumbers (LVLs of the same poplar clones. This increase may be due to materials, densification as a result of high pressure use, and the manufacturing techniques. The degree of contribution of SWs properties to the PSLs properties was lower than that of LVLs. This indicated that factors other than SWs properties played more important roles in the strength increase of PSLs.

  5. Entanglement of two hybrid optomechanical cavities composed of BEC atoms under Bell detection (United States)

    Eghbali-Arani, M.; Ameri, V.


    In this paper, firstly, we consider bipartite entanglement between each part of an optomechanical cavity composed of one-dimensional Bose-Einstein condensate (BEC). We investigate atomic collision on the behaviour of the BEC in the week photon-atom coupling constant and use Bogoliubov approximation for the BEC. Secondly under above condition, we propose a scheme for entanglement swapping protocol which involves tripartite systems. In our investigation, we consider a scenario where BECs, moving mirrors, and optical cavity modes are given in a Gaussian state with a covariance matrix. By applying the Bell measurement to the output optical field modes, we show how the remote entanglement between two BECs, two moving mirrors, and BEC-mirror modes in different optomechanical cavity can be generated.

  6. Photosynthesis, water relations, and growth of two hybrid Populus genotypes during a severe drought

    Energy Technology Data Exchange (ETDEWEB)

    Dickmann, D.I.; Liu, Zuijun; Nguyen, Phu V.; Pregitzer, K.S. (Michigan State Univ., East Lansing, MI (USA))


    During the 1988 growing season in East Lansing, Michigan, only 1.53 cm of rain fell from mid-May to mid-July, causing a severe drought. Then, a period of near record precipitation commenced; 30.4 cm of rain fell from July 19 to October 4. Growth, photosynthesis, and water relations of hybrid poplar cultivars Eugenei and Tristis, which had been established in the spring of 1987 in plastic pots buried in the ground, were measured on several sunny days during the 1988 growing season. Pots were irrigated at two different rates, and half the pots received supplemental nitrogen fertilizer. On a seasonal basis, photosynthesis and water-use efficiency in both genotypes peaked in early July and declined thereafter. Stomatal conductances were low during the drought but increased substantially when the rains commenced. Whereas nitrogen level had little effect on leaf physiology, the low water treatment produced significant reductions in photosynthesis and conductance. Diurnal measurements were made on June 17 and July 12. On both days photosynthesis and conductances were higher in Tristis than in Eugenei, especially for plants in the high water treatments and on July 12, the most extreme period of the drought. Drought produced both stomatal and mesophyll limitations to photosynthesis in both clones, though these responses were more pronounced in Eugenei. This clone also showed very low water-use efficiencies in the low water treatment on July 12. Even though the physiology of Eugenei was more impacted by drought than Tristis, it still produced two to three times more biomass over the 2-year period of the study than did Tristis. 41 refs., 10 figs., 5 tabs.

  7. Enzyme assays. (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie


    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  8. Screening for proteins interacting with MCM7 in human lung cancer library using yeast two hybrid system

    Directory of Open Access Journals (Sweden)

    Yuchen HAN


    Full Text Available Background and objective MCM7 is a subunit of the MCM complex that plays a key role in DNA replication initiation. But little is known about its interaction proteins. In this study yeast two hybrid screening was used to identify the MCM7 interacting proteins. Methods Yeast expression vector containing human full length MCM7-pGBKT7 plasmid was constructed, and with a library of cDNAs from human lung cancer-pACT2 plasmid was transformed into yeast strain AH109, and was electively grew in X-a-gal auxotrophy medium SD/-Trp-Leu-His-Ade, and the blue colonies were picked up, the plasmid of the yeast colonies was extracted , and transformed into E. Coli to extract DNA and performed sequence analysis. Results Eleven proteins were identified which could specifically interact with MCM7 proteins, among these five were cytoskeleton proteins, six were enzymes, kinases and related receptors. Conclusion The investigation provides functional clues for further exploration of MCM7 gene.

  9. Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Qing-yong Ma; Xian-kui Meng; Kang Li; Jun Cheng


    Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes. Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus (HCV) by cloning the gene of NS5ABP37 protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (α type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we made a sequence analysis by bioinformatics. Results We screened twenty-five proteins binding to NS5ABP37, including Homo sapiens cyclin Ⅰ (CCNI) gene, Homo sapiens matrix metallopeptidase 25 (MMP25) and Homo sapiens talin 1. Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV. And the biological function of NS5ABP37 may be associated with glycometabolism, lipid metabolism and apoptosis.

  10. The GacS-GacA two-component regulatory system of Pseudomonas fluorescens: a bacterial two-hybrid analysis. (United States)

    Workentine, Matthew L; Chang, Limei; Ceri, Howard; Turner, Raymond J


    The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many other aspects of bacterial physiology. Although it is known that the sensor kinases RetS and LadS feed into the GacS/GacA system, the mechanism through which this occurs is unknown, as are the protein-protein interactions in this system. To characterize and define these interactions, we utilized a bacterial two-hybrid system to study the interactions of GacS and GacA from Pseudomonas fluorescens CHA0. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in vivo was investigated. We found that the entire GacA molecule is required for GacA to interact with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. In addition, homologs of Pseudomonas aeruginosa hybrid sensor kinases, RetS and LadS, were identified in P. fluorescens, and shown to interact with GacS, but not GacA.

  11. Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

    DEFF Research Database (Denmark)

    Tchórzewski, M; Boldyreff, B; Issinger, O


    on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast...

  12. Identification of interacting proteins with aryl hydrocarbon receptor in scallop Chlamys farreri by yeast two hybrid screening. (United States)

    Cai, Yuefeng; Pan, Luqing; Miao, Jingjing; Liu, Tong


    The aryl hydrocarbon receptor (AhR) belongs to the basic-helix-loop helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. AhR has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes, as well as the mediation of the toxicity of certain xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the AhR is well-studied as a mediator of the toxicity of certain xenobiotics in marine bivalves, the normal physiological function remains unknown. In order to explore the function of the AhR, the bait protein expression plasmid pGBKT7-CfAhR and the cDNA library of gill from Chlamys farreri were constructed. By yeast two hybrid system, after multiple screening with the high screening rate medium, rotary verification, sequencing and bioinformatics analysis, the interactions of the CfAhR with receptor for activated protein kinase C 1 (RACK1), thyroid peroxidase-like protein (TPO), Toll-like receptor 4(TLR 4), androglobin-like, store-operated Ca(2+) entry (SocE), ADP/ATP carrier protein, cytochrome b, thioesterase, actin, ferritin subunit 1, poly-ubiquitin, short-chain collagen C4-like and one hypothetical protein in gill cells were identified. This study suggests that the CfAhR played fundamental roles in immune system homeostasis, oxidative stress response, and in grow and development of C. farreri. The elucidation of these protein interactions is of much importance both in understanding the normal physiological function of AhR, and as potential targets for further research on protein function in AhR interactions.

  13. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

    Directory of Open Access Journals (Sweden)

    Canard Bruno


    Full Text Available Abstract Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4. Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

  14. Spatial variability of soil carbon and nitrogen in two hybrid poplar-hay crop systems in southern Quebec, Canada (United States)

    Winans, K. S.


    Canadian agricultural operations contribute approximately 8% of national GHG emissions each year, mainly from fertilizers, enteric fermentation, and manure management (Environment Canada, 2010). With improved management of cropland and forests, it is possible to mitigate GHG emissions through carbon (C) sequestration while enhancing soil and crop productivity. Tree-based intercropped (TBI) systems, consisting of a fast-growing woody species such as poplar (Populus spp.) planted in widely-spaced rows with crops cultivated between tree rows, were one of the technologies prioritized for investigation by the Agreement for the Agricultural Greenhouse Gases Program (AAGGP), because fast growing trees can be a sink for atmospheric carbon-dioxide (CO2) as well as a long-term source of farm income (Montagnini and Nair, 2004). However, there are relatively few estimates of the C sequestration in the trees or due to tree inputs (e.g., fine root turnover, litterfall that gets incorporated into SOC), and hybrid poplars grow exponentially in the first 8-10 years after planting. With the current study, our objectives were (1) to evaluate spatial variation in soil C and nitrogen (N) storage, CO2 and nitrogen oxide (N20), and tree and crop productivity for two hybrid poplar-hay intercrop systems at year 9, comparing TBI vs. non-TBI systems, and (2) to evaluate TBI systems in the current context of C trading markets, which value C sequestration in trees, unharvested crop components, and soils of TBI systems. The study results will provide meaningful measures that indicate changes due to TBI systems in the short-term and in the long-term, in terms of GHG mitigation, enhanced soil and crop productivity, as well as the expected economic returns in TBI systems.

  15. Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Jun Cheng; Shu-Lin Zhang; Yan-Ping Huang; Lin Wang; Yan Liu; Shu-Mei Lin


    AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes.METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Nineteen colonies were selected and sequenced.Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ and H+ coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function.CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.

  16. Characterization of L1 ORF1p self-interaction and cellular localization using a mammalian two-hybrid system.

    Directory of Open Access Journals (Sweden)

    Mark Sokolowski

    Full Text Available Long INterspersed Element-1 (LINE-1, L1 is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu RNP nuclear access in the host cell.

  17. Angiogenesis Assays. (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P


    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  18. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui


    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  19. The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration. (United States)

    Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin


    Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration.

  20. Mapping the interactions of dengue virus NS1 protein with human liver proteins using a yeast two-hybrid system: identification of C1q as an interacting partner.

    Directory of Open Access Journals (Sweden)

    Emiliana M Silva

    Full Text Available Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1 is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q, which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs, such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection.

  1. Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2.

    Directory of Open Access Journals (Sweden)

    Xiangjing Fu

    Full Text Available Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6 cfu/3 µg and 2×10(9 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2 Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

  2. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan


    Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes

  3. Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus


    Pawlowski, Alice; Riedel, Kai-Uwe; Klipp, Werner; Dreiskemper, Petra; Groß, Silke; Bierhoff, Holger; Drepper, Thomas; Masepohl, Bernd


    Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, ...

  4. Progress on Application of Yeast Two-hybrid Technique%酵母双杂交技术应用进展

    Institute of Scientific and Technical Information of China (English)

    王婷; 葛怀娜; 郭宏


    Yeast two-hybrid system is one of the most powerful and widely used method to detect protein-protein interaction in living cells.Yeast two-hybrid technique is cost-effective, easy to operate, can reach the whole-genome level, and identify the interaction between different varieties.Compared with the traditional detected method, it has obvious advantages and has been applied in more and more fields.In this paper, the yeast two-hybrid technology principle and application were reviewed, including the applications on discovering new protein and proteins’new function, setting up the protein linkage map, studing of human genome DNA library, screening target for drug, and so on.The paper was expected to provide reference for the application of yeast two-hybrid system.%酵母双杂交技术是鉴定蛋白互作最有效和最广泛的分子生物学技术。该技术能直接作用于活细胞,检测细胞内蛋白质互作,具有成本低、易操作、可达到全基因组水平、能进行品种间的互作鉴定等诸多优点。较之传统的检测方法有明显优势,已在越来越多的领域得到应用。对酵母双杂交的技术原理以及应用进行了综述,介绍了该技术在发现新蛋白质、探究蛋白质功能、建立基因组蛋白连锁图、研究人类DNA文库和筛选药物作用位点等方面的重要应用,以期为该技术的广泛应用提供参考。

  5. Screening of novel proteins that interact with FLA8-C terminal from Dunaliella salina using yeast two-hybrid system%与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的新蛋白的酵母双杂交筛选

    Institute of Scientific and Technical Information of China (English)

    李靓; 崔柳青; 王瑞莉; 毛丽红; 韩康; 柴丹丹; 薛乐勋


    目的:用酵母双杂交的方法筛选与杜氏盐藻驱动蛋白Ⅱ动力亚基(FLA8)C端相互作用的蛋白.方法:设计特异性引物(上下游分别引入EcoRⅠ和BamHⅠ酶切位点),扩增FLA8 C端(433个氨基酸)cDNA,与穿梭载体pGBKT7连接以构建诱饵表达载体,然后转化酵母菌株Y187和AH109,Western blot法检测诱饵蛋白在转化酵母菌株内的表达.通过自激活实验和毒性实验检测诱饵表达载体是否适合利用酵母双杂交的方法筛选相互作用蛋白.转化诱饵质粒的酵母菌株Y187与AH109(文库菌)杂交,待三叶形合子形成后用营养缺陷型培养基和α-半乳糖苷酶活性实验筛选阳性克隆,并对阳性克隆进行测序.结果:成功构建了pGBKT7-FLA8-C双杂交诱饵载体;经检测该载体的表达产物对Y187和AH109均无自激活和毒性作用,可用于酵母双杂交实验;杂交后筛选得到2个阳性克隆,测序结果显示为鞭毛结合蛋白107(FAP107)和含蛋白结合结构域的预测蛋白.结论:成功筛选得到了与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的蛋白,分别为FAP107和预测蛋白.%Aim : To isolate proteins that interact with C-Lerminal region oi FLA8 which is the motor subunit of kinesin II from Dunaliella salina. Methods:The coding sequences of FLA8-C( 433 aa )was cloned into the pGBKT7 as bait and transformed into Y187 and AH109 yeast strains,and then the transcriptional activation and toxicity were tested. Yeast two-hybrid method was performed to screen the Dunaliella salina cDNA expression library. The positive clones were screened by auxotroph culture and assayed for α-galactosidase activity according the manufacturer s instructions. The cDNA fragments of prey proteins from positive colonies were detected by colony PCR. Results :The results showed that the FLA8-C is suitable for two-hybrid library screening. Sequence analysis on positive colonies showed that they were flagellar associated protein 107( FAP107 )and

  6. Identification of novel protein-protein interactions of Yersinia pestis type III secretion system by yeast two hybrid system.

    Directory of Open Access Journals (Sweden)

    Huiying Yang

    Full Text Available Type III secretion system (T3SS of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS.

  7. A set of host proteins interacting with papaya ringspot virus NIa-Pro protein identified in a yeast two-hybrid system. (United States)

    Gao, L; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P


    The protein-protein interactions between viral and host proteins play an essential role in plant virus infection and host defense. The potyviral nuclear inclusion protein a protease (NIa-Pro) is involved in various steps of viral infection. In this study, the host proteins interacting with papaya ringspot virus (PRSV) NIa-Pro were screened in a Carica papaya L. plant cDNA library using a Sos recruitment two-hybrid system (SRS). We confirmed that the full-length EIF3G, FBPA1, FK506BP, GTPBP, MSRB1, and MTL from papaya can interact specifically with PRSV NIa-Pro in yeast, respectively. These proteins fufill important functions in plant protein translation, biotic and abiotic stress, energy metabolism and signal transduction. In this paper, we discuss possible functions of interactions between these host proteins and NIa-Pro in PRSV infection and their role in host defense. Sos recruitment two-hybrid system; papaya ringspot virus; NIa-Pro; protein-protein interaction.

  8. Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Zhiyuan; Pardington-Purtymun, P.E.; Comeaux, J.C. [Los Alamos National Labs., NM (United States)] [and others


    The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair. Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control. Using the human RAD52 protein as the bait in a yeast two-hybrid system, we have identified a human homolog of yeast UBC9, designated UBE2I, that interacts with RAD52, RAD51, p53, and a ubiquitin-like protein UBL1. These interactions are UBE2I-specific, since another DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), does not interact with these proteins. The interaction of UBE2I with RAD52 is mediated by RAD52`s self-association region. These results suggest that the RAD52-dependent processes, cell cycle control, p53-mediated pathway(s), and ubiquitination interact through human UBE2I. 22 refs., 3 figs.

  9. Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Guisheng Chen; Xu Peng; Shugui Shi; Lusi Li; Kangning Chen; Ju Hu; Zhenhua Zhou; Jun Wu; Gaoxing Luo; Shunzong Yuan


    The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBC1D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor α2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.

  10. Use of bacterial two-hybrid system to investigate the molecular interaction between the regulators NifA and NifL of Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    廖贡献; 俞冠翘; 沈善炯; 朱家璧


    Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.

  11. Screening for the Interaction Proteins of SOC1 from Phyllostachys violascens Using Yeast Two-Hybrid System%利用酵母双杂交系统筛选雷竹 SOC1相互作用蛋白

    Institute of Scientific and Technical Information of China (English)

    潘现飞; 施泉; 林新春; 徐英武; 曹友志


    This work aims to study the target proteins of suppressor of overexpression of constans1(SOC1)in flowering way of Phyllostachys violaceus and to explore the action mechanism of SOC1. The pGBKT7-PvSOC1 vector was constructed as the bait protein,then the cDNA library of P. violascens flowering was established using SMART technology,and the interaction proteins with SOC1 were screened by yeast two-hybrid system as well as analyzed and identified. The results showed that the cDNA library for yeast two-hybrid was constructed successfully,the conversion rate of the library was about 3.0×106 converters per μg pGADT7-Rec,and the capacity of the library was up to 7.5×106 cfu/mL and the insert fragments were distributed from 0.25 kb to 2 kb. The protein interacted with the bait protein PvSOC1 was screened through the yeast two-hybrid technology. The screened target protein was identified as a protein kinase containing plentiful leucine which amino acid sequence was very conservative in different species,and had the highest affinity with rice.%在雷竹(Phyllostachys violascens)中找到 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1)在开花途径中的作用靶标,进一步探索 SOC1的作用机制。构建 pGBKT7-PvSOC1诱饵表达载体,通过 SMART 技术构建开花时期的雷竹 cDNA文库,酵母双杂交筛选与 SOC1互作的蛋白,并对其进行分析鉴定。结果显示,成功构建了可用于酵母双杂交的 cDNA 文库,文库的转化率为每微克 pGADT7-Rec 3.0×106转化子,文库滴度为7.5×106 cfu/mL,插入片段主要在250-2000 bp 之间。通过酵母双杂交实验得到与诱饵蛋白 PvSOC1相互作用的蛋白,筛选到的靶标蛋白经鉴定是一个富含亮氨酸的蛋白激酶,特征氨基酸序列在不同物种中非常保守,与水稻中的同源蛋白亲缘性最高。

  12. B cell helper assays. (United States)

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo


    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  13. 玉米弯孢叶斑病菌酵母双杂交cDNA文库的构建及评价%Construction and characterization of yeast two hybrid cDNA library of Curvularia lunata

    Institute of Scientific and Technical Information of China (English)

    荆晶; 刘铜; 陈捷


    由新月弯孢[Curvularia lunata (Wakker)Boed]引起的玉米弯孢叶斑病是我国玉米生产区的一种重要性叶斑病,曾在20世纪90年代给我国玉米生产造成较大损失.目前对该病原菌致病类型鉴定与致病相关基因、病害发生规律、综合防治等方面均有较好的研究基础[1],并且在前期的研究中发现该病菌调控毒素形成的相关基因与色素合成相关基因在表达上存在某种关联性[2],由于这2个基因均为致病相关基因,因此研究两基因表达的蛋白的关联性对于全面揭示病菌致病机理和调控网络具有重要意义.%BD SMART technique and LD-PCR were applied to synthesize double strand cDNA(ds cDNA). The ds cDNA and pGADT7-Rec were transfered into competent yeast cell to construct yeast two-hybrid cDNA library of Curvularia lunata by homologous recombination method. The results showed that library capacitance was 2.46×105 and transformation rate was 2.13×105/μg pGADT7-Rec. The plaque titer of the library was 2.5×108 pfu/mL and the recombination frequency was 88. 24%. The length of inserted cDNA fragment was ranged from 0.4 kb to 3 kb in average. This is the first time to use BD SMART technique to construct yeast two-hybrid cDNA library of C. lunata by homologous recombination.

  14. Growth cone collapse assay. (United States)

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J


    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  15. The screening of functional proteins interacting with IrrE by the yeast two-hybrid system%利用酵母双杂交系统筛选IrrE相互作用蛋白

    Institute of Scientific and Technical Information of China (English)

    田霞; 代其林; 龚元亚; 孙英坤; 谢琳; 杨娟; 王劲


    IrrE was constructed as the bait protein and then interacted with functional proteins from the Arabidopsis cDNA library by yeast two-hybrid system. The result showed that there was one interacting protein from all the positive clones which had high identity (as high identical to 98%) with the LEA14 protein (Late Embryogenesis Abundant protein 14) encoded by Arabidopsis thaliana gene Atlg01470. It was inferred that IrrE protein may play an important role in improving the tolerance to salt stress in trans-gene plants.%本研究通过酵母双杂交技术,构建了IrrE蛋白为诱饵载体,从拟南芥cDNA文库中筛选与IrrE诱饵蛋白相互作用的功能蛋白.研究结果表明,在所获得的阳性克隆中,发现了一个与拟南芥基因Atlg01470所编码的蛋白有较高同源性的蛋白,即LEA14蛋白(晚期胚胎发育富集蛋白),同源性高达98%.推测IrrE转录因子在提高植物耐盐性的过程中起了重要作用.

  16. The Health Show


    Swann, David


    Dr David Swann interviewed on The Health Show, Series 1, Episode 5, 2011 for BBC World about the award-winning 21st Century Nursing Bag. BBC World News reaches 241million people every week, available in 296 million homes, 1.8 million hotel rooms and has the highest average viewership on a weekday of any international news channel. The Health Show is a new 26-part series for BBC World News covering the most important news stories from around the world.

  17. A Fashion Show

    Institute of Scientific and Technical Information of China (English)


    <正>Story: The yearly fashion show day.The children take turns to walk on the stage and show the class their favorite clothes.Now it’s Joe’s and Phoebe’s turn.Joe walks on the stage and says,“My shorts are blue.Do you like my blue shorts?”On the other side of the stage, Phoebe is wearing her favorite pink skirt.“My skirt is pink.Do you like my pink skirt?”asks

  18. 玉米胚芽鞘负向重力生长时期的酵母双杂交文库构建及评价%Construction and Appreciation of Yeast Two Hybrid Library of Maize Coleoptile during Negtive-gravitropic Growth

    Institute of Scientific and Technical Information of China (English)

    姜川; 陈化榜


    [目的]构建玉米胚芽鞘负向重力生长时期的酵母双杂交文库,并对其进行评价.[方法]以玉米骨干自交系B73为材料,利用Trizol试剂提取玉米胚芽鞘在负向生长时期所表达的总RNA,再利用SMART技术构建酵母双杂交文库,并转化进酵母Y187中.[结果]试验得到的原始文库库容量高达1×106个单克隆;文库中插入的片段集中在0.5~1.5 kb,平均插入长度为800 bp.[结论]试验构建得到了高质量的米胚芽鞘酵母双杂交文库,为研究胚芽鞘的负向重力反应机制奠定了基础.%[Objective] The aim was to construct and appraise the yeast two hybrid library of maize coleoptile during its negative-gravitropic growth. [ Method] Using maize inbred line B73 as materials, the total RNA which expressed during negative-gravitropic growth period of maize coleoptile was extracted by Trizol, and the yeast two hybrid library was constructed by SMART technology and transformed into yeast Y187. [ Result] The results showed that the library contained 1 × 106 monoclones and the predominant size of inserted fragments in library was 0.5-1.5 kb, average size was 800 bp. [ Conclusion ] High-quailty yeast two hybrid library of maize coleoptile was constructed and it prepared the ground for the study of negative-gravitropic reaction mechanism of coleoptile.

  19. On not showing scalps

    DEFF Research Database (Denmark)

    Marselis, Randi Lorenz


    proposed by Janet Marstine, the editor of the Routledge Companion to Museum Ethics, I show how the museum succeeded in engaging users in questions of museum ethics. However, this specific debate on human remains in museums developed into an encounter between a global, museological discourse...

  20. Violence and TV Shows


    ÖZTÜRK, Yrd. Doç. Dr. Şinasi


    This study aims to discuss theories on theviolent effects of TV shows on viewers, especiallyon children. Therefore, this study includes a briefdiscussion of definitions of violence, discussionof violence theories, main results of researcheson televised violence, measuring TV violence,perception of televised violence, individualdifferences and reactions to TV violence,aggressiveness and preferences for TV violence.

  1. Honored Teacher Shows Commitment. (United States)

    Ratte, Kathy


    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  2. A Visionary Show

    Institute of Scientific and Technical Information of China (English)


    Seduction. Distinction. Relax. Pulsation. These are the "style universes" on display at Première Vision, heralded as "The World’s Premiere Fabric Show." Started more than 35 years ago by 15 French weavers, Première Vision has expanded beyond its

  3. Honored Teacher Shows Commitment. (United States)

    Ratte, Kathy


    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  4. Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

    Institute of Scientific and Technical Information of China (English)

    梅泉; 李双; 刘萍; 奚玲; 王世宣; 孟玉菡; 刘杰; 杨欣慰; 卢运萍; 汪辉


    By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...

  5. Shanghai Shows Its Heart

    Institute of Scientific and Technical Information of China (English)


    The city known as China’s economic powerhouse showed a more caring face as host of the Special Olympic Games Between October 2 and 11,the Special Olympics Summer Games were hosted in Shanghai,the first time the 40-year-old athletic com- petition for people with intellectual disabilities came to a developing country. This Special Olympics was also larger than all previous games in temps of the number of athletes.

  6. Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    Shuang LI; Ping LIU; Ling XI; Xuefeng JIANG; Jianfeng ZHOU; Shixuan WANG; Li MENG; Yunping LU; Ding Ma


    To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

  7. MOR is not enough: identification of novel mu-opioid receptor interacting proteins using traditional and modified membrane yeast two-hybrid screens. (United States)

    Petko, Jessica; Justice-Bitner, Stephanie; Jin, Jay; Wong, Victoria; Kittanakom, Saranya; Ferraro, Thomas N; Stagljar, Igor; Levenson, Robert


    The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.

  8. Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system. (United States)

    Ma, Li; Koyota, Souichi; Myoen, Yu; Yamashita, Tetsuro; Yatabe, Naoki; Koizumi, Yukio; Aosasa, Masayoshi; Nishimichi, Norihisa; Matsuda, Haruo; Sugiyama, Toshihiro


    Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.

  9. A yeast two-hybrid screen reveals a strong interaction between the Legionella chaperonin Hsp60 and the host cell small heat shock protein Hsp10. (United States)

    Nasrallah, Gheyath K


    L. pneumophila is an intracellular bacterium that replicates inside a membrane-bound vacuole called Legionella-containing vacuole (LCV), where it plentifully liberates its HtpB chaperonin. From LCV, HtpB reaches the host cell cytoplasm, where it interacts with SAMDC, a cytoplasmic protein required for synthesis of host polyamines that are important for intracellular growth of L. pneumophila. Additionally, cytoplasmic expression of HtpB in S. cerevisiae induces pseudohyphal growth, and in mammalian cells recruits mitochondria to LCV, and modifies actin microfilaments organization. This led us to hypothesize here that HtpB recruits a protein(s) from eukaryotic cells that is involved in the emergence of the aforementioned phenotypes. To identify this protein, a commercially available HeLa cDNA library was screened using a yeast two-hybrid system. Approximately 5×10(6) yeast clones carrying HeLa cDNA library plasmid were screened. Twenty-one positive clones were identified. DNA sequence analysis revealed that all of these positive clones encoded the mammalian small heat shock protein Hsp10. Based on the fact that chaperonions are required to interact with co-chaperonins to function properly in protein folding, we believe that HtpB recruits the host cell Hsp10 to appropriately interact with SAMDC and to induce the multifunction phenotypes deemed important in L. pneumophila pathogenesis.

  10. Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.

    Directory of Open Access Journals (Sweden)

    Liping Ren

    Full Text Available Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

  11. Not a "reality" show. (United States)

    Wrong, Terence; Baumgart, Erica


    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show.

  12. Showing Value (Editorial

    Directory of Open Access Journals (Sweden)

    Denise Koufogiannakis


    Full Text Available When Su Cleyle and I first decided to start Evidence Based Library and Information Practice, one of the things we agreed upon immediately was that the journal be open access. We knew that a major obstacle to librarians using the research literature was that they did not have access to the research literature. Although Su and I are both academic librarians who can access a wide variety of library and information literature from our institutions, we belong to a profession where not everyone has equal access to the research in our field. Without such access to our own body of literature, how can we ever hope for practitioners to use research evidence in their decision making? It would have been contradictory to the principles of evidence based library and information practice to do otherwise.One of the specific groups we thought could use such an open access venue for discovering research literature was school librarians. School librarians are often isolated and lacking access to the research literature that may help them prove to stakeholders the importance of their libraries and their role within schools. Certainly, school libraries have been in decline and the use of evidence to show value is needed. As Ken Haycock noted in his 2003 report, The Crisis in Canada’s School Libraries: The Case for Reform and Reinvestment, “Across the country, teacher-librarians are losing their jobs or being reassigned. Collections are becoming depleted owing to budget cuts. Some principals believe that in the age of the Internet and the classroom workstation, the school library is an artifact” (9. Within this context, school librarians are looking to our research literature for evidence of the impact that school library programs have on learning outcomes and student success. They are integrating that evidence into their practice, and reflecting upon what can be improved locally. They are focusing on students and showing the impact of school libraries and

  13. Public medical shows. (United States)

    Walusinski, Olivier


    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre.

  14. The Great Cometary Show (United States)


    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

  15. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas


    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.

  16. Colorimetric protein assay techniques. (United States)

    Sapan, C V; Lundblad, R L; Price, N C


    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  17. Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis

    Indian Academy of Sciences (India)

    Chengcheng Zhang; Lei He; Kai Kang; Heng Chen; Lei Xu; Yanming Zhang


    Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

  18. Construction of Yeast Two-hybrid cDNA Library of Verticillium dahliae%大丽轮枝菌酵母双杂交 cDNA 文库的构建

    Institute of Scientific and Technical Information of China (English)

    康静敏; 刘坤; 刘严; 张怡; 李成伟; 谭光轩


    为了分离克隆与植物互作的大丽轮枝菌致病相关蛋白基因,利用 SMART 技术构建了大丽轮枝菌酵母双杂交 cDNA 文库。结果表明,构建的文库滴度为5.2×107 cfu/mL,库容为3.9×107 cfu;文库 cDNA 插入片段长度主要分布在0.5~2.0 kb,平均为1 kb;文库重组率为96%。表明文库质量较好,为筛选与植物互作的大丽轮枝菌致病蛋白基因奠定了基础。%To seek out the V. dahliae proteins involved in the interaction of plant with V. dahliae,a yeast two-hybrid library of V. dahliae was constructed using SMART technique. Results showed that the titer of cDNA library was 5. 2 × 107 cfu/mL,the library contained 3. 9 × 107 cfu independent clones,the size distribution of insert frag-ments ranged from 0. 5 -2. 0 kb,and the recombination rate was about 96%. These data demonstrated that the li-brary could meet the requirements of standard cDNA library,which laid a foundation for screening the interaction proteins from V. dahliae.

  19. Absolute nuclear material assay (United States)

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


    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.

  20. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays. (United States)

    Tohidi, Maryam; Arbab, Parvaneh; Ghasemi, Asghar


    Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 μU/mL and -0.14 μU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

  1. Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Taddei Kevin


    Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

  2. Functional and biochemical properties of Mal de Río Cuarto virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts. (United States)

    Maroniche, Guillermo A; Mongelli, Vanesa C; Peralta, Andrea V; Distéfano, Ana J; Llauger, Gabriela; Taboga, Oscar A; Hopp, Esteban H; del Vas, Mariana


    Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  3. The Yeast Split-Ubiquitin Membrane Protein Two-Hybrid Screen Identifies BAP31 as a Regulator of the Turnover of Endoplasmic Reticulum-Associated Protein Tyrosine Phosphatase-Like B


    Wang, Bing; Pelletier, Jerry; Massaad, Michel J.; Herscovics, Annette; Shore, Gordon C


    In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. He...

  4. Cell viability assays: introduction. (United States)

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  5. Tube-Forming Assays. (United States)

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


    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.

  6. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.


    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  7. Assays for thrombopoietin

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, T.P.


    In summary, thrombopoietin levels have been determined indirectly by measuring thrombocytopoiesis in assay animals (platelet counting, measurement of isotope incorporation into newly formed platelets, changes in platelet sizes, or alterations in number and size of megakaryocytes) and by use of an immunoassay. Although much work remains, it seems clear at the present time that isotopic uptake into platelets of specially prepared assay mice (rebound-thrombocytosis) is superior to the other techniques now available for the measurement of thrombopoietin. However, the ideal assay for TSF which is specific, rapid, and inexpensive is yet to be developed. An immunoassay is in the development stage, but will require additional work before it can be utilized for the routine assay of TSF.

  8. New Rapid Spore Assay (United States)

    Kminek, Gerhard; Conley, Catharine


    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  9. 极细链格孢菌蛋白激发子PeaT1在酵母双杂交系统中转录激活活性的检测%Detection for Transcriptional Activity of Alternaria Tenuissim Protein Elicitor in Yeast Two-hybrid System

    Institute of Scientific and Technical Information of China (English)

    刘延锋; 邱德文; 曾洪梅; 杨秀芬


    The peaT1 gene fragment was amplified from pGEM-6p-1-peaT1 by PCR,and recovered target gene was cloned into pLexA vector.After digestion and sequencing,the bait vector pLexA-peaT1 was transformed into yeast strain EGY48[p8op-lacZ]by PEG/LiAC,and the transcriptional activity of bait vector was detected.The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed,for the two bands of recombinant bait plasmid in agarase gel electrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I.Therefore,the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.

  10. 酵母双杂交筛选与单剪接型2.2 kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白%Yeast two-hybrid system screening liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome

    Institute of Scientific and Technical Information of China (English)

    陈婉南; 陈金烟; 黄清玲; 林建银; 林旭


    目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.%Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.

  11. Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria

    Directory of Open Access Journals (Sweden)

    E. Zerbini


    Full Text Available El aumento de aislamientos clínicos de Mycobacterium tuberculosis resistentes a las drogas esenciales y de casos de micobacteriosis diseminadas debidas al complejo Mycobacterium avium hacen necesario investigar nuevos agentes antimicobacterianos. Los péptidos antimicrobianos son un nuevo grupo de antibióticos que poseen un mecanismo de acción particular. Algunos de ellos, como la cecropina y la melitina, han sido aislados de insectos y han demostrado buena actividad in vitro contra bacterias gram positivas y gram negativas. Híbridos sintéticos de esos péptidos han presentado mayor actividad que los péptidos individuales. En este trabajo se evaluó la actividad in vitro de dos péptidos híbridos sintéticos de melitina y cecropina contra M. tuberculosis, complejo M. avium, Mycobacterium fortuitum y Mycobacterium smegmatis. Se determinó la concentración inhibitoria mínima empleando la técnica de macrodilución en caldo. Luego se estableció la concentración bactericida mínima en medio Lowenstein Jensen. Los péptidos evaluados mostraron ser activos in vitro contra M. smegmatis, mientras que no presentaron ninguna actividad contra las otras micobacterias estudiadas.The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis

  12. Study on a rooting inducing culture system for plantlets of two hybrid Rhododendron%两种杜鹃杂交种不定芽生根的培养基配方

    Institute of Scientific and Technical Information of China (English)

    吴影倩; 耿兴敏; 罗凤霞


    以杜鹃杂交种R.simsii×R.mucronatum和R.pulchrum×R.mucronatum组培苗的不定芽为外植体,对基本培养基和蔗糖浓度采用单因子设计,对生长素种类和活性炭采用正交试验,对适宜的生根培养基配方进行了探讨.结果表明:不同的杂交组合适宜不定芽生根的培养基类型、蔗糖浓度及激素组合都有所不同.R.simsii×R.mucronatum的适宜生根培养基为1/2 WPM+ 20 g/L蔗糖+3g/L AC,R.pulchrum×R.mucronatum的合适生根培养基为WPM+ 10 g/L蔗糖+2 mg/L IBA.适宜的移栽基质均为V泥炭∶V蛭石∶V珍珠岩=1∶1∶1.%To optimize a micro propagation of hybrid Rhododendron and improve the efficiency of Rhododendron breeding,In vitro shoots were used as the explants to establish a rooting system for two hybrid Rhododendron(R.simsii × R.mucronatum and R.pulchrum × R.mucronatum).Single factor experiments were used in basic medium and sucrose concentration,orthogonal test was applied to the type of auxin and activated carbon,select the appropriate culture conditions.The results showed that the optimal rooting medium was 1/2 WPM +20 g/L sucrose +3 g/L AC for R.simsii ×R.mucronatum and the optimal rooting medium was WPM + 10 g/L sucrose + 2 mg/L IBA for R.pulchrum × R.mucronatum.Optimal transplanting medium were peat∶ vermiculite∶ perlite =1 ∶ 1 ∶ 1.

  13. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.


    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.

  14. Against vaccine assay secrecy. (United States)

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors.

  15. Against vaccine assay secrecy (United States)

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  16. 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)


    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.

  17. CTL ELISPOT assay. (United States)

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita


    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  18. Assays for calcitonin receptors

    Energy Technology Data Exchange (ETDEWEB)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.


    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  19. New oligosaccharyltransferase assay method. (United States)

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi


    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  20. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

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


    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...

  1. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.


    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  2. The corneal pocket assay. (United States)

    Ziche, Marina; Morbidelli, Lucia


    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  3. Kinetic Tetrazolium Microtiter Assay (United States)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David


    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  4. Graphene Oxides Show Angiogenic Properties. (United States)

    Mukherjee, Sudip; Sriram, Pavithra; Barui, Ayan Kumar; Nethi, Susheel Kumar; Veeriah, Vimal; Chatterjee, Suvro; Suresh, Kattimuttathu Ittara; Patra, Chitta Ranjan


    Angiogenesis, a process resulting in the formation of new capillaries from the pre-existing vasculature plays vital role for the development of therapeutic approaches for cancer, atherosclerosis, wound healing, and cardiovascular diseases. In this report, the synthesis, characterization, and angiogenic properties of graphene oxide (GO) and reduced graphene oxide (rGO) have been demonstrated, observed through several in vitro and in vivo angiogenesis assays. The results here demonstrate that the intracellular formation of reactive oxygen species and reactive nitrogen species as well as activation of phospho-eNOS and phospho-Akt might be the plausible mechanisms for GO and rGO induced angiogenesis. The results altogether suggest the possibilities for the development of alternative angiogenic therapeutic approach for the treatment of cardiovascular related diseases where angiogenesis plays a significant role.

  5. Analytical characterization of the APTIMA HPV Assay. (United States)

    Dockter, Janel; Schroder, Astrid; Eaton, Barbara; Wang, Ann; Sikhamsay, Nathan; Morales, Liezel; Giachetti, Cristina


    Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. To determine the analytical performance characteristics of the APTIMA HPV Assay. Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems. The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or low-risk HPV types. Spermicides, anti-fungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-lot, and inter-run signal variability were 99% of the data. Intra-run variability was HPV Assay showed excellent performance and robustness.


    DEFF Research Database (Denmark)


    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 a......, 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)....

  7. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

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


    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+.

  8. 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)


    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

  9. RAS - Screens & Assays - Drug Discovery (United States)

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

  10. 利用酵母双杂交系统筛选与人巨细胞病毒皮层蛋白pUL23相互作用的病毒编码蛋白%Screening of Human Cytomegalovirus Tegument Protein pUL23 Interacting Partner via Yeast Two-hybrid System

    Institute of Scientific and Technical Information of China (English)

    肖静; 李弘剑; 胡嘉淼; 肖小平; 员月明; 刘明亮; 曾宝娟; 李婧惠; 周天鸿; 冉艳红


    人巨细胞病毒(HCMV)UL23基因编码病毒皮层蛋白,该基因缺失时,病毒在人包皮成纤维细胞(HEF)中的繁殖速度加快.为进一步阐述HCMV UL23基因编码产物pUL23的功能及调控机制,采用鸟枪法构建了融合于GAL4活性区域的HCMV Towne株基因组随机表达文库.利用酵母双杂交技术,以pGBKT7-UL23为诱饵质粒,从构建的HCMV基因组表达文库中筛选到与pUL23相互作用的病毒编码蛋白pUL24.GST-pull down实验和免疫共沉淀实验进一步确认两种病毒蛋白之间的相互作用.结果表明,构建的HCMV基因组表达文库能够用于GAL40酵母双杂交系统筛选与诱饵蛋白相互作用的病毒自身编码蛋白.病毒蛋白pUL23和pUL24之间具有相互作用,这为进一步阐述pUL23在HCMV感染过程中的功能提供依据.该研究为揭示HCMV病毒感染机制奠定了基础.%Human cytomegalovirus(HCMV) UL23 gene codes a tegument protein pUL23, and the UL23 deletion HCMV mutant exhibited enhanced growth in HFF cells. In order to provide an effective tool for further study of the function and regulatory mechanism of HCMV pUL23, a random genomic library of HCMV strains Towne fused to the GAL4 activation domain was constructed by shot-gun method. Using this library, we performed yeast two-hybrid screening with a bait plasmid encoding a fusion of the GAL4 DNA binding domain (GAL4-BD) with the UL23 open reading frame. One of the interacting clones corresponds to HCMV protein pUL24. GST-pull down and co-immunoprecipitation assays confirmed the interaction between these two vial proteins. These results demonstrated that the viral genomic library could be applied to GAL4 yeast two-hybrid assay for screening HCMV proteins which interact with the bait protein. And the interaction between HCMV proteins pUL23 and pUL24 will be further characterized to elucidate the functions of pUL23 during HCMV infection. It was laid a foundation for further study of the infection mechanism of

  11. Planning a Successful Tech Show (United States)

    Nikirk, Martin


    Tech shows are a great way to introduce prospective students, parents, and local business and industry to a technology and engineering or career and technical education program. In addition to showcasing instructional programs, a tech show allows students to demonstrate their professionalism and skills, practice public presentations, and interact…

  12. Cry1Ab protein quantification in leaves, stems and grains, and effectiveness to control Spodoptera frugiperda and Helicoverpa zea on two hybrids of genetically modified corn

    Directory of Open Access Journals (Sweden)

    Geraldo Balieiro Neto


    Full Text Available A study was carried out to evaluate the infestation and associated damages to the presence of the Spodoptera frugiperda and Helicoverpa zea caterpillars, in two genetically modified (GM corn, Dekalb DKB390 and Agroceres AG8088, expressing the cry1Ab protein. For this objective, an split-splot design with two factors (hybrid x gene was carried out. Negative controls were made with the same corn hybrids without the gene cry1Ab (NoGM. The concentration of the protein Cry1Ab was determined by the ELISA (enzyme linked immuno sorbent assay technique in previously dehydrated stems, leaves and grains of GM corns. Caterpillars sampling of S. frugiperda and associated damage survey were accomplished at 15, 22, 29, 36 and 42 days after the sowing, according to a damage scale with 5 levels (0- pest absence to 5- dead plant. Countings of H. zea caterpillars and associated damage were assessed at 57, 71, 78 and 85 days after the sowing, according to a damage scale with 4 levels (0-pest absence curse to 4-gallery in the corn cob minor than 3cm. Sampled caterpillars were divided in two groups, smaller or equal to 15mm and bigger than 15mm. No insecticide application was accomplished in the GM blocks while NoGM blocks were sprayed with deltametrina (2,8%, 42 days after the sowing. The infestation level and associated damage due to S. frugiperda presence was significantly smaller (p < 0,05 in the GM corns in comparison to NoGM corns. Nevertheless, the number and associated damage of S. frugiperda caterpillars, smaller than 15 mm, were superior in the GM DKB390 corn when compared to the GM AG8088 corn. Differences were not observed in the S. frugiperda infestation and associated damage between GM corns and between NoGM corns. On average, the concentration of Cry1Ab protein was significantly superior in leaves and stems in comparison to the grain and, usually, superior in the GM AG8088 corn comparatively to GM DKB390 corn. No differences were found on level damages

  13. Bacterial assays for recombinagens. (United States)

    Hoffmann, G R


    Two principal strategies have been used for studying recombinagenic effects of chemicals and radiation in bacteria: (1) measurement of homologous recombination involving defined alleles in a partially diploid strain, and (2) measurement of the formation and loss of genetic duplications in the bacterial chromosome. In the former category, most methods involve one allele in the bacterial chromosome and another in a plasmid, but it is also possible to detect recombination between two chromosomal alleles or between two extrachromosomal alleles. This review summarizes methods that use each of these approaches for detecting recombination and tabulates data on agents that have been found to be recombinagenic in bacteria. The assays are discussed with respect to their effectiveness in testing for recombinagens and their potential for elucidating mechanisms underlying recombinagenic effects.

  14. Risk Aversion in Game Shows

    DEFF Research Database (Denmark)


    We review the use of behavior from television game shows to infer risk attitudes. These shows provide evidence when contestants are making decisions over very large stakes, and in a replicated, structured way. Inferences are generally confounded by the subjective assessment of skill in some games......, and the dynamic nature of the task in most games. We consider the game shows Card Sharks, Jeopardy!, Lingo, and finally Deal Or No Deal. We provide a detailed case study of the analyses of Deal Or No Deal, since it is suitable for inference about risk attitudes and has attracted considerable attention....

  15. Measuring performance at trade shows

    DEFF Research Database (Denmark)

    Hansen, Kåre


    Trade shows is an increasingly important marketing activity to many companies, but current measures of trade show performance do not adequately capture dimensions important to exhibitors. Based on the marketing literature's outcome and behavior-based control system taxonomy, a model is built...... that captures a outcome-based sales dimension and four behavior-based dimensions (i.e. information-gathering, relationship building, image building, and motivation activities). A 16-item instrument is developed for assessing exhibitors perceptions of their trade show performance. The paper presents evidence...

  16. Identification of calcium/calmodulin-binding receptor-like kinase GsCBRLK-interactive proteins using yeast two-hybrid system%酵母双杂交筛选与GsCBRLK相互作用的蛋白质

    Institute of Scientific and Technical Information of China (English)

    杨姗姗; 孙晓丽; 于洋; 才华; 纪巍; 柏锡; 朱延明


    GsCBRLK(calcium/calmodulin-binding receptor-like kinase from Glycine soja)在ABA及盐胁迫诱导的钙离子信号通路中起到关键的调节作用.为深入研究GsCBRLK蛋白的作用机制,文章采用膜酵母双杂交系统,以GsCBRLK为诱饵蛋白,筛选与其相互作用的蛋白质.通过构建野生大豆盐胁迫条件下的cDNA文库、膜酵母双杂交系统筛选、复筛、回转验证、生物信息学分析以及酵母体内互作验证等手段,最终获得2个(SNARE和14-3-3蛋白)与GsCBRLK诱饵蛋白相互作用的蛋白质.%GsCBRLK (calcium/calmodulin-binding receptor-like kinase from Glycine soja) links ABA (abscisic acid)-and salt-induced calcium/calmodulin signal in plant cells. In order to study the molecular mechanismes of GsCBLRK, the salt-treated Glycine soja cDNA library was screened with pB73-STE-CBRLK as bait plasmid using yeast two hybrid system. Two positive clones (SNARE and 14-3-3 protein) were identified by constructing cDNA library of wild soybean under salt treatment, membrane system yeast two hybrid screening, multiple screen, rotary validation, bioinformatic analysis and interaction identification in yeast.

  17. Tokyo Motor Show 2003; Tokyo Motor Show 2003

    Energy Technology Data Exchange (ETDEWEB)

    Joly, E.


    The text which follows present the different techniques exposed during the 37. Tokyo Motor Show. The report points out the great tendencies of developments of the Japanese automobile industry. The hybrid electric-powered vehicles or those equipped with fuel cells have been highlighted by the Japanese manufacturers which allow considerable budgets in the research of less polluting vehicles. The exposed models, although being all different according to the manufacturer, use always a hybrid system: fuel cell/battery. The manufacturers have stressed too on the intelligent systems for navigation and safety as well as on the design and comfort. (O.M.)

  18. Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs. (United States)

    de Waaij, Dewi J; Ouburg, Sander; Dubbink, Jan Henk; Peters, Remco P H; Morré, Servaas A


    This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05).

  19. Screening of Host Cell Proteins that Interact with Toxoplasma gondii ROP18 via Yeast Two-hybrid System%酵母双杂交筛选与弓形虫毒力因子ROP18相互作用的宿主蛋白

    Institute of Scientific and Technical Information of China (English)

    都建; 安然; 程里; 陈滢; 沈继龙


    目的 利用酵母双杂交方法,筛选与弓形虫毒力因子ROP18相互作用的宿主蛋白. 方法 RT-PCR扩增弓形虫RH株速殖子ROP18基因片段,扩增产物经双酶切后插入酵母双杂交诱饵载体pGBKT7中,将重组质粒导入酵母AH109菌株中,检测诱饵载体有无毒性和自激活作用,利用酵母双杂交系统从人胚胎脑cDNA文库中筛选与ROP18相互作用的宿主蛋白. 结果 构建了诱饵载体pGBKT7-ROP18,ROP18具有自激活功能.用ROP1825-251aa为诱饵,筛选获得一系列与ROP18相互作用的宿主蛋白:DNA损伤特异性结合蛋白1(DDB1)、torsin A相互作用蛋白1(TOR1AIP1)、整联蛋白β31 (integrinβ1)、溶质运载蛋白3(SLC3A2)、酪氨酸硫化转移酶2(TPST2)、Derl样域家族成员2 (DERL2)和OCIA结构域蛋白1(OCIAD1). 结论 通过酵母双杂交技术筛选出与弓形虫毒力因子ROP18相互作用的多种宿主蛋白.%Objective To screen the host cell proteins that can interact with Toxoplasma gondii ROP18 by using yeast two-hybrid system. Methods The ROP18 gene fragments were amplified by RT-PCR from mRNA of T. gondii RH strain. The product of RT-PCR was digested with double restriction enzyme and was subcloned into the bait vector pGBKT7. The recombinant plasmid was transferred into yeast AH109 strain. Its toxicity and the autonomous activating activity were tested. The human fetal brain cDNA library was screened with pGBKT7-ROP1825-25laa as the bait plasmid by yeast two-hybrid system. Results The bait was constructed and AH109/PGBKT7-ROP18 showed an autonomous activity. The yeast strain AH109/pGBKT7-ROP1825-251aa line was then mated with the Mate & PlateTM Human Fetal Brain cDNA library. Using the selection procedures, eight novel host cell proteins were obtained: damage-specific DNA binding protein 1 (DDB1), torsin A interacting protein 1 (TOR1AIP1), integrin beta 1, solute carrier family 3 (SLC3A2), tyrosylprotein sulfotransferase (TPST2), OCIA domain containing 1 (OCIAD1

  20. EDTA interference in electrochemiluminescence ACTH assay. (United States)

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer


    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  1. Risk Aversion in Game Shows

    DEFF Research Database (Denmark)

    Andersen, Steffen; Lau, Morten I.; Rutström, E. Elisabet


    , and the dynamic nature of the task in most games. We consider the game shows Card Sharks, Jeopardy!, Lingo, and finally Deal Or No Deal. We provide a detailed case study of the analyses of Deal Or No Deal, since it is suitable for inference about risk attitudes and has attracted considerable attention....

  2. Phyllodes tumor showing intraductal growth. (United States)

    Makidono, Akari; Tsunoda, Hiroko; Mori, Miki; Yagata, Hiroshi; Onoda, Yui; Kikuchi, Mari; Nozaki, Taiki; Saida, Yukihisa; Nakamura, Seigo; Suzuki, Koyu


    Phyllodes tumor of the breast is a rare fibroepithelial lesion and particularly uncommon in adolescent girls. It is thought to arise from the periductal rather than intralobular stroma. Usually, it is seen as a well-defined mass. Phyllodes tumor showing intraductal growth is extremely rare. Here we report a girl who has a phyllodes tumor with intraductal growth.

  3. Pembrolizumab Shows Promise for NSCLC. (United States)


    Data from the KEYNOTE-001 trial show that pembrolizumab improves clinical outcomes for patients with advanced non-small cell lung cancer, and is well tolerated. PD-L1 expression in at least 50% of tumor cells correlated with improved efficacy.

  4. Create a Polarized Light Show. (United States)

    Conrad, William H.


    Presents a lesson that introduces students to polarized light using a problem-solving approach. After illustrating the concept using a slinky and poster board with a vertical slot, students solve the problem of creating a polarized light show using Polya's problem-solving methods. (MDH)

  5. The interaction between LHBs and homo sapiens pancreatic elastase 2A protein using mammalian two-hybrid%哺乳动物双杂交技术验证HBV表面抗原大蛋白与胰腺弹性蛋白酶2A的相互作用

    Institute of Scientific and Technical Information of China (English)

    刘文祥; 张楠; 汪涛; 侯鹏


    目的 验证乙型肝炎病毒(hepatitis B virus,HBV)表面抗原大蛋白(hepatitis B virus large surface protein,LHBs)与人胰腺弹性蛋白酶2A(elastase 2A)蛋白在细胞内是否存在明确的相互作用关系.方法 构建真核表达质粒pACT-Elastase 2A,设立阳性对照组、实验组、阴性对照组和空细胞组,用哺乳动物双杂交技术验证LHBs与人胰腺Elastase 2A蛋白之间的相互作用.结果 实验组与3个阴性对照组之间,均数都存在统计学差异(其P值分别为0.031、0.006和0.007).结论 哺乳动物双杂交技术证实了LHBs与人胰腺Elastase 2A蛋白在Capan2细胞内存在相互作用关系.%Objective To validate the intra-cellular interaction between LHBs and homo sapiens elastase 2A protein.Methods At first,we constructed eukaryotic expression vector pACT-Elastase 2A and confirmed the interaction between LHBs and homo sapiens elastase 2A protein by mammalian two-hybrid experiment.We set up positive control group,experimental group,negative control group and empty cell group.Results We constructed successfully eukaryotic expression vector pACT-Elastase 2A.The results of mammalian two-hybrid experiment showed that there were significant difference in means between experimental group and negative controls(experimental group vs group 1:P =0.031 ;experimental group vs group 2:P =0.006 ;experimental group vs group3:P =0.007).There were significantly statistics difference between experimental group and each negative control group.Conclusions LHBs can directly interact with homo sapiens elastase 2A protein in intra-cell.

  6. Herbicide resistance screening assay. (United States)

    Peterson, Joan M


    Herbicide resistance screening is a method that can be used not only to determine presence of the enzyme, phosphinothricin acetyltransferase, encoded by either the Bar or the Pat gene in transgenic maize, but also to assess the inheritance ratio of those genes in a segregating population. Herbicide screening can also be used to study linkage of a transgene of interest that was cotransformed with the herbicide resistance marker gene. By combining the herbicide screen assay with a PCR-based screen of leaf tissue DNA for the presence of both the Bar or the Pat gene marker and a cotransformed transgene of interest from the same seedling tissue and maintaining that seedling identity, the researcher can identify linkage or the possible breakdown in linkage of the marker gene and the transgene of interest. Further, the occurrence of "DNA silencing" can be evaluated if an individual seedling that was susceptible to the applied herbicide nonetheless gave PCR data that indicated presence of the gene responsible for herbicide resistance. Similarly, "DNA silencing" of the gene of interest may be investigated if the seeds can be screened and scored for that phenotypic trait in a nondestructive manner prior to planting.

  7. Reality show: um paradoxo nietzschiano

    Directory of Open Access Journals (Sweden)

    Ilana Feldman


    Full Text Available

    O fenômeno dos reality shows - e a subseqüente relação entre imagem e verdade - assenta-se sobre uma série de paradoxos. Tais paradoxos podem ser compreendidos à luz do pensamento do filósofo alemão Friedrich Nietzsche, que, através dos usos de formulações paradoxais, concebia a realidade como um mundo de pura aparência e a verdade como um acréscimo ficcional, como um efeito. A ficção é então tomada, na filosofia de Nietzsche, não em seu aspecto falsificante e desrealizador - como sempre pleiteou nossa tradição metafísica -, mas como condição necessária para que certa espécie de invenção possa operar como verdade. Sendo assim, a própria expressão reality show, através de sua formulação paradoxal, engendra explicitamente um mundo de pura aparência, em que a verdade, a parte reality da proposição, é da ordem do suplemento, daquilo que se acrescenta ficcionalmente - como um adjetivo - a show. O ornamento, nesse caso, passa a ocupar o lugar central, apontando para o efeito produzido: o efeito-de-verdade. Seguindo, então, o pensamento nietzschiano e sua atualização na contemporaneidade, investigaremos de que forma os televisivos “shows de realidade” operam paradoxalmente, em consonância com nossas paradoxais práticas culturais.

  8. 猪瘟病毒酵母双杂交 pGBK T7-NS3诱饵载体的构建与鉴定%Construction and Identification of CSFV pGBKT7-NS3 Bait Vector in Yeast Two-hybrid System

    Institute of Scientific and Technical Information of China (English)

    宋江伟; 田宏; 吴锦艳; 陈妍; 尚佑军; 刘湘涛


    The NS3 gene of classical swine fever virus was amplified by RT-PCR and confirmed by sequen-cing and enzyme digestion .The NS3 gene was then subcloned into pGBKT7 to construct the bait vector ac-cording to the reading frame .The reconstructed bait vector pGBKT7-NS3 was transformed into Y2H Gold yeast strain ,its self-activation and toxic effect were tested by the phenotype assay .The expression of pG-BKT7-NS3 was analysed by Western blot .As a result ,the successfully reconstructed bait vector pGBKT7-NS3 would not self-activitate the reporter gene and had no toxic effect to the yeast ,which laid the founda-tion for using yeast two-hybrid assay to screen interaction proteins .%利用 RT-PCR技术扩增获得猪瘟病毒NS3基因片段,将其克隆至酵母双杂交系统诱饵载体pGBKT7中,经酶切,测序验证其正确插入后,将重组诱饵质粒转化酵母菌Y2H Gold ,检测其在酵母中毒性和自我激活作用,并利用Western blot分析诱饵载体在酵母中的转化情况。结果表明,pGBKT7-NS3诱饵载体在Y2H Gold酵母细胞中可以表达,此载体在酵母细胞Y2H Gold中无毒性和自我激活能力,研究结果为使用酵母双杂交技术筛选相互作用蛋白奠定了基础。

  9. Picasso on Show in Shanghai

    Institute of Scientific and Technical Information of China (English)


    A staff member of the National Picasso Museum of France checks one of the great Spanish artist Pablo Picasso’s works at the China Pavilion inside the site of the 2010 World Expo in Shanghai on October 12.Sixty-two priceless paintings and statues selected from the works of the renowned artist have been brought to the pavilion for an upcoming exhibition to premiere on October 18.Besides these representative masterpieces,50 valuable photographs showing the artist’s whole life will also be presented.The exhibition’s estimated value is 678 million euros ($934 million).It will be held until January 10,2012.

  10. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

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


    assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......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......% tested positive on all four. Agreement was lower in women aged ≥ 30 years (30%, vs. 49% at samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30...

  11. Reality shows: uma abordagem psicossocial

    Directory of Open Access Journals (Sweden)

    Marília Pereira Bueno Millan

    Full Text Available Desde os primórdios da civilização, o ser humano mostra necessidade de representar cenicamente seus dramas pessoais e vicissitudes existenciais. O "reality show" é uma das versões pós-modernas da encenação da vida humana. Este artigo, por meio de uma pesquisa bibliográfica, analisa criticamente as relações existentes entre o "reality show" e aspectos psicossociais do comportamento humano. Conclui-se que tais programas televisivos são o retrato da contemporaneidade, ou seja, revelam a morte do sujeito, a fugacidade das experiências vividas, a desvalorização da história e o culto à imagem e à superficialidade. Por meio da sedução do espectador, mobilizam-se aspectos primitivos de seu psiquismo, fazendo com que ele se sinta narcisicamente poderoso e onipotente e se acredite dono do destino dos participantes do programa. Sugerem-se novos estudos que contribuam para a reflexão crítica e maior conscientização.

  12. "Medicine show." Alice in Doctorland. (United States)


    This is an excerpt from the script of a 1939 play provided to the Institute of Social Medicine and Community Health by the Library of Congress Federal Theater Project Collection at George Mason University Library, Fairfax, Virginia, pages 2-1-8 thru 2-1-14. The Federal Theatre Project (FTP) was part of the New Deal program for the arts 1935-1939. Funded by the Works Progress Administration (WPA) its goal was to employ theater professionals from the relief rolls. A number of FTP plays deal with aspects of medicine and public health. Pageants, puppet shows and documentary plays celebrated progress in medical science while examining social controversies in medical services and the public health movement. "Medicine Show" sharply contrasts technological wonders with social backwardness. The play was rehearsed by the FTP but never opened because funding ended. A revised version ran on Broadway in 1940. The preceding comments are adapted from an excellent, well-illustrated review of five of these plays by Barabara Melosh: "The New Deal's Federal Theatre Project," Medical Heritage, Vol. 2, No. 1 (Jan/Feb 1986), pp. 36-47.

  13. 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


    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.

  14. Casimir experiments showing saturation effects

    CERN Document Server

    Sernelius, Bo E


    We address several different Casimir experiments where theory and experiment disagree. First out is the classical Casimir force measurement between two metal half spaces; here both in the form of the torsion pendulum experiment by Lamoreaux and in the form of the Casimir pressure measurement between a gold sphere and a gold plate as performed by Decca et al.; theory predicts a large negative thermal correction, absent in the high precision experiments. The third experiment is the measurement of the Casimir force between a metal plate and a laser irradiated semiconductor membrane as performed by Chen et al.; the change in force with laser intensity is larger than predicted by theory. The fourth experiment is the measurement of the Casimir force between an atom and a wall in the form of the measurement by Obrecht et al. of the change in oscillation frequency of a 87 Rb Bose-Einstein condensate trapped to a fused silica wall; the change is smaller than predicted by theory. We show that saturation effects can exp...

  15. Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test. (United States)

    Hagio, Soichiro; Furukawa, Satoshi; Abe, Masayoshi; Kuroda, Yusuke; Hayashi, Seigo; Ogawa, Izumi


    Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy or in situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumi et al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other.

  16. Construction and evaluation of yeast two-hybrid cDNA library of Diaphorina citri (Hemiptera: Psyllidae)%柑橘木虱酵母双杂交cDNA文库的构建及评价

    Institute of Scientific and Technical Information of China (English)

    马晓芳; 陈国庆; 张学潮; 徐海君


    In order to study the interacting proteins between the Asian citrus psyllid ( Diaphorina citri) and Candidatus Liberibacter asiaticus ( CLas) which is the pathogenic bacterium causing Huanglongbing, yeast two-hybrid cDNA library of D. citri was constructed using the Switching Mechanism at 5' End of the RNA Transcript (SMART) technique. The total RNA was isolated from the citrus psyllid adults bred in the laboratory and subjected to reverse transcription, and the double-strand cDNAs ( ds cDNAs) were synthesized. The ds cDNAs were ligated with homologous adapter and purified by the chromatography column. By using homologous recombination reaction, the ds cDNAs were transformed into the Y187 competent cell with the library plasmid pGADT7-Rec to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained more than 106 independent clones, the titer of the amplified library was 2. 23 × 10 cfu/mL, and the average size of inserts was above 750 bp in the cDNA library. These results demonstrate that the library meets the requirements of the standard cDNA library. Moreover, two membrane proteins, ORF420 and ORF3420, from ( CLas) were used as bait proteins to screen the interacting proteins in the library, but no positive clone was screened in the tests. The yeast two-hybrid cDNA library of D. citri will be useful for the research on the interaction between insect vectors and C. Liberibacter asiaticus in the future.%为了探索研究柑橘木虱Diaphorina citri与柑橘黄龙病(Huanglongbing,HLB)病原菌的相互作用蛋白,本研究运用RNA转录中5 '末端转换机制(Switching Mechanism at 5'End of the RNA Transcript,SMART)技术构建了柑橘木虱的酵母双杂交cDNA文库.以实验室饲养的柑橘木虱为材料,提取总RNA,经反转录后合成ds cDNA,两端添加同源重组序列,并用层析柱纯化;ds cDNA与文库质粒pGADT7-Rec在酵母Y187感受态细胞内发生同源重组,柑橘木虱cDNA重组到文库质粒

  17. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay. (United States)

    Chang, A; Dusing, S


    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  18. From Antenna to Assay (United States)

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.


    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  19. Transporter assays and assay ontologies: useful tools for drug discovery. (United States)

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P


    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  20. The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B. (United States)

    Wang, Bing; Pelletier, Jerry; Massaad, Michel J; Herscovics, Annette; Shore, Gordon C


    In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31. BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis. Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein. PTPLB turns over rapidly through degradation by the proteasome system. Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.

  1. The establishment of a library screening method based on yeast two-hybrid system and its use to determine the potential interactions of liver proteins in ayu, Plecoglossus altivelis. (United States)

    Shi, Y H; Chen, J; Li, C H; Yang, H Y; Lu, X J


    Knowledge of specific protein-protein interaction (PPI) is an important component in understanding biological processes and regulatory mechanisms. A library to library screening method (LLS) was established based on yeast two-hybrid (YTH) system in this research, and applied to study the PPIs in ayu liver. In total, 23 out of 55 interaction pairs were found positive through phenotypic identification, with a positive rate of 41.8%. Of the 11 unique PPIs, 9 interactions including FGB/FGG, CaM/Spna2, C9/Apo-AI-1, α₂M/Ft, RPL10/RPL5, C8α/C9, FGG/Apo-AI-1, LECT2/Tf, and Apo-AI-2/C9 were previously reported. The other two PPIs including FGG/CLR and Wap65/C3 are novel, and in vitro co-immunoprecipitation (co-IP) experiments further confirmed these interactions. FGG/CLR interaction might play a role in regulating the inflammatory response. The interaction between Wap65 and C3 hints that Wap65 might function through the complement activation pathways when microbial infection occurs.

  2. A simultaneous disulfide bond cleavage, N,S-bialkylation/N-protonation and self-assembly reaction: syntheses, structures and properties of two hybrid iodoargentates with thiazolyl-based heterocycles. (United States)

    Liu, Guang-Ning; Li, Ke; Fan, Qing-Shun; Sun, Hui; Li, Xin-Yu; Han, Xiao-Nan; Li, Yu; Zhang, Zhen-Wei; Li, Cuncheng


    Solvothermal reactions of AgI with the vulcanization accelerator 2,2'-dibenzothiazolyl disulfide and hydroiodic acid in alcohols afford two hybrid iodoargentates with one-dimensional structures, namely (Et2mbt)[Ag2I3] (1, Hmbt = 2-mercaptobenzothiazole) and {(Hmbt)[AgI]}n (2). The syntheses of both 1 and 2 involve unprecedented multiple in situ reactions. Specifically, a simultaneous disulfide bond cleavage, N and S donor atoms bialkylation, and self-assembly reaction lead to 1 that contains a discrete N,S-biethylated cation (Et2mbt)(+) and a rare inorganic (Ag2I3)(-) anionic chain, while a simultaneous disulfide bond cleavage, N-protonation and self-assembly reaction affords 2 which features zwitterionic Hmbt molecules coordinating with the opposite side Ag atoms of a neutral inorganic (AgI)n chain via forming Ag-S coordination bonds. The simultaneous alkylation on a thiazolyl-N donor and a thiol-S donor atom for a thiazolyl-based heterocycle using inexpensive alcohols and haloids under solvothermal conditions instead of traditional two-step organic synthesis was found for the first time. The two compounds have band gaps of 2.95 eV for 1, and 2.78 eV for 2 exhibiting an observed blue shift compared with bulk AgI. Also, 1 and 2 can be used as effective heterogeneous photocatalysts for methyl orange dye treatment under UV light irradiation.



    Bishnoi Kapil*, , ,; Kataria Mahesh; Singhal Vipin; Gupta Deepika


    Micronutrients added to foods are analyzed using various procedures depending on their nature and properties. The microbiological assays are better than chemical method because any suitable change in vitamin molecule which may not be detected by chemical method will be revealed by change in microbial activity. The microbiological assay of vitamins is based upon the comparison of the stimulation of growth of bacteria by measured concentration of vitamin with that produced by known concentratio...

  4. Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies (United States)

    Hanson, Sonya M.; Ekins, Sean; Chodera, John D.


    All experimental assay data contains error, but the magnitude, type, and primary origin of this error is often not obvious. Here, we describe a simple set of assay modeling techniques based on the bootstrap principle that allow sources of error and bias to be simulated and propagated into assay results. We demonstrate how deceptively simple operations—such as the creation of a dilution series with a robotic liquid handler—can significantly amplify imprecision and even contribute substantially to bias. To illustrate these techniques, we review an example of how the choice of dispensing technology can impact assay measurements, and show how large contributions to discrepancies between assays can be easily understood and potentially corrected for. These simple modeling techniques—illustrated with an accompanying IPython notebook—can allow modelers to understand the expected error and bias in experimental datasets, and even help experimentalists design assays to more effectively reach accuracy and imprecision goals.

  5. Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies. (United States)

    Hanson, Sonya M; Ekins, Sean; Chodera, John D


    All experimental assay data contains error, but the magnitude, type, and primary origin of this error is often not obvious. Here, we describe a simple set of assay modeling techniques based on the bootstrap principle that allow sources of error and bias to be simulated and propagated into assay results. We demonstrate how deceptively simple operations--such as the creation of a dilution series with a robotic liquid handler--can significantly amplify imprecision and even contribute substantially to bias. To illustrate these techniques, we review an example of how the choice of dispensing technology can impact assay measurements, and show how large contributions to discrepancies between assays can be easily understood and potentially corrected for. These simple modeling techniques--illustrated with an accompanying IPython notebook--can allow modelers to understand the expected error and bias in experimental datasets, and even help experimentalists design assays to more effectively reach accuracy and imprecision goals.

  6. Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples. (United States)

    Danylo, Alexis; Courtemanche, Chantal; Pelletier, René; Boudreault, Alexandre A


    Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

  7. Screening of Putative Proteins That are Interacted with NBS-LRR Protein Pik-h by the Yeast Two-Hybrid System%水稻NBS-LRR类抗稻瘟病蛋白Pik-h的互作蛋白筛选

    Institute of Scientific and Technical Information of China (English)

    王加峰; 刘浩; 王慧; 陈志强


    信号蛋白、4个参与光合作用的叶绿体蛋白、1个含有U-BOX结构域的蛋白及4个未知功能蛋白。【结论】成功构建了适宜于研究抗病基因(R基因)介导反应途径的酵母双杂交cDNA文库,筛选出Pik-h的互作蛋白,为进一步研究Pik-h或其他抗稻瘟病基因介导的抗病机制打下了基础。%[Objective] To further investigate the signaling pathways activated directly by the resistance proteins Pikh-1 and Pikh-2 from the resistance gene Pik-h, yeast two-hybrid assay was performed to screen a rice leaf cDNA library using Pikh-1 and Pikh-2 as bait, respectively.[Method] All rice leaves of IRBL8 (Pik-hNIL) from the time points (12 and 24 h after inoculation with GD0193) were harvested for total RNA preparation. The rice leaf cDNA library was further constructed with the Make Your Own Mate&Plate Library System kit (Clontech). Bait plasmids pGBKT7-Pikh1 and pGBKT7-Pikh2 were constructed using the rapid homologous recombination method. The expression of the two proteins in the Y2H Gold strain was further detected with Western blot and the self-activation and cytotoxicity were also tested. Proteins interacting with the baits Pikh1 and Pikh2 were screened on SD/-Ade/-His/-Leu/-Trp/X-α-Gal plates from the rice leaf cDNA library with the mating strategy. Then the plasmids were prepared from the blue interacting clones and retransformed back into Y2H Gold strain with the corresponding bait. The plasmids that passed the reconfirmation were sequenced and the DNA sequences were further used to blast rice genome annotation project database. The functions of the interaction proteins were further analyzed with gene ontology (GO) annotation.[Result] The rice leaf cDNA library contained 2.2×106 independent clones. The sizes of most inserts were above 400 bp in the cDNA library. Bait plasmids (pGBKT7-Pikh1 and pGBKT7-Pikh2) expressed the corresponding proteins (BD-Pikh-1 and BD-Pikh-2) well and showed no self-activation and cytotoxicity

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

    Directory of Open Access Journals (Sweden)

    Carina Ladeira


    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.

  9. Screening of proteins interacting with HSPC016 by yeast two-hybridization technique%用酵母双杂交技术筛选与HSPC016相互作用的蛋白

    Institute of Scientific and Technical Information of China (English)

    宋志强; 孙丽华; 郝飞


    目的 采用酵母双杂交技术筛选毛乳头细胞中与造血干细胞(hematopoietic stem/progenitor cells,HSPC)分化相关基因HSPC016相互作用的蛋白,了解其参与调控毛乳头细胞凝集性生长的分子机制.方法 应用酵母双杂交系统,将构建的pGBKT7-HSPC016诱饵质粒与含原代人毛乳头细胞cDNA文库质粒的酵母Y187进行配合,筛选与HSPC016相互作用的蛋白,通过回复性杂交试验验证其可靠性,并对阳性克隆进行测序和生物信息学分析.结果 筛选出4个与HSPC016有相互作用的蛋白,包括转录因子叉头框蛋白O1(forkhead box O1,FOXO1)、丝裂原活化蛋白激酶11、磷酸次黄苷酸激酶3调节亚单位3(PIK3R3)、肝X受体,它们均与细胞内能量代谢、转录调节相关.结论 HSPC016可能通过参与细胞内活性氧水平调节,并与细胞内参与能量和转录调节的相关信号分子相互作用,调节毛乳头细胞的凝集性生长状态.%Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization,HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid (named as pGBKT7-HSPC016) in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then,the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH 109. Four proteins,including forkhead family of transcription factors (FOXO 1 ), mitogen

  10. 肝细胞生成素在睾丸组织中的相互作用蛋白%Identification of Hepatopoietin Interacting Proteins in Human Testis by Yeast Two-hybrid System

    Institute of Scientific and Technical Information of China (English)

    陈筱潇; 李勇; 邱宗荫; 贺福初


    Hepatopoietin (HPO) has diverse functions in the regulation of cell growth and differentiation. Its high level expression in testis suggests that HPO may have important role for testicular physiological functions. Using HPO as "bait", a yeast two-hybrid library screen of human testis was performed. By screening and selecting the positive colonies, retesting interactions in yeast, amplifying the AD/library inserts, sequencing and sequence comparing, four HPO-interacting proteins were identified: NADH dehydrogenase 1, Na+/K+ ATPase beta-3 subunit (ATP1β3), phospholipase C delta 1 and epididymal secretory protein. The significance of identification of these proteins may provide mechanistic insight into the biologic role of HPO in testis.%肝细胞生成素(HPO)具有复杂的生理功能,在睾丸中的高表达提示其在生殖活动中的重要性,而不仅局限于肝再生.构建了酵母表达载体pGBKT7-HPO,采用酵母双杂交系统,以HPO为诱饵蛋白,从人睾丸cDNA文库中寻找能够与HPO相互作用的蛋白质.经过筛选、验证阳性克隆,并进行PCR、测序和序列比对,得到4种相互作用蛋白质:NADH脱氢酶1、钠/钾ATP酶β3亚基、磷脂酶C δ1以及附睾分泌蛋白.提示HPO可能参与了细胞的蛋白质合成,能量代谢等.通过对候选蛋白的研究,为探讨HPO对睾丸组织细胞功能的调节机制提供了重要的线索.

  11. A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays. (United States)

    Hsu, Denise C; Zaunders, John J; Plit, Marshall; Leeman, Craig; Ip, Susanna; Iampornsin, Thatri; Pett, Sarah L; Bailey, Michelle; Amin, Janaki; Ubolyam, Sasiwimol; Avihingsanon, Anchalee; Ananworanich, Jintanat; Ruxrungtham, Kiat; Cooper, David A; Kelleher, Anthony D


    A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.

  12. Construction, expression and identification of yeast two-hybrid bait vector pGBKT7-tH of peste des petits ruminants virus%小反刍兽疫病毒酵母双杂交诱饵载体pGBKT7-tH的构建、表达和鉴定

    Institute of Scientific and Technical Information of China (English)

    蒙学莲; 朱学亮; 翟军军; 窦永喜; 才学鹏


    The fragment of tH was amplified by PCR,and was cloned into the bait expression vector pGBKT7. After being verified by restriction digestion and sequencing, the bait vector was transformed into yeast cells AH109. The toxicity,leakage and self-transcriptional activation of PPRV-tH protein were firstly detected. Then the expression of was analyzed by Western blotting. Results showed that PPRV-tH was amplified and cloned into pGBKT7 successfully. The bait protein vector was transformed into AH109 as well and the toxicity,leakage and self- transcriptional activation of PPRV-tH protein were not observed. The PPRV-tH protein could be effectively expressed in transformed yeast. It plays a critical part in works of screening receptor gene by yeast two-hybrid system.%利用PCR技术扩增获得PPRV-tH基因片段,将其克隆至酵母双杂交系统诱饵载体pGBKT7中,经酶切、测序验证其正确插入后,将重组诱饵质粒转化酵母菌AH109中,检测其在酵母中有无渗漏、自我激活作用和毒性.利用western blotting分析诱饵蛋白在酵母中的表达情况,以鉴定其作为诱饵蛋白的可行性.结果表明,成功扩增到了PPRV-tH,并正确构建了pGBKT7-tH诱饵表达载体,此载体在酵母细胞AH109中无毒性、渗漏和自我激活能力,且能正确表达tH蛋白.

  13. Assay optimization for molecular detection of Zika virus (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


    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

  14. Barcoded microchips for biomolecular assays. (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu


    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  15. Radioreceptor assay method for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Mori, K.F.; Wood, R.J. (Bureau of Drug Research, Health and Welfare Canada, Ottawa, Ontario. Health Protection Branch)


    A sensitive practical radioreceptor assay method for pharmaceutical insulin products has been developed with partially purified rat liver plasma membranes and the optimal conditions under which the best overall assay performance is obtainable have been defined. Intra- and inter-assay variations of the method averaged 7.3 and 12.2% of the man, respectively, when expressed as the coefficient of variation. Potency estimates of an insulin product obtained with the proposed method correlated well with those determined by the mouse convulsion bioassay method. Liver membranes prepared according to the method could be stored for up to ten weeks at 4/sup 0/C and for 6 months or more at -18/sup 0/C without losing insulin-binding ability.

  16. Diagnostic Certified Assay: Neuromuscular and Cardiac Assessments

    Directory of Open Access Journals (Sweden)

    Rea Valaperta


    Full Text Available The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%. Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results.


    Institute of Scientific and Technical Information of China (English)

    王自文; 黄兵; 董辉; 赵其平; 夏伟丽; 朱顺海; 门启婓; 李聪; 朱雪龙; 韩红玉


    为了筛选出柔嫩艾美耳球虫钙依赖蛋白激酶(EtCDPK4)在虫体内发挥作用时的作用受体即相互作用蛋白,构建了能够应用于筛选柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库的诱饵重组质粒pGBKT7-EtCDPK4,本研究以柔嫩艾美耳球虫子孢子为材料,提取了总RNA,经反转录得到cDNA第一链,利用PCR的方法扩增获得了该基因大小为1660 bp的EtCDPK4的功能区片段,并将其连接于酵母BD诱饵pGBKT7载体上,经双酶切鉴定和序列分析,以及检测了pGBKT7-EtCDPK4在酵母双杂交系统中的自激活、细胞毒性和表达情况。结果显示,成功构建了酵母双杂交诱饵重组质粒pGBKT7-EtCDPK4,在酵母双杂交系统中没有自激活活性和细胞毒性,且在酵母细胞Y2HGold中能够进行表达。表明所构建的诱饵质粒pGBKT7-EtCDPK4可以应用于酵母双杂交系统中,用于筛选柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库,获得可能与EtCDPK4具有相互作用的蛋白质。%In order to screen for the interacting protein with CDPK4 of Eimeriatenella, the recombinant bait plasmid pGBKT7-EtCDPK4 was constructed, which can be used to screen for Eimeriatenella yeast two-hybrid cDNA library. The total RNA of E.tenellasporozoite was extracted, then the first strand of cDNA was obtained by reverse transcriptase. The 1660 bp fragment of EtCDPK4 was amplified by PCR and cloned into yeast BD bait vector pGBKT7, then the recombinant plasmid pGBKT7-EtCDPK4 was identified by double enzyme digestion and sequenced. The self-activation, toxic action and protein expression of the recombination bait plasmid pGBKT7-EtCDPK4 were tested, respectively. These results show that the recombinant pGBKT7-EtCDPK4 plasmid was constructed successfully and proved to be no self-activation and toxic action. Moreover, it was expressed in the Y2HGold yeast cell. So the recombinant plasmid pGBKT7-EtCDPK4 can be used to screen for Eimeriatenella

  18. 人星状病毒非结构蛋白酵母双杂交诱饵表达载体构建及酵母转化子特性鉴定%Construction of Yeast Two-Hybrid Bait Vector of HAstV Non-Struc-tural Protein and Characterization of Yeast Transformants

    Institute of Scientific and Technical Information of China (English)

    李攀; 崔成成; 杨思达; 黄芬; 曾韦锟; 井申荣


    Objective To construct a yeast two-hybrid bait vector of non-structure protein 1a/3 (nsP1a/3) of human astrovirus (HAstV), and to characterize the yeast transformants after elec-trotransformation of the recombinant plasmid into the yeast competent cells .Methods The coding sequence of HAstV nsP 1 a/3 obtained by PCR was inserted into the modified plasmid pGST 7.The re-combinant vector nsP1a/3-pGBST7 confirmed by DNA sequencing was transformed into the Y 2 HGo-ld yeast competent cells .The cytotoxicity of the expression products of the recombiant plasmid nsP1a/3-pGBST7 and the autoactivation of the report gene in yeast transformants were exam-ined.Results The sequencing of the nsP 1 a/3-pGBST7 vector showed that the recombinant bait plasmid was successfully constructed and the open reading frame of nsP 1a/3 insert was correct.After electrotransformation, it was found that the expression products of the nsP 1a/3-pGBST7 vector had no cytotoxicity to yeast transformants and also they did not activate the report gene .Conclusion The yeast two-hybrid bait vector of HAstV nsP 1 a/3 was successfully constructed , which laid a foun-dation for further studying the interaction of HAstV nsP 1 a/3 with target proteins and the replication mechanism of HAstV in host cells .%目的:构建人星状病毒非结构蛋白3( non-structure protein 1a/3, nsP1a/3)基因酵母双杂交诱饵重组质粒,电转化酵母感受态细胞后鉴定酵母转化子特性。方法聚合酶链反应扩增人星状病毒nsP1a/3编码的基因序列,定向插入到穿梭表达载体pGBKT7改造后的pGBST7中,测序确定无误后将其电转化到Y2HGold酵母感受态细胞中,对细胞特性即nsP1a/3-pGBST7诱饵质粒表达产物的毒性和报告基因自激活作用进行鉴定。结果测序结果表明诱饵重组质粒构建正确,阅读框无误。电转化酵母后,其表达产物对酵母细胞无毒性作用,对报告基因亦无激活作用。结论成功构建了

  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:;; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)


    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. Bioluminescence-Sensing Assay for Microbial Growth Recognition

    Directory of Open Access Journals (Sweden)

    Heba Ramadan Eed


    Full Text Available The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A bioluminescent recombinant E. coli strain was used with luciferase extracted from transformed bacteria. Results showed that there is a direct correlation between the bioluminescence intensity of the ATP bioluminescence-sensing assay and the microbial viability. Bacterial counts from food samples were detected using the developed sensing assay and validated by the traditional plate-counting method. Compared with the plate-counting method, ATP bioluminescence-sensing assay is a more rapid and efficient approach for detecting microbial viability.

  1. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.


    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.

  2. Fishing TaSC Interacting Protein in Wheat Using Split Ubiquitin Yeast Two Hybrid System%利用分裂泛素酵母双杂交技术钓取小麦TaSC互作蛋白质

    Institute of Scientific and Technical Information of China (English)

    蔺芳芳; 杨旭; 武小翠; 刘晓梅; 葛荣朝; 赵宝存


      TaSC (Triticum asetivum L. salt-tolerance related gene, GenBank 登录号为 AY956330)是从小麦耐盐突变体RH8706-49中克隆的高盐诱导表达的耐盐相关基因.以该基因编码的蛋白质TaSC为诱饵,运用分裂泛素酵母双杂交技术从小麦 cDNA 表达文库中钓取互作蛋白质,筛选到一个编码小麦未知功能蛋白质的基因(GenBank 登录号为AK336035),命名为TaSCIP1(TaSC interaction protein 1).双分子荧光互补(BiFC)实验证实TaSCIP1与TaSC存在互作.该互作蛋白的分离有利于进一步研究TaSC基因的耐盐机制.%The TaSC (Triticum asetivum L. salt-tolerance related gene, GenBank accession number AY956330) has been cloned from wheat salt tolerant mutant RH 8706-49, which is induced by high salinity. To fish interacting proteins of this gene, we used TaSC protein as bait to hybrid with the wheat cDNA expression library using the split ubiquitin yeast two hybrid system in this experiment. A novel wheat gene (GenBank accession number AK336035) encoding a protein with unknown function was isolated, which was designated TaSCIP1 (TaSC interaction protein 1). Bimolecular fluorescence complementation (BiFC) experiment con-firmed the interaction between proteins TaSCIP1 and TaSC. The separation of TaSC-interacting protein is beneficial to disclose the mechanism of salt tolerance gene TaSC.

  3. 应用酵母双杂交技术筛选猪Calsarcin-2蛋白结合蛋白%Screening of Proteins Interacting with Porcine Calsarcin-2 by Yeast Two-hybrid Techniques

    Institute of Scientific and Technical Information of China (English)

    辛磊磊; 朱文娟; 杨锷; 陈大雷; 张宁波; 唐中林; 杨述林; 李奎


    Calsarcins是钙调磷酸酶肌小节特异性结合蛋白家族,本实验旨在研究Calsarcin-2(CS2)在猪骨骼肌纤维形成和信号通路调控中的功能.应用酵母双杂交方法筛选猪骨骼肌组织中CS2蛋白结合蛋白,从猪骨骼肌文库中筛选出180个阳性克隆,经分析得到12个相互作用的蛋白,研究发现骨骼肌α-肌动蛋白1(ACTA1),肌联蛋白(TTN)及钙调素1(CALM1)为CS2互作蛋白.ACTA1参与肌肉收缩过程,TTN调节肌小节动力传导及Z线装配,CALM1参与钙离子信号传导.结果表明,CS2在维持Z线结构稳定和参与钙离子信号传导方面起重要作用.%To elucidate the function of calsarcin-2 (CS2) in the formation of skeletal muscle fiber and signaling pathway regulation, we used yeast two-hybrid approach to look for CS2 interacting proteins. The porcine skeletal library screen gave 180 positive interactions, in which 12 interacting proteins were obtained. Bioinformation analysis found that actin alpha l(ACTAl), titin(TTN) and calmodulin 1 (CALM 1) were CS2 interaction partners. ACTA1 involved in the process of muscle contraction, TTN regulated the dynamics conduction of sarcomere and Z disc assembly, CALM 1 paticipated in calcium signal transduction. These results indicate that CS2 is implicated in the transduction of calcium signals and maintaining the structure of Z disc.

  4. Hunting for Novel Protein Factors in G-protein Pathway with Yeast Two-hybrid System%应用酵母双杂交研究G蛋白通路中的蛋白因子

    Institute of Scientific and Technical Information of China (English)

    鲁宁; 李旌军; 黄秉仁


    Objective To explore the protein factors that could interact with Gβ subunit within the G protein sig nal transducing pathway. Methods The highly sensitive protein-protein interaction system——Yeast Two-hybrid System was applied to screen the human cDNA library with constructed “Bite plasmid” contain ing Gβ subunit gene fragment. And then the faulse positive test was adapted. Results Three positive gene fragments were obtained. One codes for “Actin bundling protein”. The other two are new ones and their GeneBank accession numbers are AF288405 and AF288406 respectively. Conclusions It is the first time to find that among human brain tissue, Gβ subunit becomes a structural or functional unit interacts with actin bundling protein and the other two unknown protein factors to activate the following pathway. This re sult may be important to understand the relationship between the actin cytoskeleton and G proteins.%目的探寻G蛋白信号传导途径中与Gβ亚基相互作用的下游蛋白因子,揭示G蛋白信号传导通路的机制。方法采用敏感性较高的酵母双杂交体系,构建含Gβ亚基基因的饵质粒筛选人脑cDNA文库。结果获得了肌动蛋白集束调控蛋白(actin bundling protein)的编码基因和两个新基因片段,GeneBank登录号分别为AF288405(427 bp)及AF288406(2832 bp)。结论提示在人脑组织中Gβ亚基作为结构和功能单位可能通过与actin bundling protein 及另两种未知的蛋白因子间的相互作用而介导了信号的传导,对于揭示G蛋白信号传递通路与actin细胞骨架之间的关系起重要的提示作用

  5. Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

    Directory of Open Access Journals (Sweden)

    Projan Steve


    Full Text Available Abstract Background Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H, is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost in the amplified library. Results Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. Conclusion Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the

  6. Chromatographic assessment of two hybrid monoliths prepared via epoxy-amine ring-opening polymerization and methacrylate-based free radical polymerization using methacrylate epoxy cyclosiloxane as functional monomer. (United States)

    Wang, Hongwei; Ou, Junjie; Lin, Hui; Liu, Zhongshan; Huang, Guang; Dong, Jing; Zou, Hanfa


    Two kinds of hybrid monolithic columns were prepared by using methacrylate epoxy cyclosiloxane (epoxy-MA) as functional monomer, containing three epoxy moieties and one methacrylate group. One column was in situ fabricated by ring-opening polymerization of epoxy-MA and 1,10-diaminodecane (DAD) using a porogenic system consisting of isopropanol (IPA), H2O and ethanol at 65°C for 12h. The other was prepared by free radical polymerization of epoxy-MA and ethylene dimethacrylate (EDMA) using 1-propanol and 1,4-butanediol as the porogenic solvents at 60°C for 12h. Two hybrid monoliths were investigated on the morphology and chromatographic assessment. Although two kinds of monolithic columns were prepared with epoxy-MA, their morphologies looked rather different. It could be found that the epoxy-MA-DAD monolith possessed higher column efficiencies (25,000-34,000plates/m) for the separation of alkylbenzenes than the epoxy-MA-EDMA monolith (12,000-13,000plates/m) in reversed-phase nano-liquid chromatography (nano-LC). Depending on the remaining epoxy or methacrylate groups on the surface of two pristine monoliths, the epoxy-MA-EDMA monolith could be easily modified with 1-octadecylamine (ODA) via ring-opening reaction, while the epoxy-MA-DAD monolith could be modified with stearyl methacrylate (SMA) via free radical reaction. The chromatographic performance for the separation of alkylbenzenes on SMA-modified epoxy-MA-DAD monolith was remarkably improved (42,000-54,000 plates/m) when compared with that on pristine epoxy-MA-DAD monolith, while it was not obviously enhanced on ODA-modified epoxy-MA-EDMA monolith when compared with that on pristine epoxy-MA-EDMA monolith. The enhancement of the column efficiency of epoxy-MA-DAD monolith after modification might be ascribed to the decreased mass-transfer resistence. The two kinds of hybrid monoliths were also applied for separations of six phenols and seven basic compounds in nano-LC. Copyright © 2014 Elsevier B.V. All

  7. Assessment of plaque assay methods for alphaviruses. (United States)

    Juarez, Diana; Long, Kanya C; Aguilar, Patricia; Kochel, Tadeusz J; Halsey, Eric S


    Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a "semisolid-variant method" using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.

  8. Clinical Comparisons of Two Free Light Chain Assays to Immunofixation Electrophoresis for Detecting Monoclonal Gammopathy

    Directory of Open Access Journals (Sweden)

    Han-Sung Kim


    Full Text Available Free light chains (FLCs are useful biomarkers for the diagnosis and monitoring of various plasma cell dyscrasias. One hundred fifty-seven samples from 120 patients for screening or monitoring of monoclonal gammopathy (MG were included. The new N Latex FLC assays (Siemens Healthcare Diagnostics GmbH, Germany were compared with the Freelite FLC assays (The Binding Site Ltd., UK and the results were analyzed with those of immunofixation electrophoresis (IFE. The Freelite FLC assay showed significantly wider assay ranges than the N Latex FLC assay. The correlation coefficients of the two FLC kappa (κ assays, lambda (λ assays, and the κ/λ ratio were 0.9792, 0.8264, and 0.9064, respectively. The concordance rate was 84.7% for the FLC κ assays, 79.6% for FLC λ, and 89.2% for the κ/λ ratio. The clinical sensitivity and specificity of the κ/λ ratios were 72.2% and 93.6% for the Freelite assay and 64.6% and 100% for the N Latex FLC assay. Two FLC assays showed good correlations and concordance. However, the clinical sensitivity of the κ/λ ratio was higher in the Freelite FLC assays; clinical specificity was higher in the N Latex FLC assay. Both FLC assays seem to have limited clinical utility in detecting MG in certain clinical settings.

  9. 21 CFR 225.158 - Laboratory assays. (United States)


    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  10. Gclust Server: 132782 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available ds Sin3p in a two-hybrid assay 1 1.00e-99 0.0 0.0 0.0 0.0 0.0 12.5 Show 132782 Cluster ID 132782 Sequence ID... Sequence length 949 Representative annotation Protein that binds Sin3p in a two-hybrid assay

  11. 利用酵母双杂交筛选与苹果褪绿叶斑病毒CP互作的寄主因子%Screening of the Host Factors Interacting with CP ofApple chlorotic leaf spot virusby Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    何乙坤; 钟敏; 胡同乐; 王树桐; 段豪; 丁丽; 王亚南; 曹克强


    Objective] The objective of this study is to screen the host factors interacting with coat protein (CP) ofApple chlorotic leaf spot virus (ACLSV) by yeast two-hybrid system and analyze their function in the interaction between host and virus by the method of bioinformatics.[Method] Firstly, the bait vector of pGBKT7-CP was constructed and transformed into Y2H gold. The growth of the transformed yeast was assayed on the culture plate of SD/-Trp, SD/-Trp/-His and SD/-Trp/-Ade-X-α-Gal to make clear the self-activating effect of pGBKT7-CP. The plasmids of pGBKT7-CPand pGBKT7 were transformed intoY2H gold, the OD 600 of which was tested by ultraviolet spectrophotometer at different stages. The effect of pGBKT7-CP on the yeast was analyzed. Secondly, the interactive proteins of CP were screened from cDNA library ofMalus sylvestris cv. R12740-7A constructed early by yeast two-hybrid sequencing transformation. At last, the positive clones were selected from the culture plate of SD/-Ade/-His/-Leu/-Trp/X-α-Gal and sequenced. BLAST analysis was made for the host factors in the GenBank and the function in the interaction between host and virus was deduced based on gene annotation.[Result] The bait vector of pGBKT7-CP was constructed. The transformed yeast Y2H gold was able to grow on the plate of SD/-Trp, but not able to grow on the plate of SD/-Trp/-His and SD/-Trp/-Ade-X-α-Gal, which implied that the recombined plasmid had no ability to activate the reporter gene of His and Ade.The growth of pGBKT7-CPand the control was similar, which predicated that the recombined plasmid had no virulence to the yeast. Sixty-nine interactive factors were obtained, which were divided into 10 types of proteins based on their functions, including transcription factor activity, carbon fixation activity, cellular iron ion homeostasis, defense response, hydrolase activity, oxidase activity, oxidoreductase activity, photosystem II assembly and protein binding activity. Eight host genes (Malus3

  12. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

    Directory of Open Access Journals (Sweden)

    Chen Zhang


    Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

  13. Application of radioreceptor assay of benzodiazepines for toxicology

    Energy Technology Data Exchange (ETDEWEB)

    Aaltonen, L.; Scheinin, M. (Department of Pharmacology and the Department of Internal Medicine, University of Turku, Finland)


    A radioreceptor assay (RRA) for determining benzodiazepines (BZ) has been developed and applied to toxicological analysis of serum samples from 21 patients with acute BZ overdosage. The method was sensitive (e.g., lorazepam 17 nM, diazepam 41 nM), and specific for pharmacologically active BZ derivatives. The reproducibility of the results was good (intra-assay variation < 8%, inter-assay variation < 10%). Concentrations measured by the RRA showed a good correlation with those obtained by gas-liquid chromatographic analysis of the same samples. The quantitative results represent the sum of one or several parent substances and all biologically active metabolites, in proportion to their receptor binding affinities.

  14. Comet Assay in Cancer Chemoprevention. (United States)

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


    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.

  15. The skin-blanching assay. (United States)

    Smit, P; Neumann, H A M; Thio, H B


    The skin-blanching assay is used for the determination and bioequivalence of dermatologic glucocorticoids (GCs). The exact mechanism of the production of blanching is not fully understood, but it is considered that local vasoconstriction of the skin microvasculature and the consequent blood-flow reduction cause this phenomenon. Several factors influence skin blanching, including drug concentration, duration of application, nature of vehicle, occlusion, posture and location. The intensity of vasoconstriction can be measured in several ways: visual or quantitative methods, such as reflectance spectroscopy, thermography, laser Doppler velocimetry and chromametry. In literature, contradicting results in the correlation of the skin-blanching assay with different tests to determine GC sensitivity have been reported, limiting its clinical usefulness.

  16. Bioluminescence assay for cell viability. (United States)

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N


    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  17. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

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


    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 or less of hyaluronate, and is unaffected by the presence of proteins.

  18. Development and Validation of a HPV-32 Specific PCR Assay

    Directory of Open Access Journals (Sweden)

    Leigh Janet


    Full Text Available Abstract Background Human Papillomavirus-32 (HPV-32 has traditionally been associated with focal-epithelial-hyperplasia (FEH. It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects. Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

  19. Purification and kinase assay of PKN. (United States)

    Mukai, Hideyuki; Ono, Yoshitaka


    PKN is a serine/threonine protein kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC) in the carboxyl-terminal region and three repeats of the antiparallel coiled coil (ACC) domain in the amino-terminal region. Mammalian PKN has three isoforms each derived from different genes, PKN1 (PKNalpha/PRK1/PAK1), PKN2 (PRK2/PAK2/PKNgamma), and PKN3 (PKNbeta). PKN isoforms show different enzymatic properties and tissue distributions and have been implicated in various distinct cellular processes (reviewed in Mukai [2003]). This chapter discusses methods to prepare purified enzymes and to assay substrate phosphorylation activities.

  20. Mass-based readout for agglutination assays (United States)

    Chunara, Rumi; Godin, Michel; Knudsen, Scott M.; Manalis, Scott R.


    We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63-630nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry.

  1. Micronuclei assay: A potential biomonitoring protocol in occupational exposure studies. (United States)

    Palanikumar, L; Panneerselvam, N


    As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. This particularly concerns the use of MN as a biomarker ofgenotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. The present paper reviews the use of the MN assay in biomonitoring of occupational exposure studies.

  2. Heritability of nociception IV: neuropathic pain assays are genetically distinct across methods of peripheral nerve injury. (United States)

    Young, Erin E; Costigan, Michael; Herbert, Teri A; Lariviere, William R


    Prior genetic correlation analysis of 22 heritable behavioral measures of nociception and hypersensitivity in the mouse identified 5 genetically distinct pain types. In the present study, we reanalyzed that dataset and included the results of an additional 9 assays of nociception and hypersensitivity, with the following goals: to replicate the previously identified 5 pain types; to test whether any of the newly added pain assays represent novel genetically distinct pain types; and to test the level of genetic relatedness among 9 commonly used neuropathic pain assays. Multivariate analysis of pairwise correlations between assays shows that the newly added zymosan-induced heat hypersensitivity assay does not conform to the 2 previously identified groups of heat hypersensitivity assays and cyclophosphamide-induced cystitis, the first organ-specific visceral pain model examined, is genetically distinct from other inflammatory assays. The 4 included mechanical hypersensitivity assays are genetically distinct and do not comprise a single pain type as previously reported. Among the 9 neuropathic pain assays including autotomy, chemotherapy, nerve ligation and spared nerve injury assays, at least 4 genetically distinct types of neuropathic sensory abnormalities were identified, corresponding to differences in nerve injury method. In addition, 2 itch assays and Comt genotype were compared to the expanded set of nociception and hypersensitivity assays. Comt genotype was strongly related only to spontaneous inflammatory nociception assays. These results indicate the priority for continued investigation of genetic mechanisms in several assays newly identified to represent genetically distinct pain types.

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


    Carina Ladeira; Susana Viegas; Manuel C. Gomes


    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  4. Immunoradiometric assay for quantification of prostate-specific antigen (PSA)

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz, Miriam [Institute of Nuclear Sciences and Technology, Havana (Cuba). Dept.of Radiochemistry]. E-mail:


    To develop an immunoradiometric assay (IRMA) for quantification of total PSA in serum, we carried out the evaluation of a panel of 12 monoclonal antibodies (MAbs). The mAbs (CB-PSA 1 to CB-PSA 10) were developed in the Center of Genetic Engineering and Biotechnology (CIGB) Havana, Cuba and mAbs (CMC-H9, CMC-A5) in the Center of Immunology and Biologics (CENIB), Camaguey, Cuba. The inhibition assays were carried out using as reference a commercial equimolar assay for total PSA. For the evaluation, the mAbs were labeled with {sup 125} I by the method of Chloramine T and the capture mAbs were used conjugated with biotin. For separation, it was used the avidin coupled to magnetic particles. To validate the assay we evaluated the sensibility, inter and intraassay precision and the correlation with reference assay in 65 samples of patient under suspicion of prostate cancer. The partner CB-PSA 4 / CB-PSA 9 (capture / tracer) showed the best results in the IRMA. The developed assay presented a detection limit of 0.025 mg/L, a good intra and interassay precision and a high correlation with the reference assay. (author)

  5. Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System%甘蓝SCR识别与结合SRK胞外域核心编码区DNA序列的酵母双杂交检测

    Institute of Scientific and Technical Information of China (English)

    薛丽琰; 曾静; 杨昆; 高启国; 李成琼; 任雪松; 王小佳; 罗兵; 朱利泉; 杨永军; 张贺翠; 常登龙; 陈松; 彭一波; 杨红


    The S-locus cystein-rich protein (SCR) is the male-determining factor of self-incompatibility in Brassica. In this study, the SCR fragments with different lengths amplified from Brassica oleracea L. were ligated with pGBKT7 to construct recombi-nant bait plasmids, which were then transformed into yeast Y2HGold cells for detecting their interaction with the S-locus receptor kinase extracellular domain (eSRK) in Brassica oleracea by using the yeast two-hybrid system. The results showed that recombi-nant vectors were not activated autonomously. The full length SCR could interact with eSRK, and the core region in the SCR was located between 97 and 186 bp. Moreover, the result also indicated that the splicing site of signal peptides of this haplotype SCR and its several adjacent amino acid residues could affect the interaction. These conclusions add some novel insights into the mechanism research of self- incompatibility in Brassica.%SCR是芸薹属自交不亲和性的雄性决定因子.为研究SCR与SRK之间相互作用的核心区段,通过构建结球甘蓝的包含系列不同长度的SCR的cDNA序列的pGBKT7融合载体,利用酵母双杂交系统检测SCR与SRK之间的相互作用.结果显示,构建的载体均未出现自激活现象,所获得SCR全长与其相对应SRK胞外域具有相互作用,且相互作用核心编码区位于SCR编码基因的第97~186 bp处.实验结果还显示,此单倍型的SCR信号肽剪切位点及相邻的几个氨基酸残基对相互作用实验结果有干扰作用.以上结论为包括甘蓝在内的芸薹属自交不亲和机制的研究提供了新的实验依据.

  6. Bacterial assay of contact lens wearers. (United States)

    Hart, D E; Hosmer, M; Georgescu, M; Farris, R L


    The goal of the project was to determine the quantity of bacteria on the contact lens and adjacent areas of the eye. This paper is a quantitative study of the contact lens and ocular aerobic microbiota in a mixed group of daily and extended wear disposable contact lens users. The contact lens, the lower fornix, tears collecting at the lower fornix, and edge of the lower lid at the Meibomian gland margin were assayed for the quantity of bacterial colony forming units (CFU). Eighteen patients wearing 49 disposable high water content hydrogel contact lenses were assayed and the mean lens age was 8.8 +/- 4.6 days. Three patients wore their lenses on a daily wear basis and 15 on an extended wear schedule. Tear samples were obtained with sterile microbial loops and the lens was macerated into small particles with a tissue grinder. The samples were poured onto the surface of chocolate agar plates and incubated at 35 degrees C for 48 h in 5% Co2. The lid margin revealed the greatest bacterial presence (mean = 9.7 CFU; median = 2 CFU; mode = 0 CFU). The lens showed the next greatest presence of CFU (mean = 4.5 CFU; median = 1 CFU; mode = 0). The fornix and tears revealed the least bacterial presence (fornix: mean = 2.6 CFU; median = 0 CFU; mode = 0 CFU). The bacteria were coagulase-negative staphylococci. The bacterial assay of disposable lens wearing contact lens subjects indicates that the lid margins are the greatest source of bacteria with the tears being the lowest. These studies support the concept that in the eye, the lens typically does not possess a large number of bacteria under normal conditions.

  7. 21 CFR 864.7525 - Heparin assay. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  8. A colorimetric assay for cytokinin oxidase. (United States)

    Libreros-Minotta, C A; Tipton, P A


    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  9. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert;


    ) 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...

  10. 21 CFR 866.3210 - Endotoxin assay. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a) Identification. An endotoxin assay is a device that uses serological techniques in whole blood. The device...

  11. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

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


    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...

  12. Development of a STAT5 phosphorylation assay as a rapid bioassay to assess interleukin-7 potency. (United States)

    Zumpe, C; Engel, K; Wiedemann, N; Metzger, A U; Pischetsrieder, M; Bachmann, C L


    Interleukin (IL)-7 is a cytokine inducing the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. As a consequence of IL-7 activating this pathway, STAT5 is phosphorylated. In pharmaceutical quality control, the potency of biopharmaceuticals is commonly assessed by proliferation assays. This is also possible for IL-7 conjugates. However, the disadvantage of these classical "endpoint-assays" is that they require very long incubation times, up to several days, since they measure the downstream events of a cellular response. As an alternative to this, we developed a rapid intracellular phosphorylation assay, measuring IL-7 induced STAT5 phosphorylation in Kit 225 cells. The Kit 225 human T cell line expresses the IL-7 receptor and is responsive to IL-7, therefore making it a good candidate cell line for assay development. Like the Kinase receptor activation (KIRA) assay, developed by Sadick et al. [1], the STAT5 phosphorylation assay was performed using two separate microtiter plates: the first one for cell stimulation and lysis, the second one for enzyme-linked immuno sorbent assay (ELISA). The assay showed a high accuracy and precision with a mean recovery of 102% and a mean coefficient of variation of 9%. In comparison to the classical proliferation assay, the phosphorylation assay is much faster. Thus, the assay procedure time can at least be reduced from six to three days by using STAT5 phosphorylation instead of proliferation as an endpoint due to the shorter incubation time with IL-7. Moreover, the phosphorylation assay shows a wider dynamic range and higher signal to noise ratios and is thus more robust than the proliferation assay.mAs a consequence, this assay could serve as reliable, accurate, precise and fast alternative to the classical proliferation assay for IL-7. This study also serves as an example for the typical steps during development and qualification / validation of a potency assay for quality control testing.

  13. Assaying environmental nickel toxicity using model nematodes. (United States)

    Rudel, David; Douglas, Chandler D; Huffnagle, Ian M; Besser, John M; Ingersoll, Christopher G


    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  14. Assaying environmental nickel toxicity using model nematodes.

    Directory of Open Access Journals (Sweden)

    David Rudel

    Full Text Available Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water, we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  15. Assaying environmental nickel toxicity using model nematodes (United States)

    Rudel, David; Douglas, Chandler; Huffnagle, Ian; Besser, John M.; Ingersoll, Christopher G.


    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegansand P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  16. Steroid assays in paediatric endocrinology. (United States)

    Honour, John W


    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  17. Detection of Streptococcus pyogenes using rapid visual molecular assay. (United States)

    Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing


    Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis.

  18. Activity assay of membrane transport proteins

    Institute of Scientific and Technical Information of China (English)

    Hao Xie


    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  19. 酵母双杂交系统筛选HBV PreS1相互作用蛋白%Screening the hepatitis B virus PreS1 associated proteins in yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    叶峰; 张曦; 邸莹; 王小清; 刘小静; 孔颖; 赵英仁; 陈天艳; 刘敏


    Objective To screen the interacted protein hepatitis B virus (HBV) PreSl with human hepatocytes from normal human liver cDNA library by sos-recruitment system (SRS) and explore the mechanism of HBV endocylosis. Methods PCR was performed to amplify the gene of HBV PreSl from the plasmid PCP10/ HBV ayw subtype containing the whole fragment of HBV;the PCR product was cloned into yeast expression plasmid pSos. and reconstituted plasmid was tested by auto-sequencing assay and named pSos- PreSl. The pSos-PreSl fusion protein expressed in the yeast cells was confirmed by Western blot after pSos- PreSl was transfected into the yeast cell cdc25.Self-activation of the bait protein was determined by cotransformation of pSos- PreSl and pMyr-Lamin C, and the cytoplasmic localization of the bait protein was verified by cotransformation of pSos- PreSl and pMyr SB. Yeast cells co-transfected with pSos- PreSl and the normal human liver cDNA library grew in selective nutrition and temperature. The true positive clones were submitted for sequencing. The results were submitted to the BLAST notebook of NCBI to seek homologous sequence. Results The yeast expression vector of HBV PreSl gene was constructed successfully and its expression in yeast was verified. The recombinant bait plasmid did not have self-activation and toxicity to yeast cdc25H cells. Furthermore, the cytoplasmic localization of the bait protein was verified correctly. After yeast cells were co-transfected with pSos-PreSl and the normal human liver cDNA library, 5 clones were positive and showed high homology with KCMF1, cyt C, vitamin D binding protein, Homo sapiens albumin (ALB) and Homo sapiens asialoglycoprotein receptor (ASGPR). Conclusion We obtained 5 proteins which may interact with HBV PreSl protein by SRS. This is helpful for exploring the mechanism of HBV endocytosis.%目的 利用Sos招募系统(SRS),构建含HBV PreS1基因的酵母双杂交诱饵载体,筛选人肝细胞与HBV PreS1蛋白相互作用的

  20. Accurate non-invasive image-based cytotoxicity assays for cultured cells

    Directory of Open Access Journals (Sweden)

    Brouwer Jaap


    Full Text Available Abstract Background The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. Results Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. Conclusions This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

  1. 巴西橡胶树胶乳均一化酵母双杂交cDNA文库构建%Construction of a normalized yeast two-hybrid cDNA library of the latex of rubber tree (Hevea brasiliensis Müll. Arg.)

    Institute of Scientific and Technical Information of China (English)

    余海洋; 张宇; 王萌; 覃碧


    In this study, the latex of rubber tree (Hevea brasiliensis) clone ‘Reyan 7-33-97’ was used as plant material. SMART® cDNA synthesis technology was used to generate the ifrst strand cDNA, and then long distance PCR (LD-PCR) was used to amplify double-strand cDNA (ds-cDNA). The ds-cDNA was normalized by duplex-speciifc nuclease (DSN). The normalized cDNA was puriifed by running CHROMA SPIN+TE-400 column to remove short cDNA fragments. The puriifed cDNA and linear vector pGADT7-Rec were co-trans-formed into competent Y187 yeast cell to generate a normalized yeast two-hybrid cDNA library. The results show that the cDNA of the latex of rubber tree was wide range of fragment sizes and with uneven abundance before normalization. After normalized by DSN and puriifed using CHROMA SPIN+TE-400 column, cDNA below 500 bp had been removed efficiently, and high abundance of cDNA had been reduced significantly. Moreover, RT-PCR revealed that the transcripts of two housekeeping genes18S rRNA andβ-actinwere decreased signiifcantly after normalization. The harvested library had 1.26×106 independent clones. The titer of the library was up to 3.23×107 cfu·mL-1, the recombination rate was 87%, and the average insert size was more than 1.0 kb. In this study, a normalized yeast two-hybrid cDNA library from the latex of rubber tree has been successfully established, which provides a reference for studying natural rubber biosynthetic pathway and its molecular regulation mechanism in rubber tree.%以巴西橡胶树无性系‘热研7-33-97’胶乳为材料,采用SMART® cDNA合成技术反转录合成cDNA第一链,并通过LD-PCR合成双链cDNA (ds-cDNA),采用双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理,并经过CHROMA SPIN+TE-400柱子去除短片段的cDNA,纯化后的cDNA和线性化载体pGADT7-Rec共转化酵母Y187感受态细胞构建均一化酵母双杂交cDNA文库。结果显示:均一化之前橡胶树胶乳cDNA片段分布较

  2. Development of a rapid ATP bioluminescence assay for biocidal susceptibility testing of rapidly growing mycobacteria. (United States)

    Kapoor, Renuka; Yadav, Jagjit S


    An ATP-based biocide susceptibility assay for mycobacteria was developed by optimizing the cell lysis and assay conditions. Compared to the conventional agar plating method, the assay was rapid (1.5 h) and showed high sensitivity and specificity as determined by receiver operating characteristic (ROC) analysis. The test species, Mycobacterium immunogenum, M. chelonae, and M. abscessus, showed various susceptibilities to the glutaraldehyde- and isothiazolone-based test biocides.

  3. Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A. (United States)

    Bowyer, Annette E; Van Veen, Joost J; Goodeve, Anne C; Kitchen, Steve; Makris, Michael


    The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.

  4. Optimized and comparative antioxidant assays and its applications in herbal and synthetic drug analysis as an antioxidants. (United States)

    Nile, Shivraj Hariram; Khobragade, C N; Park, Se Won


    Drug development in the recent times often relies on use of natural and synthetic drugs that are promising candidates as therapeutic agents for prevention of diseases and disorders. They possess different chemical structures with wide range of therapeutic activities. Many natural and synthetic drugs act as antioxidant agents in various metabolic processes. Increasing epidemiological, clinical and experimental studies have shown that intake of antioxidants drugs provide protection against various disorders and diseases related to oxidative stress. The factors responsible for this oxidative stress are mainly free radicals, reactive nitrogen species (RNS) and reactive oxygen species (ROS). The antioxidant drugs act as free radical scavenging, reducing and metal chelating substances; Antioxidants also show inhibition of various metabolic enzymes and factors responsible for inflammation. The present paper reviews different In vitro assays for determination of antioxidant activities (Table 1). The basic assays include DDPH assay, OH Scavenging assay, Reducing activity assay, TEAC assay, FCR assay, PRTC assay, ABTS assay, FRAP assay, ORAC assay, Ferric thiocynate assay, TRAP assay, Chemiluminescence assay, NBT assay, CUPRAC Assay.

  5. Evaluation of three commercial progesterone receptor assays in a single tamoxifen-treated breast cancer cohort. (United States)

    Kornaga, Elizabeth N; Klimowicz, Alexander C; Guggisberg, Natalia; Ogilvie, Travis; Morris, Don G; Webster, Marc; Magliocco, Anthony M


    Estrogen receptor and progesterone receptor status are routinely assessed using immunohistochemistry assays to assist in patient prognosis and clinical management. Three commonly utilized autostainer vendors-Dako, Leica and Ventana-provide ready-to-use progesterone receptor assays; however, they have never been directly compared in a single breast cancer cohort. We looked at three immunohistochemical progesterone receptor assays, in addition to original ligand-binding assay results, in a single retrospective, tamoxifen-treated breast cancer cohort to investigate inter- and intra-observer agreement, concordance, prognostic ability and measures of test performance. All immunohistochemical assays utilized the manufacturer's specified protocols. Five-year disease-free survival was the endpoint of interest, and multivariate models were adjusted for lymph node status, tumor grade, tumor size and human epidermal growth factor 2 status. All assays showed substantial to almost perfect agreement between the three observers (Dako κ=0.69-0.90; Leica κ=0.70-0.89; and Ventana κ=0.78-0.94) and concordance (Dako/Leica κ=0.81; Dako/Ventana κ=0.78; and Leica/Ventana κ=0.82). Univariate survival analyses showed that only the ligand-binding assay, Dako and Ventana assays achieved statistical significance. No statistically significant results were seen in multivariate models, although a strong trend was seen with the Ventana progesterone receptor assay. All assays performed similarly with regards to measures of test performance with ligand-binding assay set as the reference, and all immunohistochemical assays outperformed the ligand-binding assay in regards to 5-year disease-free survival. Despite similar agreement and concordance with the progesterone receptor assays, clear differences were noted with regards to 5-year disease-free survival. Additional survival analyses suggest that clinical utility of estrogen receptor assays vary when investigated in combination with

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

    Directory of Open Access Journals (Sweden)

    Grace Ka Yan Chan

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

  7. Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

    Directory of Open Access Journals (Sweden)

    Kazutoyo Miura

    Full Text Available Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA. The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH Harmonised Tripartite Guideline Q2(R1 details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability. We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in

  8. Fabrics China Creation Show Hold in Shanghai

    Institute of Scientific and Technical Information of China (English)


    @@ On August 5-6th.the 3rd Fabrics China Creation Show(one series events of Reach & Touch),organized by China National Textile & Apparel Council and National Textile Development Center,was held in Shanghai,aiming to providing textile producers and designers a platform to show their inspirations and creative ideas in fabric design.

  9. Serving Up Activities for TV Cooking Shows. (United States)

    Katchen, Johanna E.

    This paper documents a presentation given on the use of English-language television cooking shows in English-as-a-Second-Language (ESL) and English-as-a-Foreign-Language (EFL) classrooms in Taiwan. Such shows can be ideal for classroom use, since they have a predictable structure consisting of short segments, are of interest to most students,…

  10. Development and application of assays for serotonin

    Energy Technology Data Exchange (ETDEWEB)

    Gow, I.F.


    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.

  11. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay. (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk


    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P HBcrAg assays.

  12. Quantitative Microplate Assay for Real-Time Nuclease Kinetics


    Eriksson, Jonas; Langel, Ülo


    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

  13. The identification and structure of an N-terminal PR domain show that FOG1 is a member of the PRDM family of proteins.

    Directory of Open Access Journals (Sweden)

    Molly K Clifton

    Full Text Available FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being.

  14. Voyeurismo Televisivo, Reality Shows e Brasilidade Televisiva

    Directory of Open Access Journals (Sweden)

    Suzana Kilpp


    Full Text Available In the last years we watched a boom of reality shows in the media and also in the academic production specialized in this subject. It remains, however, a epistemological gap related to the aesthetic and techniques (which are related to the televisions grammars that TV uses in these programs to enunciate ethics directions to its own voyeurism, that goes far beyond reality shows, having repercussions on social imaginary of transparency and surveillance, and the redesign of public and private spaces. In this gap, the article points out the debate of Brazilian reality shows in the perspective of the televisions grammars.

  15. Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity

    Directory of Open Access Journals (Sweden)

    Dessy Natalia


    Full Text Available Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC is a versatile catalyst for the organic synthesis of chiral α-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the α-hydroxy ketone (R-2,2′-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations.

  16. Fecal Transplant Shows Early Promise Against Autism (United States)

    ... 163263.html Fecal Transplant Shows Early Promise Against Autism Small study found giving healthy gut bacteria to ... study suggests a novel treatment for kids with autism: Give these young patients a fresh supply of ...

  17. Poverty Harder on Women's Hearts, Research Shows (United States)

    ... page: Poverty Harder on Women's Hearts, Research Shows Poor females ... reduce the burden of cardiovascular disease around the world," Peters said. The study findings were published online ...

  18. Diabetes Drug Shows Promise Against Parkinson's (United States)

    ... fullstory_167612.html Diabetes Drug Shows Promise Against Parkinson's Byetta improved symptoms of motor disease in small, ... may do double duty as a treatment for Parkinson's disease, a new study suggests. "This is a ...

  19. UV Photography Shows Hidden Sun Damage (United States)

    ... mcat1=de12", ]; for (var c = 0; c UV photography shows hidden sun damage A UV photograph gives ... developing skin cancer and prematurely aged skin. Normal photography UV photography 18 months of age: This boy's ...

  20. Study Shows How Zika Attacks Infant Brain (United States)

    ... gov/news/fullstory_162514.html Study Shows How Zika Attacks Infant Brain Virus can copy itself thousands ... New research paints a chilling portrait of how Zika ravages the infant brain. Scientists from the U.S. ...

  1. Fabrics China Creation Show Held in Shanghai

    Institute of Scientific and Technical Information of China (English)


    On August 5-6th,the 3rd Fabrics China Creation Show (one series events of Reach & Touch),organized by China National Textile & Apparel Council and National Textile Development Center,was held in Shanghai,

  2. Voyeurismo Televisivo, Reality Shows e Brasilidade Televisiva


    Suzana Kilpp


    In the last years we watched a boom of reality shows in the media and also in the academic production specialized in this subject. It remains, however, a epistemological gap related to the aesthetic and techniques (which are related to the televisions grammars) that TV uses in these programs to enunciate ethics directions to its own voyeurism, that goes far beyond reality shows, having repercussions on social imaginary of transparency and surveillance, and the redesign of public and private s...

  3. Liposomes and MTT cell viability assay: an incompatible affair. (United States)

    Angius, Fabrizio; Floris, Alice


    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to evaluate the cytotoxicity potential of drugs vehicled by liposomes. However, liposome delivering drugs could produce inconsistent values of MTT absorbance. On the basis of previous experiments demonstrating the MTT affinity for lipid droplets, this paper aims to show that empty-liposomes interfere, per se, on MTT assay due to its lipidic nature. This brings into question the use of MTT testing cytotoxicity when liposomes are involved in delivering drugs.

  4. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis. (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman


    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  5. A radioactive assay for the degradation of neuropeptide Y

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, R.; Lucius, R.; Mentlein, R. [Kiel Univ. (Germany)


    Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian central nervous system. Like other neuropeptides, NPY is inactivated by specialized neuro-peptidases. To trace the degradation of NPY, an assay was established using biotinylated NPY. Biotinyl-NPY was radiolabeled with Na{sup 125}I by the chloramine-T method and bound to a streptavidin-agarose matrix. The amount of radiolabeling was analyzed by reverse-phase HPLC. The assay was carried out with five peptidases and inhibitors to demonstrate different specific activity. Measurable amounts of radioactivity were released by treatment with endopeptidase-24.18, plasmin, and trypsin, whereas dipetidylpeptidase IV (DPPIV) and angiotensin-converting enzyme (ACE) showed no activity in this assay. In the case of DPPIV this is due to a resistance of the assay to aminopeptidase attack. The assay is useful to study the specific degradation of NPY particularly by endopeptidases in all kinds of biological samples. (authors). 31 refs., 6 figs.

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

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

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

  7. Online Italian fandoms of American TV shows

    Directory of Open Access Journals (Sweden)

    Eleonora Benecchi


    Full Text Available The Internet has changed media fandom in two main ways: it helps fans connect with each other despite physical distance, leading to the formation of international fan communities; and it helps fans connect with the creators of the TV show, deepening the relationship between TV producers and international fandoms. To assess whether Italian fan communities active online are indeed part of transnational online communities and whether the Internet has actually altered their relationship with the creators of the original text they are devoted to, qualitative analysis and narrative interviews of 26 Italian fans of American TV shows were conducted to explore the fan-producer relationship. Results indicated that the online Italian fans surveyed preferred to stay local, rather than using geography-leveling online tools. Further, the sampled Italian fans' relationships with the show runners were mediated or even absent.

  8. 2008 LHC Open Days Physics: the show

    CERN Document Server


    A host of events and activities await visitors to the LHC Open Days on 5 and 6 April. A highlight will be the physics shows funded by the European Physical Society (EPS), which are set to surprise and challenge children and adults alike! School children use their experience of riding a bicycle to understand how planets move around the sun (Copyright : Circus Naturally) Participating in the Circus Naturally show could leave a strange taste in your mouth! (Copyright : Circus Naturally) The Rino Foundation’s experiments with liquid nitrogen can be pretty exciting! (Copyright: The Rino Foundation)What does a bicycle have in common with the solar system? Have you ever tried to weigh air or visualise sound? Ever heard of a vacuum bazooka? If you want to discover the answers to these questions and more then come to the Physics Shows taking place at the CERN O...

  9. Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay. (United States)

    Gajski, Goran; Gerić, Marko; Oreščanin, Višnja; Garaj-Vrhovac, Vera


    In the present study the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay were used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes (PBLs) of 50 healthy children from the general population in Croatia (age, 11.62±1.81 years). Mean values of tail length, tail intensity and tail moment, as comet assay parameters, were 12.92±0.10, 0.73±0.06 and 0.08±0.01, respectively. The mean frequency of micronuclei (MN) for all subjects was 2.32±0.28 per 1000 bi-nucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.72±0.24 and of nuclear buds (NBUDs) 1.44±0.19. The mean nuclear division index (NDI) was 1.70±0.05. When comet-assay parameters were considered, higher mean values for all three were found for the female population. According to the Mann-Whitney U test applied on the results of the comet assay, the only statistically significant difference between the male and female populations was found for tail length. Similar to the results obtained by the comet assay, girls showed higher mean values of all three measured parameters of the CBMN Cyt assay. This difference was statistically significant for total number of NPBs only. In the case of the NDI, a higher mean value was also obtained in girls, but this difference was not statistically significant. The results obtained present background data that could be considered as normal values for healthy children living in urban areas, and can later on serve as baseline values for further toxicological monitoring. Additionally, the usefulness of both techniques in measuring cytogenetic damage during bio-monitoring of children is confirmed.

  10. Liquid Crystal Research Shows Deformation By Drying (United States)


    These images, from David Weitz's liquid crystal research, show ordered uniform sized droplets (upper left) before they are dried from their solution. After the droplets are dried (upper right), they are viewed with crossed polarizers that show the deformation caused by drying, a process that orients the bipolar structure of the liquid crystal within the droplets. When an electric field is applied to the dried droplets (lower left), and then increased (lower right), the liquid crystal within the droplets switches its alignment, thereby reducing the amount of light that can be scattered by the droplets when a beam is shone through them.

  11. A Talk Show from the Past. (United States)

    Gallagher, Arlene F.


    Describes a two-day activity in which elementary students examine voting rights, the right to assemble, and women's suffrage. Explains the game, "Assemble, Reassemble," and a student-produced talk show with five students playing the roles of leaders of the women's suffrage movement. Profiles Elizabeth Cady Stanton, Lucretia Mott, Susan…

  12. The Last Great American Picture Show

    NARCIS (Netherlands)

    Elsaesser, Thomas; King, Noel; Horwath, Alexander


    The Last Great American Picture Show brings together essays by scholars and writers who chart the changing evaluations of the American cinema of the 1970s, sometimes referred to as the decade of the lost generation, but now more and more recognized as the first New Hollywood, without which the cinem

  13. Gene Therapy Shows Promise for Aggressive Lymphoma (United States)

    ... page: Gene Therapy Shows Promise for Aggressive Lymphoma Over one-third ... TUESDAY, Feb. 28, 2017 (HealthDay News) -- An experimental gene therapy for aggressive non-Hodgkin lymphoma beat back more ...

  14. Development, management and economy of show caves.

    Directory of Open Access Journals (Sweden)

    Cigna Arrigo A.


    Full Text Available The problems concerning the development of show caves are here considered by taking into account different aspects of the problem. A procedure to carry out an Environmental Impact Assessment (EIA has been established in the last decade and it is now currently applied. Such an assessment starts with a pre-operational phase to obtain sufficient information on the undisturbed status of a cave to be developed into a show cave. Successively a programme for its development is established with the scope to optimise the intervention on the cave at the condition that its basic environmental parameters are not irreversibly modified. The last phase of the assessment is focussed to assure a feedback through a monitoring network in order to detect any unforeseen difference or anomaly between the project and the effective situation achieved after the cave development. Some data on some of the most important show caves in the world are reported and a tentative evaluation of the economy in connection with the show caves business is eventually made.

  15. Show Them You Really Want the Job (United States)

    Perlmutter, David D.


    Showing that one really "wants" the job entails more than just really wanting the job. An interview is part Broadway casting call, part intellectual dating game, part personality test, and part, well, job interview. When there are 300 applicants for a position, many of them will "fit" the required (and even the preferred) skills listed in the job…

  16. TV-Show Retrieval and Classification

    NARCIS (Netherlands)

    Musto, C.; Narducci, F.; Lops, P.; Semeraro G.; Gemmis, M. de; Barbieri, M.; Korst, J.H.M.; Pronk, S.P.P.; Clout, R.A.W.


    Recommender systems are becoming popular tools to aid users in finding interesting and relevant TV-shows and other digital video assets,based on implicitly learned user preferences. In this context, a common assumption is that user preferences can be specified by program types (movie, sports, ...) a

  17. The British Show in Australia, 1985

    Directory of Open Access Journals (Sweden)

    Anthony Bond


    Full Text Available In 1984–85, The British Show, an exhibition largely made up of New British Sculpture, was curated for Australia and New Zealand. This essay discusses the context and effects of the exhibition on art in Australia. It also seeks to define the sources of originality and innovation of the artists included.

  18. Laser entertainment and light shows in education (United States)

    Sabaratnam, Andrew T.; Symons, Charles


    Laser shows and beam effects have been a source of entertainment since its first public performance May 9, 1969, at Mills College in Oakland, California. Since 1997, the Photonics Center, NgeeAnn Polytechnic, Singapore, has been using laser shows as a teaching tool. Students are able to exhibit their creative skills and learn at the same time how lasers are used in the entertainment industry. Students will acquire a number of skills including handling three- phase power supply, operation of cooling system, and laser alignment. Students also acquire an appreciation of the arts, learning about shapes and contours as they develop graphics for the shows. After holography, laser show animation provides a combination of the arts and technology. This paper aims to briefly describe how a krypton-argon laser, galvanometer scanners, a polychromatic acousto-optic modulator and related electronics are put together to develop a laser projector. The paper also describes how students are trained to make their own laser animation and beam effects with music, and at the same time have an appreciation of the operation of a Class IV laser and the handling of optical components.

  19. Liquid chromatographic assay for dicloxacillin in plasma. (United States)

    Alderete, Oscar; González-Esquivel, Dinora F; Del Rivero, L Misael; Castro Torres, Nelly


    A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.


    NARCIS (Netherlands)



    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

  1. Radioreceptor assay: theory and applications to pharmacology

    Energy Technology Data Exchange (ETDEWEB)

    Perret, G. (U.E.R. de Medecine, Sante et Biologie Humaine, 93 - Bobigny (France)); Simon, P. (Faculte de Medecine Pitie-Salpetriere, 75 - Paris (France))

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, ..beta..-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising.

  2. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    DEFF Research Database (Denmark)

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred


    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...

  3. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit (United States)

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok


    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  4. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay. (United States)

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana


    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  5. Assays for Determination of Protein Concentration. (United States)

    Olson, Bradley J S C


    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

  6. La mujer, en los talk shows

    Directory of Open Access Journals (Sweden)

    Antrop. José Gamboa Cetina


    Full Text Available Dentro de los medios de comunicación, uno de los que más ha impactado a la población latinoamericana ha sido la televisión, y en la barra programática de las televisoras ha surgido un tipo de programas denominados talk shows. En los últimos meses, la sociedad mexicana ha vivido el "boom" de los talk shows, que en poco tiempo han saturado la barra de la programación vespertina de las dos principales empresas televisivas de la república mexicana. Este fenómeno puede estudiarse desde diversas perspectivas. Sin embargo, por motivos de espacio, en esta ocasión analizaremos su impacto en las mujeres, desde diferentes dimensiones.

  7. 2009 Hands Across Pacific Show Held

    Institute of Scientific and Technical Information of China (English)


    <正>The 2009 Hands Across the Pacific Show co-sponsored by the Canadian International Cultural Exchanges (CICEX) and Hunan Provincial People’s Association for Friendship with Foreign Countries(HPPAFFC) was held respectively in the Tower Hall of Tokyo,Japan from July 2 to 7 and in the Hunan Provincial Museum from August 1 to 5.Jing Dunquan,Vice President of the

  8. The People's Show: A Critical Analysis


    Robin Francis


    The 1990s heralded a new form of museum exhibition: "The People's Show." A light-hearted celebration of popular culture, the concept has had phenomenal success throughout the United Kingdom. Beneath the humour, however, are more complex and radical agendas relating to cultural rights. The paper explores the issues associated with the rise and possible wane of this museum-based popular cultural phenomenon.

  9. Reality, ficción o show

    Directory of Open Access Journals (Sweden)

    Sandra Ruíz Moreno


    Full Text Available Para tener un punto de vista claro y objetivo frente a la polémica establecida en torno al programa “Protagonistas de novela” y la tendiente proliferación de los reality show en las parrillas de programación de la televisión colombiana, se realizó un análisis de texto y contenido de dicho programa, intentando definirlo desde sus posibilidades de realidad, ficción y show. Las unidades de análisis y el estudio de su tratamiento arrojaron un alto contenido que gira en torno a las emociones del ser humano relacionadas con la convivencia, tratadas a manera de show y con algunos aportes textuales de ficción, pero sin su elemento mediador básico, el actor, quitándole toda la posibilidad de tener un tratamiento con la profundidad, distancia y ética que requieren los temas de esta índole. El resultado es un formato que sólo busca altos índices de sintonía y que pertenece más a la denominada televisión “trash”, que a una búsqueda de realidad del hombre y mucho menos de sociedad.

  10. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp. (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  11. Improved benzodiazepine radioreceptor assay using the MultiScreen (R) Assay System

    NARCIS (Netherlands)

    Janssen, MJ; Ensing, K; de Zeeuw, RA


    In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time, The filtration in this method was performed by using the MultiScreen(R) Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom,

  12. A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay. (United States)

    Emter, Roger; Natsch, Andreas


    The KeratinoSens™ assay was the first cell-based in vitro test in the skin sensitisation adverse outcome pathway to be endorsed by an ECVAM statement. It includes a cell viability assessment, which serves two purposes: It forms part of the prediction model to exclude false-positive irritants and cytotoxicity provides some information on sensitizer potency of chemicals, which can feed into a multivariate potency model. In the KeratinoSens™ protocol, Nrf2-dependent luciferase induction and the MTT-viability assay are performed in parallel plates. Resazurin-based viability assays do not require cell lysis and are compatible with luciferase measurements in the same cells. Here, we performed detailed comparison of the tetrazolium-based MTT assay and the PrestoBlue® assay on 35 reference chemicals tested in the full KeratinoSens™ protocol. Log-transformed IC50 and IC30 values measured with both methods correlate with an R(2) of 0.97 and 0.95. A single chemical showed divergent results and analysis by four different viability assays indicated the PrestoBlue® read-out to be correct. The new more rapid and resource efficient approach has clear advantages: Dose-response curves show lower variability and the two endpoints are measured on the same cells. This approach is a valid addition to or replacement of the MTT-readout in the KeratinoSens™ assay and it is recommended as a general tool for luciferase-based reporter assays.

  13. Bite my art to show your love


    Kim, Hye yeon


    Two years ago, I came to the States for MFA program in UCSD. At first, I started making instructions for performance to communicate with people in my clumsy English, but it soon became my general process of working. In this show, I present a room surrounded by five video projections and drawings. They are documentation of performances concerning the balance of relationship. I performed two with Josh Aaron, other two with Josh Tonies, and one by myself. The performance done by myself reveals t...

  14. Showing and Saying. An Aesthetic Difference

    Directory of Open Access Journals (Sweden)

    Vicente Sanfélix Vidarte


    Full Text Available Wittgenstein’s distinction between saying and showing and the associated thesis, what can be shown cannot be said, were crucial to his first philosophy, persisted throughout the evolution of his whole thought and played a key role in his views on aesthetics. The objective of art is access to the mystical, forcing us to become aware of the uniqueness of our own experience and life. When art is good is a perfect expression and the work of art becomes like a tautology. An important consequence of this understanding of art is the irreducibility of the aesthetic to the scientific perspective.

  15. Latest European coelacanth shows Gondwanan affinities. (United States)

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan


    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  16. The optimal condition of performing MTT assay for the determination of radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Semie; Kim, Il Han [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)


    The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. Four human cancer cell lines - PCI-1, SNU-1066, NCI-H63O and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy, For clonogenic assay, cells in 25 cm{sup 2} flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10-14 days, For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R{sup 2} value of 0.975-0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was

  17. Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D. (United States)

    Farrell, Christopher-John L; Soldo, Joshua; McWhinney, Brett; Bandodkar, Sushil; Herrmann, Markus


    Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice. 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD₂ recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD₃ cross-reactivity and precision of the automated assays were evaluated. The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD₂ recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD₂ under-recovery, a trend consistent with 3-epi-25-OHD₃ cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD₃ cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations. Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.

  18. A high-throughput chemically induced inflammation assay in zebrafish

    Directory of Open Access Journals (Sweden)

    Liebel Urban


    Full Text Available Abstract Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article:

  19. 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: [VITO NV, Boeretang 200, BE-2400 Mol (Belgium)


    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.

  20. Mobile non-destructive assay system

    Energy Technology Data Exchange (ETDEWEB)

    Colarusso, A.P.; Audas, J.H.; Bieri, J.M.; Herrera, G.C.; Hastings, R.D.; Horton, W.S.; Kuckertz, T.H.; Kunz, W.E.; Medvick, P.A.; Vogel, P.A.


    A mobile system for non-destructive assay (NDA), developed at the Los Alamos National Laboratory, provides accurate and sensitive measurements for transuranic (TRU) isotopes contained in 208-iota drums of miscellaneous nuclear wastes. The NDA unit consists of four major subsystems: an assay chamber, counting and digital electronics, data acquisition, and a neutron generator. It performs both active and passive neutron waste measurements. The former determines the amount of fissile isotopes at a sensitivity level of 1 mg plutonium. The latter determines spontaneous fission and ..cap alpha..,n) isotopes at a comparable level. A complete assay consists of sequential active and passive measurements. The assay measurement and other supporting data are incorporated in a commercial spreadsheet program (Lotus 1,2,3) for further analysis, which includes various matrix corrections and a determination of whether or not the drum exceeds the 100-nCi/g threshold for TRU wastes. Field tests have been performed on three separate occasions, accomplishing more than 1800 waste drum assays. These waste drum assays are discussed, especially those comparing passive and active neutron measurements with independent segmented gamma scan assays. Results obtained with a set of 15 drums containing plutonium prepared from standards and actual hot waste matrices are also reviewed.

  1. Figure of Beijing ABP Showing up

    Institute of Scientific and Technical Information of China (English)


    @@ Last year, there were many good news from Beijing Advanced Business Park(ABP): on October 29, 2005, Xu Weiping, Chairman of the Board of ABP, was chosen as the"2005 Ten Major Chinese Administrative Person in News";On November 8,2005,CCIM (American Certified Commercial Investment Member) invited 40 world famous real estate investors to visit ABS and they showed will to cooperate; On November 22, 2005, Johnson Controls, Inc., one of World Top 500, signed the contract with Zhongguan Village Fengtai Garden that the headquarter of JCI energy administration China Branch will move to ABP to improve the energy administration and radiate all the nation; on December 10,2005, "CEO Salon" co-organized by Phoenix Satellite Television and ABP, was held in the meeting room of ABP, over 120CEOs met here to discuss the trend of China economic development and the development opportunities brought by 11th Five-Year Plan for enterprises.

  2. Lemurs and macaques show similar numerical sensitivity (United States)

    Jones, Sarah M.; Pearson, John; DeWind, Nicholas K.; Paulsen, David; Tenekedjieva, Ana-Maria; Brannon, Elizabeth M.


    We investigated the precision of the approximate number system (ANS) in three lemur species (Lemur catta, Eulemur mongoz, and Eulemur macaco flavifrons), one Old World monkey species (Macaca mulatta) and humans (Homo sapiens). In Experiment 1, four individuals of each nonhuman primate species were trained to select the numerically larger of two visual arrays on a touchscreen. We estimated numerical acuity by modeling Weber fractions (w) and found quantitatively equivalent performance among all four nonhuman primate species. In Experiment 2, we tested adult humans in a similar procedure, and they outperformed the four nonhuman species but showed qualitatively similar performance. These results indicate that the ANS is conserved over the primate order. PMID:24068469

  3. Star Shows It Has The Right Stuff (United States)


    Astronomers have used an observation by NASA's Chandra X-ray Observatory to make the best case yet that a star can be engulfed by its companion star and survive. This discovery will help astronomers better understand how closely coupled stars, and perhaps even stars and planets, evolve when one of the stars expands enormously in its red giant phase. The binary star system known as V471 Tauri comprises a white dwarf star (the primary) in a close orbit -- one thirtieth of the distance between Mercury and the Sun -- with a normal Sun-like star (the secondary). Chandra's data showed that the hot upper atmosphere of the secondary star has a deficit of carbon atoms relative to nitrogen atoms. "This deficit of carbon atoms is the first clear observational evidence that the normal star was engulfed by its companion in the past," according to Jeremy Drake of the Smithsonian Astrophysical Observatory in Cambridge, MA, who coauthored an article on V471 in The Astrophysical Journal Letters with Marek Sarna of the N. Copernicus Astronomical Center in Poland. The white dwarf star was once a star several times as massive as the Sun. Nuclear fusion reactions in the core of such a star convert carbon into nitrogen over a period of about a billion years. When the fuel in the core of the star is exhausted, the core collapses, triggering more energetic nuclear reactions that cause the star to expand and transform into a red giant before eventually collapsing to become a white dwarf. The carbon-poor material in the core of the red giant is mixed with outer part of the star, so its atmosphere shows a deficit of carbon, as compared with Sun-like stars. The X-ray spectra of a red giant star (top panel) and a Sun-like star (bottom panel) show the large difference in the peaks due to carbon atoms in the two stars. Theoretical calculations indicate that a red giant in a binary system can completely envelop its companion star and dramatically affect its evolution. During this common envelope

  4. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

    Johnson, Sarah Stewart; Hebsgaard, Martin B; Christensen, Torben R


    Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over......-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  5. 蛋白质相互作用的分析:利用酵母两性杂交系统探索蛋白质功能%Analysis of Protein Interactions:Probing the Function of Proteins with Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)


    酵母两性杂交系统是近年来发展起来的广泛应用于蛋白质相互作用研究的分子遗传学方法.它的研究过程包括诱捕质粒的构建,融合蛋白质粒文库的筛选,假阳性的消除和真阳性的探测.这个实验方法有利于利用已知蛋白去探测和它相互作用的未知蛋白及编码未知蛋白的cDNA.越来越多的研究证明酵母两性杂交系统是探索动植物蛋白质功能的有用技术.这一技术正被广泛应用于世界上许多重要的实验室.该文综述了酵母两性杂交技术在蛋白质相互作用研究中的最新进展.%The yeast two-hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion protein, elimination of false positives and delection analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest. More and more studies have demonstrated that the two-hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants. This method has been used to identify new interaction protein in many laboratories. The recently reported yeast tri-brid system, should allow the investigation of more complex protein-protein interactions. The aim of this review is to outline the recent progress made in protein interactions by using yeast two-hybrid system.

  6. Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

    Directory of Open Access Journals (Sweden)

    Luciano B. Lopes


    Full Text Available This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia, according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.

  7. Polymerase chain reaction assay for avian polyomavirus.


    Phalen, D.N.; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By us...

  8. Fluoride assay methodology for carbonated beverages. (United States)

    Heilman, Judith R; Levy, Steven M; Wefel, James S; Patterson, Kristine Y; Cutrufelli, Rena; Pehrsson, Pamela R; Holden, Joanne M


    The purpose of this paper was to review different methodological techniques used for the assessment of fluoride in carbonated beverages, and compare results using a fluoride ion electrode direct read method with and without a prior decarbonation treatment. The carbonated beverages in this study were either purchased locally at grocery stores in Iowa City, Iowa, or purchased as part of a national representative sampling approach included in the National Fluoride Database and Intake Assessment Study (NFDIAS). The samples were compared with and without a decarbonating process. Soda pop and beer samples were analyzed by removing a 1-ml sample and adding a 1-ml buffer solution. The fluoride concentration of the sample and buffer combination was then determined using a fluoride ion specific electrode. There was no significant difference in the fluoride concentration of the samples with or without prior decarbonation. The mean absolute difference between the soda pop group with and without decarbonation was 0.01 ppm F, while results from the beer samples showed variation of 0.00 to 0.02 parts per million fluoride (ppm F). These differences were not statistically significant for the soda pop or beer groups (P=.50 and P=.74, respectively). Whether or not decarbonation was conducted prior to analysis, the fluoride assay results were the same. Therefore, decarbonation of soda pop and beer was deemed unnecessary prior to fluoride analysis.

  9. Prandiology of Drosophila and the CAFE assay (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


    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

  10. Establishment of indirect immunofluorescence assay for rotavirus. (United States)

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y


    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production.

  11. Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases. (United States)

    Riester, Daniel; Hildmann, Christian; Haus, Patricia; Galetovic, Antonia; Schober, Andreas; Schwienhorst, Andreas; Meyer-Almes, Franz-Josef


    Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.

  12. Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay. (United States)

    Sarkar, Anupam; Bhagat, Jacky; Ingole, Baban S; Rao, Durga P; Markad, Vijaykumar L


    This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2 ) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl2 . In vitro exposure of hydrogen peroxide (H2 O2 ; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment.

  13. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    Directory of Open Access Journals (Sweden)

    Buch Karl


    Full Text Available Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98 were included in the experiment to study its principal and general applicability.

  14. NASA GIBS Use in Live Planetarium Shows (United States)

    Emmart, C. B.


    The American Museum of Natural History's Hayden Planetarium was rebuilt in year 2000 as an immersive theater for scientific data visualization to show the universe in context to our planet. Specific astrophysical movie productions provide the main daily programming, but interactive control software, developed at AMNH allows immersive presentation within a data aggregation of astronomical catalogs called the Digital Universe 3D Atlas. Since 2006, WMS globe browsing capabilities have been built into a software development collaboration with Sweden's Linkoping University (LiU). The resulting Uniview software, now a product of the company SCISS, is operated by about fifty planetariums around that world with ability to network amongst the sites for global presentations. Public presentation of NASA GIBS has allowed authoritative narratives to be presented within the range of data available in context to other sources such as Science on a Sphere, NASA Earth Observatory and Google Earth KML resources. Specifically, the NOAA supported World Views Network conducted a series of presentations across the US that focused on local ecological issues that could then be expanded in the course of presentation to national and global scales of examination. NASA support of for GIBS resources in an easy access multi scale streaming format like WMS has tremendously enabled particularly facile presentations of global monitoring like never before. Global networking of theaters for distributed presentations broadens out the potential for impact of this medium. Archiving and refinement of these presentations has already begun to inform new types of documentary productions that examine pertinent, global interdependency topics.

  15. The great American medicine show revisited. (United States)

    Tomes, Nancy


    Since the late 1800s, changes in the advertising and marketing of medicinal drugs have produced heated debates in the United States. With the emergence of the modern prescription drug between 1938 and 1951, concerns that once focused primarily on patients' use of over-the-counter drugs were broadened to include physicians and their "doctors' drugs" as well. The medical profession's growing control over their patients' drug choices inevitably heightened the scrutiny of their own performance as consumers. Although deeply divided over issues of the patient's role in medical decision making, consumer activists and physician reformers expressed similar concerns about the impact of aggressive pharmaceutical marketing and advertising on the doctor-patient relationship, and starting in the late 1950s they employed strikingly similar strategies to counter the new corporate "medicine show." Yet their efforts to promote a more rational use of prescription drugs have usually been too little and too late to offset the effectiveness of pharmaceutical advertising and mar-keting activities.

  16. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase. (United States)

    Thöny, Beat; Calvo, Ana C; Scherer, Tanja; Svebak, Randi M; Haavik, Jan; Blau, Nenad; Martinez, Aurora


    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

  17. The earliest published electrocardiogram showing ventricular preexcitation. (United States)

    Von Knorre, Georg H


    When in 1930, Wolff, Parkinson, and White published what is today known as the WPW, or preexcitation syndrome, they, and subsequently others, found few comparable cases in the preceding literature. Among these the report of Cohn and Fraser, published in 1913, was the earliest. However, another even earlier documentation in a 1909 article by Hoffmann escaped notice till now. The ECG of a patient with paroxysmal tachycardia reveals a short PR interval and a delta-wave-induced widening of the QRS complex, even though the reproduced tachycardia was not preexcitation related. The interpretation of this poorly reproduced ECG can be confirmed by another and more detailed description of the patient in an electrocardiography textbook published in 1914 by the same author. Thus, the earliest publication of an ECG showing ventricular preexcitation now can be dated back to 1909. Moreover, the Hoffmann monograph contains two additional examples of the WPW syndrome not noticed until now. All three cases published by Hoffmann had their first ECG recordings in 1912 or earlier.

  18. Ribavirin shows immunomodulatory effects on activated microglia. (United States)

    Savic, Danijela; Stojiljkovic, Mirjana; Lavrnja, Irena; Parabucki, Ana; Bjelobaba, Ivana; Nedeljkovic, Nadezda; Herdegen, Thomas; Pekovic, Sanja


    Abstract Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.

  19. Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

    DEFF Research Database (Denmark)

    Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine


    Selective serotonin reuptake inhibitors are known to have a range of disorders that are often linked to the endocrine system e.g. hormonal imbalances, breast enlargement, sexual dysfunction, and menstrual cycle disorders. The mechanisms behind most of these disorders are not known in details....... In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were......M for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine...

  20. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan


    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  1. Antimutagenic activity of casein against MNNG in the E. coli DNA repair host-mediated assay.

    NARCIS (Netherlands)

    Boekel, van M.A.J.S.; Goeptar, A.R.; Alink, G.M.


    The effect of caseinate and soy protein in the diet on the mutagenicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was assessed in-vivo and ex-vivo in the DNA-repair host-mediated assay and liquid suspension assay, respectively. Of the two proteins only casein showed a strong antimutagen

  2. Optimization of a homogeneous assay for kinase inhibitors in plant extracts. (United States)

    Dufau, Isabelle; Lazzari, Anne; Samson, Arnaud; Pouny, Isabelle; Ausseil, Frederic


    To identify natural and original kinase inhibitors from plant extracts, we have developed and compared a heterogeneous enzyme-linked immunosorbent assay (ELISA) and a homogeneous time-resolved fluorescence (HTRF, Cisbio International, Bagnols/Cèze, France) assay. Kinase affinity for the ATP substrate was determined in both assays, and the same [ATP]/ATP Km ratio was used in each case to enable the identification of ATP competitive and noncompetitive inhibitors. Assays were then used to screen the same collection of chemical compounds and plant extracts. The intra-assay correlation analysis of each technology showed a very good screening precision in HTRF and an acceptable one in ELISA. When the two methods were compared, a poor correlation was obtained with a higher hit rate in the ELISA. We then performed a detailed study of the ELISA hits and showed that they also presented a strong antioxidant activity, associated with high adsorption into microplate wells, which interfered with the horseradish peroxidase-based detection system. These hits were then flagged as false-positives. We also showed that many plant extracts presented this kind of activity and that this interference could explain the lack of correlation between the assays. These findings suggest that assay design should be carefully adapted to the substances to be screened and that interferences should be extensively considered before any assay development process and comparison studies. In spite of a few interferences, our results showed that a homogeneous-phase assay like the HTRF assay could be more efficiently used for plant extract screening than a heterogeneous-phase assay like ELISA.

  3. Isolation, production, purification, assay and characterization of ...

    African Journals Online (AJOL)

    Isolation, production, purification, assay and characterization of fibrinolytic ... are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample. ... recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp.

  4. 21 CFR 864.7250 - Erythropoietin assay. (United States)


    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass)...

  5. ApoHRP-based Assay to Measure Intracellular Regulatory Heme (United States)

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A.; Dhahbi, Joseph M.


    The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells. PMID:25525887

  6. The comet assay: a heavenly method! (United States)

    Collins, Andrew R


    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey.

  7. Electrochemical Assay of Gold-Plating Solutions (United States)

    Chiodo, R.


    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  8. Variables Affecting Two Electron Transport System Assays


    Burton, G. Allen; Lanza, Guy R.


    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methan...

  9. MRI shows clodronate-liposomes attenuating liverinjuryinratswithsevereacutepancreatitis

    Institute of Scientific and Technical Information of China (English)

    Jian-Xin Zhang; Sheng-Chun Dang; Yong Zhang; Xin Sha; Li-Rong Zhang; Chuan-She Wei; Min Chen; De-Li Jiang


    BACKGROUND: Studies have revealed that macrophages play an important role in the development of severe acute pancreatitis (SAP). Activated macrophages can lead to a systemic inlfammatory response, induce lipid peroxidation, impair membrane structure, result in injury to the liver and the other extrahepatic organs, and eventually result in multiple organ dysfunction syndrome by promoting excessive secretion of cytokines. Liver injury can further aggravate the systemic inlfammatory response and increase mortality by affecting the metabolism of toxins and the release of excessive inlfammatory mediators. Clodronate is a synthetic bisphosphonate, which is often used for treating bone changes caused by osteoporosis and other factors. In the current study, we created liposomes containing superparamagnetic iron oxide particles (SPIOs) for macrophage labeling and magnetic resonance imaging, using a novel method that can bind the clodronate to induce apoptosis and deplete macrophages. METHODS: Superparamagnetic Fe3O4 nanoparticles were prepared by chemical coprecipitation. SPIO-containing liposomes and SPIO-clodronate-containing liposomes were prepared by the thin iflm method. SAP models were prepared by injection of sodium taurocholate (2 ml/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group, a SAP plus SPIO-liposome group, and a SAP plus SPIO-clodronate-containing group. Two and six hours after SAP models were available, T2-weighted MRI scans (in the same plane) of the livers of rats in each group were performed. At the end of the scans, 2 ml of blood was taken from the superior mesenteric vein to measure the levels of serum amylase, ALT, AST, TNF-α, and IL-6. Pathological changes in the liver and pancreas were assessed. RESULTS: Transmission electron microscopy showed that the liposomes had a uniform size. No pathological changes in the pancreata of rats in the control group were noted. The

  10. Evaluation of environmental genotoxicity by comet assay in Columba livia. (United States)

    González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I


    The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

  11. Assay Stability, the missing component of the Error Budget. (United States)

    Mackay, Mark; Hegedus, Gabe; Badrick, Tony


    Gaining a better understanding of Quality Control (QC) processes is a key requirement to improving performance and reducing patient risk. Detecting analytical error is dependent on a QC strategy that reliably detects a critical shift in a result away from the true value. Recently the concept of Six Sigma has been used by diagnostic laboratories to assess the performance of assays and to assist in the selection of QC rules. The sigma metric is one measure of an assay's ability to perform within specification. However an additional dimension to managing an assay is its stability in bias over time. The concept of long term stability is the same as measured QC drift (SEdrift) which is the effect of numerous calibrations, changes in reagent lots and other conditions i.e. a long term effect. This implies that the standard error budget is wrong because it is modelled on short term QC and misses this SEdrift stability component. We show that SEdrift provides a measure of Assay Stability that should be included in Quality Planning and that by including an allowance for this drift, determining target imprecision appropriate for matched QC algorithms that provide high error detection is as simple as dividing the Allowable Performance Specification by 4, 5 or 6. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  12. Multiple MTS Assay as the Alternative Method to Determine Survival Fraction of the Irradiated HT-29 Colon Cancer Cells. (United States)

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


    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.

  13. Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil. (United States)

    Maeda, Akihisa; Takahashi, Kei; Tsuchiyama, Hiromi; Oshida, Keiyu


    The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Allergenicity assay of allergen from Dermatophagoides farinae in transgenic tobacco

    Institute of Scientific and Technical Information of China (English)

    TANG Mingjuan; SHEN Ye; HU Yuanlei; CAO Lei; NI Ting; ZHANG Hongyu; LIN Zhongping


    Derf2 gene for one of mite allergens in Dermatophagoides farinae has been cloned and expressed under regulation of 35S promoter in transgenic tobacco. The transcriptional analysis showed that this mite complete gene structure in genomic sequence could be spliced at prediction site. Allergenicity assay with immunological sera indicated that the extracts from the transgenic tobacco gave obvious positive IgE binding reaction with specific serum pool. This work would be of potential use in allergenicity assessment of genetically modified food.

  15. A novel fluorogenic probe for monoamine oxidase assays

    Institute of Scientific and Technical Information of China (English)

    You You Lu; Yu Guang Wang; Bin Dai; Yi Qi Dai; Zhao Wang; Zheng Wei Fu; Qing Zhu


    Monoamine oxidase is flavoenzymes, widely distributed in mammals. It is well recognized that MAOs serve an important role in metabolism that they have close relationship with health .Along with the discoveries between MAOs and neurotic disease, more and more studies have been jumped in .In this paper, we design a new probe for assaying the activities of MAOs. The results showed that the probe [7-(3-aminopropoxy)coumarin] is simple, effective and sensitive for MAOB.

  16. Reactive glia show increased immunoproteasome activity in Alzheimer's disease. (United States)

    Orre, Marie; Kamphuis, Willem; Dooves, Stephanie; Kooijman, Lieneke; Chan, Elena T; Kirk, Christopher J; Dimayuga Smith, Vanessa; Koot, Sanne; Mamber, Carlyn; Jansen, Anne H; Ovaa, Huib; Hol, Elly M


    The proteasome is the major protein degradation system within the cell, comprised of different proteolytic subunits; amyloid-β is thought to impair its activity in Alzheimer's disease. Neuroinflammation is a prominent hallmark of Alzheimer's disease, which may implicate an activation of the immunoproteasome, a specific proteasome variant induced by immune signalling that holds slightly different proteolytic properties than the constitutive proteasome. Using a novel cell-permeable proteasome activity probe, we found that amyloid-β enhances proteasome activity in glial and neuronal cultures. Additionally, using a subunit-specific proteasome activity assay we showed that in the cortex of the APPswePS1dE9 plaque pathology mouse model, immunoproteasome activities were strongly increased together with increased messenger RNA and protein expression in reactive glia surrounding plaques. Importantly, this elevated activity was confirmed in human post-mortem tissue from donors with Alzheimer's disease. These findings are in contrast with earlier studies, which reported impairment of proteasome activity in human Alzheimer's disease tissue and mouse models. Targeting the increased immunoproteasome activity with a specific inhibitor resulted in a decreased expression of inflammatory markers in ex vivo microglia. This may serve as a potential novel approach to modulate sustained neuroinflammation and glial dysfunction associated with Alzheimer's disease.

  17. Coagulation tests show significant differences in patients with breast cancer. (United States)

    Tas, Faruk; Kilic, Leyla; Duranyildiz, Derya


    Activated coagulation and fibrinolytic system in cancer patients is associated with tumor stroma formation and metastasis in different cancer types. The aim of this study is to explore the correlation of blood coagulation assays for various clinicopathologic factors in breast cancer patients. A total of 123 female breast cancer patients were enrolled into the study. All the patients were treatment naïve. Pretreatment blood coagulation tests including PT, APTT, PTA, INR, D-dimer, fibrinogen levels, and platelet counts were evaluated. Median age of diagnosis was 51 years old (range 26-82). Twenty-two percent of the group consisted of metastatic breast cancer patients. The plasma level of all coagulation tests revealed statistically significant difference between patient and control group except for PT (p50 years) was associated with higher D-dimer levels (p=0.003). Metastatic patients exhibited significantly higher D-dimer values when compared with early breast cancer patients (p=0.049). Advanced tumor stage (T3 and T4) was associated with higher INR (p=0.05) and lower PTA (p=0.025). In conclusion, coagulation tests show significant differences in patients with breast cancer.

  18. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins


    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.

  19. 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.


    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

  20. A systematic comparison of three commercial estrogen receptor assays in a single clinical outcome breast cancer cohort. (United States)

    Kornaga, Elizabeth N; Klimowicz, Alexander C; Guggisberg, Natalia; Ogilvie, Travis; Morris, Don G; Webster, Marc; Magliocco, Anthony M


    Breast cancers are routinely assessed for estrogen receptor status using immunohistochemical assays to assist in patient prognosis and clinical management. Specific assays vary between laboratories, and several antibodies have been validated and recommended for clinical use. As numerous factors can influence assay performance, many laboratories have opted for ready-to-use assays using automated stainers to improve reproducibility and consistency. Three commonly used autostainer vendors-Dako, Leica, and Ventana-all offer such estrogen receptor assays; however, they have never been directly compared. Here, we present a systematic comparison of three platform-specific estrogen receptor ready-to-use assays using a retrospective, tamoxifen-treated, breast cancer cohort from patients who were treated in Calgary, Alberta, Canada from 1985 to 2000. We found all assays showed good intra-observer agreement. Inter-observer pathological scoring showed some variability: Ventana had the strongest agreement followed closely by Dako, whereas Leica only showed substantial agreement. We also analyzed each estrogen receptor assay with respect to 5-year disease-free survival, and found that all performed similarly in univariate and multivariate models. Determination of measures of test performance found that the Leica assay had a lower negative predictive value than Dako or Ventana, compared with the original ligand-binding assay, while other measures-sensitivity, specificity, positive predictive value, and accuracy-were comparable between the three ready-to-use assays. When comparing against disease-free survival, the difference in negative predictive value between the vendor assays were not as extreme, but Dako and Ventana still performed slightly better than Leica. Despite some discordance, we found that all ready-to-use assays were comparable with or superior to the ligand-binding assay, endorsing their continued use. Our analysis also allowed for exploration of estrogen receptor

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

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    Bas-jan Van Der Leede


    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.

  2. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

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    Chao Shan


    Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  3. Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results. (United States)

    van der Leede, Bas-Jan; Doherty, Ann; Guérard, Melanie; Howe, Jonathan; O'Donovan, Mike; Plappert-Helbig, Ulla; Thybaud, Véronique


    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 the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively comet assay and the regulatory acceptance of the current ICH S2 guidance.

  4. SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries. (United States)

    Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P


    The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.

  5. Urolithins display both antioxidant and pro-oxidant activities depending on assay system and conditions. (United States)

    Kallio, Tuija; Kallio, Johanna; Jaakkola, Mari; Mäki, Marianne; Kilpeläinen, Pekka; Virtanen, Vesa


    The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

  6. Optimising automation of a manual enzyme-linked immunosorbent assay

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    Corena de Beer


    Full Text Available Objective: Enzyme-linked immunosorbent assays (ELISAs are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib kit (MK016 from The Binding Site Company was optimised to be used on an automated BioRad PhD™ system in the Immunology Laboratory (National Health Laboratory Service in Tygerberg, South Africa.Methods: An automated ELISA system that uses individual well incubation was compared to a manual method that uses whole-plate incubation.Results: Results were calculated from calibration curves constructed with each assay. Marked differences in calibration curves were observed for the two methods. The automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. A comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. All incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. Several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. Calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid.Conclusion: Proper validation is vital before converting manual ELISA assays to automated or semi-automated methods. 

  7. Comparison of antibacterial activities of root-end filling materials by an agar diffusion assay and Alamar blue assay

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    Tsui-Hsien Huang


    Conclusion: We concluded that both the agar diffusion test and Alamar blue assay gave comparable findings of assessing the antimicrobial activity present in root-end filling materials. No antimicrobial activity was detected for mineral trioxide aggregate, calcium silicate cement, or amalgam after coming into contact with S. mutans, S. sanguinis and E. coli. IRM showed high antimicrobial activity against both S. sanguinis and E. coli.


    Directory of Open Access Journals (Sweden)



    differences between the hybrids were observed on the first cycle, and it was showed a difference on amino acids concentration for DINA 10 with hardening, on the 6th and last day of the second cycle. The K+ concentration did not change neither during the water stress nor between the hybrids. These results showed only differences on the capacity of solute accumulation of DINA 10, for amino acids on the second water stress cycle. However, IAC 8222 maintained a higher psihf, accentuated by the hardening. This response can be due to an adjustment in the coefficient of cell wall extensibility.

  9. Inferring Protein Associations Using Protein Pulldown Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.


    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  10. Comparative investigation of various cellulase assay procedures

    Energy Technology Data Exchange (ETDEWEB)

    Canevascini, G.; Gattlen, C.


    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).

  11. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays (United States)

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


    Summary The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( 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. PMID:26867747

  12. 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.


    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( 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.

  13. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma


    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.

  14. Research highlights: digital assays on chip. (United States)

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino


    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  15. Role of Quantiferon TB gold assays in monitoring the efficacy of antituberculosis therapy

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    N. Helmy


    Conclusion: The analysis of QFT-G assay results showed that in the majority of our TB patients there was a correlation between clinical treatment outcome and changes of IFN-γ response to M. tuberculosis-specific antigens.

  16. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara


    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  17. Fungicide resistance assays for fungal plant pathogens. (United States)

    Secor, Gary A; Rivera, Viviana V


    Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

  18. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

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    Lyerly Herbert K


    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  19. Tissue and method specificities of phenylalanine ammonia-lyase assay. (United States)

    Kováčik, Jozef; Klejdus, Bořivoj


    A large number of studies have estimated phenylalanine ammonia-lyase (PAL) activity because it strongly reacts to various stimuli. Activity of this enzyme has been assayed mainly by means of spectrophotometry, but the precision of this method is poorly known. We compared assays of PAL activity using spectrophotometry and high performance liquid chromatography (HPLC) in two species (Matricaria chamomilla and Arabidopsis thaliana). Additionally, copper-exposed M. chamomilla plants and buffer with additive were also tested. Our data indicate that spectrophotometry both overestimates (leaves of M. chamomilla) and underestimates (leaves and roots of A. thaliana) PAL activity in comparison with HPLC, suggesting interference of UV-absorbing metabolites. HPLC also showed more accurate detection of cinnamic acid in Cu-exposed chamomile roots. Addition of dithiothreitol to the extraction buffer enhanced PAL activity but reduced proteins, indicating an artificial negative effect. A comparison of PAL activity in selected species is also provided.

  20. A modified method for the turbidimetric assay of nisin

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    Simone Hickmann Flôres


    Full Text Available Nisin is an antibacterial peptide produced by Lactococcus lactis subsp. lactis that shows a broad spectrum of inhibitory activity against gram-positive microorganism including bacterial spores. Methods for nisin activity estimation in solution is redefined. In this work a new turbidimetric assay method has been described making possible to assay nisin satisfactorily in total incubation period of approximately six hours.Nisina é um peptídeo antibacteriano produzido por Lactococcus lactis subsp lactis que apresenta um grande espectro antimicrobiano para microrganismos gram-positivos incluindo esporos bacterianos. Alguns métodos para deteminação da atividade de nisina foram definidos. Neste trabalho foi proposto uma modificação no método para determinação de nisina no qual obteve-se resultados satisfatórios em aproximadamente 6 horas.

  1. A system for performing high throughput assays of synaptic function.

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    Chris M Hempel

    Full Text Available Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

  2. [Plasma assay of methadone enantiomers with high performance liquid chromatography]. (United States)

    Batista, R; Badré-Sentenac, S; Bardin, C; Chast, F


    6-Dimethylamino-4,4-diphenylheptane-3-one (methadone) is a synthetic opioid. Presence of an assymetrical carbon in its structure explains existence of two enantiomers. Levogyral enantiomer or R-methadone exhibits an 25 fold superior analgesic activity to the dextrogyral enantiomer S-methadone. In order to run separately a plasma assay of these two enantiomers, a chromatographic chiral separation method coupled with an ultraviolet detection has been performed. It allows selective assay of each enantiomer. This method, although an analytical interference with d-propoxyphene, is sensitive, reproductible, specific and shows a convenient resolution for the analysis of both the stereospecific and racemic forms. This method can be applied for pharmacokinetic study of the drug in patients treated by methadone.

  3. Synthesis and reducing power assay of methyl semicarbazone derivatives

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    Manmohan Singhal


    Full Text Available In the present study we have designed a new pharmacophore ‘Chalconesemicarbazone’ by pharmacophore hybridization approach of drug design. A series of novel chalconesemicarbazones was synthesized and evaluated for their antioxidant activity by reducing power assay. Most of the compounds were found to be potent antioxidants. Free radicals play an important role in various pathological and xenotoxic effects so antioxidant may have protective role in these pathological conditions. Based on the results of reducing power assay 1-[1-(2,4-dihydroxyphenyl-3-(2-hydroxyphenylallylidene]-4-(4-methylphenylsemicarbazide (compound 18 and 1-[1-(2,5-dihydroxyphenyl-3-(6-hydroxyphenylallylidene]-4-(4-methylphenylsemicarbazide (compound 21 were the most active lead compounds. It was found that methoxy and hydroxyl substituted chalconesemicarbazones exhibited potent reducing power and unsubstituted compound showed less reducing potential.

  4. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO


    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  5. Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients. (United States)

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


    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 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 comet assay in peripheral blood cells is perfectly reflected by the MN assay on oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions.

  6. Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment. (United States)

    Berckmans, P; Leppens, H; Vangenechten, C; Witters, H


    There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

  7. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal (United States)

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page:

  8. 芥菜开花调控蛋白SVP与FLC酵母表达载体的构建及其相互作用研究%Determination of Interactions Between SVP and FLC in Brassica juncea Coss. by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    汤青林; 李念祖; 丁宁; 陈竹睿; 宋明; 王志敏


    为深入研究芥菜开花信号整合子的两个核心调节因子SHORTVEGETATIVEPHASE(SVP)与FLOWERINGLOCUSC(FLC)相互作用的分子机理,通过PCR扩增,从芥菜材料‘QJ’中分别克隆含EcoRI/BamHI双酶切位点的SVP和FLC编码区全长,并利用酵母双杂交体系,将FLC与GAL4报告基因DNA激活域融合(pGADT7FLC),SVP与GAL4报告基因DNA结合域融合(pGBKT7SVP)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现白激活和毒性现象。融合的二倍体酵母(pGADT7FLC×pGBKT7SVP)能在选择性固体培养基QDODUA(SD/-Ade/-His/-Leu/-Trp/X-α-GaFAbA)上生长,并且菌落呈蓝色。将诱饵质粒(pGBKT7SVP)与猎物质粒(pGADT7FLC)载体互换(pGADT7SVP、pGBKTTFLC),再次转化酵母后仍能融合成二倍体酵母(pGADT7SVP×pGBKT7FLC),并同时激活报告基因AURI-C、HIS3、ADE2、MELl,由此表明SVP与FLC蛋白能够相互结合。%The fate of the flowering signal integrators is determined by SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC) . For further study on the mechanism of the mutual recognition between SVP and FLC in Brassicajuncea Coss. (Mustard) variety' QJ', the coding sequences of SVP and FLC with digestion sites of EcoR I/BamH I were respectively amplified via PCR, and the interactions between SVP and FLC were detected by the yeast two-hybrid system. The full-length FLC was fused to the GAL4 DNA activation domain, which was designated as pGADT7FLC and then transformed into Y187 yeast stain. While SVP was fused to the GAL4 DNA binding domain, which was designated as pGBKT7SVP and then transformed into Y2HGold yeast stain. The two transformed yeaststains did not exhibit autoactivation and toxicity. The yeast stains of pGADT7FLC and pGBKT7SVP could mate into yeast diploids. The zygote diploids grew on selective agar plates QDO/X/A (SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA) with blue stains. The results

  9. Swietenia mahagony extract shows agonistic activity to PPARγ and gives ameliorative effects on diabetic db/db mice

    Institute of Scientific and Technical Information of China (English)

    Dan-dan LI; Xu SHEN; Hua-liang JIANG; Jun-hua CHEN; Qing CHEN; Guo-wei LI; Jing CHEN; Jian-min YUE; Min-li CHEN; Xiao-ping WANG; Jian-hua SHEN


    Aim: To search the peroxisome proliferator-activated receptor γ (PPARγ) agonists from Swietenia mahagony extract (SmE) and observe the possible ameliorative effects of SmE on diabetic db/db mice. Methods: The PPARγ agonistic activity of SmE was screened by yeast-two hybrid system. The blood glucose levels of diabetic db/db mice were measured using a blood glucose level monitor and the data were statistically analyzed by NDST8.8W software. Results: By using the clinical drug rosiglitazone as a positive control, it was found that the PPARγ agonistic activity of SmE at a concentration of 50 μg/L was approximately half that of 35.7 μg/L (0.1 μmol/L) of rosiglitazone. At the dose of 1000 mg/kg, SmE remark ably decreased the blood glucose concentration of db/db mice from (15.26±2.98) to (7.58±2.20) mmol/L, and reduced the blood glucose levels by 55.49% compared with the control group (P<0.01). Conclusion: SmE shows agonistic activity to PPARγ and can ameliorate the blood glucose levels of diabetic db/db mice. SmE may be thus used as a potential agent for diabetes therapy.

  10. Show Horse Welfare: Horse Show Competitors' Understanding, Awareness, and Perceptions of Equine Welfare. (United States)

    Voigt, Melissa A; Hiney, Kristina; Richardson, Jennifer C; Waite, Karen; Borron, Abigail; Brady, Colleen M


    The purpose of this study was to gain a better understanding of stock-type horse show competitors' understanding of welfare and level of concern for stock-type show horses' welfare. Data were collected through an online questionnaire that included questions relating to (a) interest and general understanding of horse welfare, (b) welfare concerns of the horse show industry and specifically the stock-type horse show industry, (c) decision-making influences, and (d) level of empathic characteristics. The majority of respondents indicated they agree or strongly agree that physical metrics should be a factor when assessing horse welfare, while fewer agreed that behavioral and mental metrics should be a factor. Respondent empathy levels were moderate to high and were positively correlated with the belief that mental and behavioral metrics should be a factor in assessing horse welfare. Respondents indicated the inhumane practices that most often occur at stock-type shows include excessive jerking on reins, excessive spurring, and induced excessive unnatural movement. Additionally, respondents indicated association rules, hired trainers, and hired riding instructors are the most influential regarding the decisions they make related to their horses' care and treatment.

  11. Expansion of a SNaPshot assay to a 55-SNP multiplex: Assay enhancements, validation, and power in forensic science. (United States)

    Wang, Qian; Fu, Lihong; Zhang, Xiaojing; Dai, Xinyu; Bai, Mei; Fu, Guangping; Cong, Bin; Li, Shujin


    A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty-four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single-base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father-child-mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10(-22) and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.


    Institute of Scientific and Technical Information of China (English)

    杜润滋; 邓璐霞; 黄宁; 罗朝志


    [Objective] To screen proteins binding with α-defensin (HNP-1) mature peptide from placenta cDNA libraty by yeast two hybrid technique. [Methods] The cDNA fragment encoding HNP-1 mature peptide was amplified by polymera-sechain reaction (PCR) and constructed into pGBK-T7 vector as the bait plasmid in yeast two hybrid system 3. Subsequently , the RNA from fetal adrenal gland was obtained and then transformed into cDNA library using SMART technology. The fetal adrenal cDNA library was screened with pGBKT7-HNP-1 as bait plasmid by yeast-two hybrid system Matchmaker Lexa. Finally, the positive clone was obtained by PCR and then identified by sequence. Then the interaction between them was determined by GST pull down in vitro and coimmunoprecipitates experiments in vivo. [Results] Bait and cDNA library have been constructed successfully and transformed into yeast. Then the interaction protein was found-melanocortin 2 receptor (ACTHR), CCAAT-enhancer-binding proteins (C/EBP), Tramembrane trafficking protein (TMP21), low density lipoprotein receptor-related protein 6 (LRP6). Therefore, melanocortin 2 receptor (ACTH-R) was determined into the major subjects. And bands which can demonstrate the relationship between HNP-1 and ACTH-R was obtained in GST pull down and coimmunoprecipitates experiments. [Conclusion] ACTH-R can bind to HNP-1 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-1 mature peptide.%[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子.[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白.最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证

  13. A Cell Lysis and Protein Purification - Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins (United States)

    Nakyama, Tetsuya; Tabata, Kazuhito; Noji, Hiroyuki; Yokokawa, Ryuji

    We have developed two devices applicable to evaluate genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification - assay device. A motor protein, F1-ATPase expressed in E.coli, was focused in this report as a target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were demonstrated in order to detect F1-ATPase and to evaluate its activity. The specific bonding between his-tag in F1-ATPase and Ni-NTA coated on a glass surface was utilized for the purification process. After immobilization of F1-ATPase, avidin-coated microspheres and adenosine tri-phosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, at the optimum condition, extracts enough amount of protein for single molecule assay. Once cell lysate was injected to the purification - assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentratioin provides an optimal concentration for the assay i.e. the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected to the device. The optimum concentration was achieved by our cell lysis device not by the conventional method by ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification - assay device, and the results were compatible to that of conventional assay in which F1-ATPase was purified in bulk scale. In conclusion, we have demonstrated on-chip cell lysis and assay appropriate for the sequential analysis without any pretreatment. On-chip devices replacing conventional bioanalytical methods will be

  14. Drugs and brain death: drug assay perspectives. (United States)

    Morris, R G


    The ability to make any meaningful interpretation of a drug assay result is very dependent upon a knowledge of the limitations of the method(s) used (sensitivity, specificity etc.), and the concentration that may be measured in plasma and its relationship to CNS effects. We need more information about 'critical' concentrations for each drug and sedation in the setting of the brain-injured patient before meaningful interpretation can be applied to such data. While the above discussion is critical of screen-type assays, the alternative specific assays are not easily provided for, as obviously the resourcing of laboratories to be able to deliver such specialized services for a range of therapeutic drugs, in addition to 'social' drugs or other toxins (e.g. glues, pesticides, solvents, environmental substances etc), becomes an increasingly complex issue in the current economic climate. Hence, the analytical laboratory can offer valuable support to the clinical team however, the interpretation of such results must be assessed in the light of many limitations of such assay methods and not seen as the 'gold standard' for assessment of brain function.

  15. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays. (United States)

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W


    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  16. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero


    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.

  17. Comet assay on mice testicular cells

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar


    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...

  18. In vitro solubility assays in drug discovery. (United States)

    Kerns, Edward H; Di, Li; Carter, Guy T


    The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.

  19. Benzodiazepine Synthesis and Rapid Toxicity Assay (United States)

    Fletcher, James T.; Boriraj, Grit


    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…

  20. High throughput comet assay to study genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Naouale El Yamani


    per slide. Subsequent incubation with FPG revealed damage not seen with the basic assay for strand breaks (without FPG (Harris et al., 2015. Statistical evaluation showed that oleic acid coated Fe3O4 and TiO2 NMs are genotoxic, in the experimental conditions used. No differences were seen between cell lines representing a range of different tissues – demonstrating the general usefulness of in vitro models and the ability of cells to classify NMs as genotoxic and non-genotoxic (Cowie et al., 2015. We are currently studying the effects of 20 NMs in the NANoREG project using A549, BEAS B2 and TK6 cells - again demonstrating the usefulness of the HTP comet assay for nanogenotoxicity testing.

  1. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

    Directory of Open Access Journals (Sweden)

    Neufeld Gera


    Full Text Available Abstract Background The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. Methods We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. Results The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2 is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. Conclusion Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on

  2. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus (United States)

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy


    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  3. Production and assay of forskolin antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ho, L.T.; Ho, R.J.


    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  4. Evaluation of the reliability of maize reference assays for GMO quantification. (United States)

    Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel


    A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb

  5. European sero-epidemiology network: standardisation of the results of diphtheria antitoxin assays. (United States)

    von Hunolstein, C; Aggerbeck, H; Andrews, N; Berbers, G; Fievet-Groyne, F; Maple, P A; Olander, R M; Raux, M; Tischer, A


    A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

  6. A molecular inversion probe assay for detecting alternative splicing

    Directory of Open Access Journals (Sweden)

    Palm Curtis


    Full Text Available Absract Background A sensitive, high-throughput method for monitoring pre-mRNA splicing on a genomic scale is needed to understand the spectrum of alternatively spliced mRNA in human cells. Results We adapted Molecular Inversion Probes (MIPs, a padlock-probe based technology, for the multiplexed capture and quantitation of individual splice events in human tissues. Individual MIP capture probes can be quantified using either DNA microarrays or high-throughput sequencing, which permits independent assessment of each spliced junction. Using our methodology we successfully identified 100% of our positive controls and showed that there is a strong correlation between the data from our alternative splicing MIP (asMIP assay and quantitative PCR. Conclusion The asMIP assay provides a sensitive, accurate and multiplexed means for measuring pre-mRNA splicing. Fully optimized, we estimate that the assay could accommodate a throughput of greater than 20,000 splice junctions in a single reaction. This would represent a significant improvement over existing technologies.

  7. Micronucleus assays with Tradescantia pollen tetrads: an update. (United States)

    Misík, M; Ma, T-H; Nersesyan, A; Monarca, S; Kim, J K; Knasmueller, S


    Micronucleus (MN) assays with early pollen tetrad cells of Tradescantia (Trad-MN assays) are at present the most widely used bioassays with plants for the detection of genotoxins in the environment. So far, ∼ 160 chemicals have been tested and ∼ 100 articles that concern complex environmental mixtures were published. This article summarises the results of Trad-MN studies, which have been carried out during the last 15 years with individual compounds and investigations concerning the pollution of environmental compartments (soil, water and air). The evaluation shows that the effects of certain genotoxins such as heavy metals, radionuclides, pesticides and air pollutants can be easily detected with this test. Comparisons with results obtained in MN studies with mitotic (root tip) cells indicate that meiotic tetrad cells are in general more sensitive. Important issues for future research concern the evaluation of the suitability of wildlife Tradescantia species that are sometimes used instead of specific clones (such as #4430 for which standardised protocols have been developed) as well as the assessment of the predictive value of Trad-MN results in regard to the prediction of cancer hazards in humans and adverse effects at the ecosystem level. The fact that the genotoxic effects of certain compound such as metals, which can be detected with plant bioassays, in particular with the Trad-MN assay but not in other commonly used bioassays (e.g. in bacterial tests) makes them an essential element in the batteries for environmental monitoring.

  8. Hydroponic Growth and the Nondestructive Assay for Dinitrogen Fixation 1 (United States)

    Imsande, John; Ralston, Edward J.


    Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pKa 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day. PMID:16662112

  9. Bio-barcode gel assay for microRNA (United States)

    Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min


    MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

  10. Simplex optimization of acoustic assay for plasminogen activators. (United States)

    Ghazali, Mirnader; Hayward, Gordon L


    This article discusses the optimization of a newly developed method for measuring the activity of plasminogen activators using a thickness-shear-mode acoustic sensor. A variable-size simplex algorithm was used for optimization. Preliminary tests were performed to design the first simplex. A desirability function was defined to translate each performance value to a membership value of 0 to 1. If there was more than one performance variable, their membership values were translated to an aggregated membership value using another function that considers their individual influence on sensor performance. Two rounds of optimization were carried out for streptokinase followed by a single optimization for tissue-type plasminogen activator. In the last optimization, ratios of control variables were used in order to reduce the number of parameters and to formulate easily adjustable assay conditions. The results showed the usefulness of the simplex method for optimizing this type of assay, and the importance of preliminary tests and prior knowledge in providing rapid convergence using fewer experiments. The optimized plasminogen activator assay can be considered a reference method for measurement of all members of this drug class.

  11. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip (United States)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  12. A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    Full Text Available Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1 and streptococcal (SpeA and SpeC toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA, 3 pg/ml (SEB, 25 pg/ml (TSST-1, 6 ng/ml (SpeA, and 100 pg/ml (SpeC. These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

  13. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    Directory of Open Access Journals (Sweden)

    Dabisch-Ruthe Mareike


    Full Text Available Abstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP, were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml and RSV (103 copies/ml. The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS samples (n = 100 of mechanically ventilated patients in winter (n = 50 and summer (n = 50. In winter, respiratory viruses were detected in 32 TS samples (64% by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32% and PIV-2 (20%. Multiple infections were detected

  14. Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay. (United States)

    Wollin, Klaus-M; Gorlitz, Bernd-D


    The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

  15. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity. (United States)

    Mohan, Vijee; Gupta, Soni; Thomas, Sherinmol; Mickey, Hanjabam; Charakana, Chaitanya; Chauhan, Vineeta Singh; Sharma, Kapil; Kumar, Rakesh; Tyagi, Kamal; Sarma, Supriya; Gupta, Suresh Kumar; Kilambi, Himabindu Vasuki; Nongmaithem, Sapana; Kumari, Alka; Gupta, Prateek; Sreelakshmi, Yellamaraju; Sharma, Rameshwar


    Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

  16. Drug interference in the Bradford and 2,2'-bicinchoninic acid protein assays. (United States)

    Marshall, T; Williams, K M


    The interference of a range of drugs and related substances has been investigated in the Bradford Coomassie brilliant blue (CBB) protein dye-binding assay and the 2,2'-bicinchoninic acid (BCA) protein assays. Chlorpromazine was the only substance to interfere in the CBB assay but the interference was slight. In contrast, the BCA reagent interacted strongly with chlorpromazine, the penicillins, vitamin C, and paracetamol and the mode of interference varied with the test substance. The chlorpromazine produced turbidity and an atypical color. The penicillins show a slow but normal color response while vitamin C and paracetamol gave an immediate and intense response.

  17. The chemical-in-plug bacterial chemotaxis assay is prone to false positive responses

    Directory of Open Access Journals (Sweden)

    Ward Mandy J


    Full Text Available Abstract Background Chemical-in-plug assays are commonly used to study bacterial chemotaxis, sometimes in the absence of stringent controls. Results We report that non-chemotactic and non-motile mutants in two distinct bacterial species (Shewanella oneidensis and Helicobacter pylori show apparent zones of accumulation or clearing around test plugs containing potential attractants or repellents, respectively. Conclusions Our results suggest that the chemical-in-plug assay should be used with caution, that non-motile or non-chemotactic mutants should be employed as controls, and that results should be confirmed with other types of assays.

  18. Lump corrections for radioactive waste assay. (United States)

    Miller, T J


    Previous studies have shown that automated radioactive waste assay techniques, such as segmented gamma scanner (SGS) and automated qualitative and quantitative (AQ2), have severely underestimated fissile material due to either the malfunction or absence of appropriate lump correction routines. This paper examines the application of manual techniques, such as Monte Carlo N particle (MCNP) and spectral non-destructive assay platform (SNAP) software, to lump corrections in plutonium (Pu), enriched uranium (EU) and depleted uranium (DU) waste streams. Excellent results have been obtained when comparing MCNP with SNAP and applying the SNAP lump correction routine to a range of simulated and typical wastes containing various Pu and EU lump sizes. It has been concluded that the need for lump corrections was relatively rare and usually apparent from abnormal gamma ray peak area ratios, since most AWE waste streams are only lightly shielded.

  19. Transient expression assays in tobacco protoplasts. (United States)

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain


    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  20. Posttranslational Modification Assays on Functional Protein Microarrays. (United States)

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng


    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  1. Comparative assay of Vipera ammodytes antivenom potency. (United States)

    Capitanescu, Cristian; Macovei Oprescu, Anca Monica; Supeanu, Alexandru; Coculescu, Bogdan Ioan; Strambu, Victor; Macovei, Radu Alexandru; Manole, Gheorghe


    The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

  2. Kinetic viability assays using DRAQ7 probe. (United States)

    Wlodkowic, Donald; Akagi, Jin; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew


    Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy-to-use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death-inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single-color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.

  3. A new fluorescent assay for sialyltransferase. (United States)

    Kajihara, Y; Kamitani, T; Sakakibara, T


    A new fluorescent assay for the sialyltransferase reaction was established. After incubation of the sialyltransferase reaction, the sialyloligosaccharide obtained was treated by acid hydrolysis, and then the NeuAc that was released was labeled with 1,2-diamino-4,5-methylenedioxibenzene. The fluorescent-labeled NeuAc could be estimated by HPLC (excitation: 373 nm; emission: 448 nm) and a Lineweaver-Burk plot could be plotted with the data from this analysis.

  4. Delivery of High-Quality Biomarker Assays

    Directory of Open Access Journals (Sweden)

    Brian N. Swanson


    Full Text Available Biomarker measurements now support key decisions throughout the drug development process, from lead optimization to regulatory approvals. They are essential for documenting exposure-response relationships, specificity and potency toward the molecular target, untoward effects, and therapeutic applications. In a broader sense, biomarkers constitute the basis of clinical pathology and laboratory medicine. The utility of biomarkers is limited by their specificity and sensitivity toward the drug or disease process and by their overall variability. Understanding and controlling sources of variability is not only imperative for delivering high-quality assay results, but ultimately for controlling the size and expense of research studies. Variability in biomarker measurements is affected by: biological and environmental factors (e.g., gender, age, posture, diet and biorhythms, sample collection factors (e.g., preservatives, transport and storage conditions, and collection technique, and analytical factors (e.g., purity of reference material, pipetting precision, and antibody specificity. The quality standards for biomarker assays used in support of nonclinical safety studies fall under GLP (FDA regulations, whereas, those assays used to support human diagnostics and healthcare are established by CLIA (CMS regulations and accrediting organizations such as the College of American Pathologists. While most research applications of biomarkers are not regulated, biomarker laboratories in all settings are adopting similar laboratory practices in order to deliver high-quality data. Because of the escalation in demand for biomarker measurements, the highly-parallel (multi-plexed assay platforms that have fueled the rise of genomics will likely evolve into the analytical engines that drive the biomarker laboratories of tomorrow.

  5. Methods and devices for protein assays (United States)

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee


    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.

  6. Immunoreagents and competitive assays to fludioxonil


    Abad Fuentes, Antonio; Agulló, Consuelo; Esteve Turrillas, Francesc Albert; Abad Somovilla, Antonio; Mercader Badia, Josep Vicent


    Fludioxonil is a new-generation fungicide widely used for postharvest fruit protection. The aim of this study was to produce hitherto unreported immunoreagents for Fludioxonil analysis by immunoassay. Derivatives of this agrochemical were synthesized with different linker tethering sites. Those functionalized haptens were activated, and the purified active esters were efficiently conjugated to different carrier proteins for immunogen and assay antigen preparation. Antibodies to Fludioxonil we...

  7. Cell based assay for hypoglycemic drugs screening

    Institute of Scientific and Technical Information of China (English)

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  8. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space. (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S


    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  9. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay. (United States)

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel


    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  10. An application of the RFQ Linac: Nuclear waste assay characterization (United States)

    Lamkin, K.; Schultz, F.; Womble, P.; Humphrey, D.; Vourvopoulos, G.


    A collaboration between Oak Ridge National Laboratory and Western Kentucky University examines the problem of characterization and assay of nuclear waste with high intrinsic neutron and gamma-ray fields. This waste is defined as Remote Handled-Transuranic waste (RH-TRU). A Radiofrequency Quadrupole Linac is used to produce pulses of neutrons, which impinge on the drum that contains the nuclear waste. The neutrons, after being thermalized in the matrix of the drum, are captured by the fissile material (239Pu or 235U), which releases fast neutrons upon fission. Experimental results will be presented to show the versatility of employing the RFQ with the Differential Die-away Technique.

  11. Polymerase chain reaction assay for avian polyomavirus. (United States)

    Phalen, D N; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  12. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier


    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.

  13. [Novobiocin inhibits angiogenesis and shows synergistic effect with vincristine]. (United States)

    Yang, Jun; Jiang, Min; Zhen, Yong-su


    To study the anti-angiogenic activity of novobiocin and its mechanism of action. The anti-angiogenic activity of novobiocin was determined using chick embryo chorioallantoic membrane(CAM) assay. MTT assay, zymography and related assays were used to observe the effects of drugs on bovine aorta endothelial cells and human pulmonary carcinoma PG cells. Novobiocin at the doses of 100 and 200 micrograms/egg inhibited angiogenesis by 31.6% and 68.7% in CAM, respectively. The combination of novobiocin and vincristine enhanced the anti-angiogenic effect. Novobiocin inhibited the proliferation of bovine aortic endothelial cells in a concentration-dependent manner. In addition, novobiocin suppressed MMP-2 secretion, migration, and tube formation of endothelial cells. As determined by MTT assay, novobiocin in combination with vincristine displayed synergistic effect on the proliferation of PG cells, This study demonstrates that novobiocin is active in suppressing angiogenesis and the anti-angiogenic activity may be enhanced by combination with vincristine. The anti-angiogenic activity of novobiocin may be related, at least in part, to its inhibition of cell proliferation, cell migration, tube formation and secretion of matrix metalloproteinases.

  14. A single step protein assay that is both detergent and reducer compatible: The cydex blue assay. (United States)

    Rabilloud, Thierry


    Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single-step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay.

  15. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay. (United States)

    Sonavane, Manoj; Creusot, Nicolas; Maillot-Maréchal, Emmanuelle; Péry, Alexandre; Brion, François; Aїt-Aïssa, Selim


    Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish.

  16. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay. (United States)

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz


    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  17. Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II. (United States)

    Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera


    Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

  18. Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay. (United States)

    Tang, Tuo-Xian; Xiong, Wen; Finkielstein, Carla V; Capelluto, Daniel G S


    The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

  19. Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator. (United States)

    Chen, Chia-Hung; Sarkar, Aniruddh; Song, Yong-Ak; Miller, Miles A; Kim, Sung Jae; Griffith, Linda G; Lauffenburger, Douglas A; Han, Jongyoon


    We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.

  20. Adapted cytokinesis-block micronucleus assay (CBMn) for mouse embryonic stem cells




    Authors: Hamid Kalantari, Hamid Gourabi & Hossein Baharvand ### Abstract Our observation showed the addition of cytochalasin-B to mouse embryonic stem cells (mESC) culture for CBMn analysis led to the induction of apoptosis in these cells. On the other hand, addition of cyt-B is the most critical part of the cytokinesis-block micronucleus assay (CBMn) technique that cannot be omitted. Thus, modification of the traditional CBMn assay seems to be necessary. In this paper, we attempt...

  1. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays


    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Marcotte, Edward M.


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential ap...

  2. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification. (United States)

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


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

  3. A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology. (United States)

    Abderrahim, Mohamed; Arribas, Silvia M; Condezo-Hoyos, Luis


    Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H2O2) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL(-1)), HRP (1UmL(-1)) and H2O2 (250µmolL(-1)) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL(-1)) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H2O2 as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL(-1) (R(2)=0.993) and very sensitive with limits of detection (LOD) of 0.005UmL(-1) and quantitation (LOQ) of 0.01UmL(-1). PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples.

  4. Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load (United States)

    Ng, Kim Tien; Chook, Jack Bee; Oong, Xiang Yong; Chan, Yoke Fun; Chan, Kok Gan; Hanafi, Nik Sherina; Pang, Yong Kek; Kamarulzaman, Adeeba; Tee, Kok Keng


    Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5′-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2–7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control. PMID:27721388

  5. The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. (United States)

    Møller, Peter


    Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammalian cell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring at its present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as additional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basal level of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise, air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA in leukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictive value of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is important that the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment the comet assay can be used in characterization of hazards.

  6. Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice. (United States)

    Steinbach, Jessica M; Garza, Elizabeth T; Ryan, Bryce C


    Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

  7. Development of an opioid self-administration assay to study drug seeking in zebrafish. (United States)

    Bossé, Gabriel D; Peterson, Randall T


    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria. (United States)

    Llewellyn, David; Miura, Kazutoyo; Fay, Michael P; Williams, Andrew R; Murungi, Linda M; Shi, Jianguo; Hodgson, Susanne H; Douglas, Alexander D; Osier, Faith H; Fairhurst, Rick M; Diakite, Mahamadou; Pleass, Richard J; Long, Carole A; Draper, Simon J


    The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

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

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku


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

  10. A novel prothrombin time assay for assessing the anticoagulant activity of oral factor Xa inhibitors. (United States)

    Barrett, Yu Chen; Wang, Zhaoqing; Knabb, Robert M


    Conventional prothrombin time (PT) assays have limited sensitivity and dynamic range in monitoring the anticoagulant activity of direct factor Xa inhibitors. Hence, new assays are needed. We modified a PT assay by adding calcium chloride (CaCl2) to the thromboplastin reagent to increase assay dynamic range and improve sensitivity. Effects of calcium and sodium ion concentrations, and sample handling, were evaluated to optimize assay performance. Increasing concentrations of calcium ions produced progressive increases in PT across the factor Xa inhibitor concentrations of 0 to 2500 nmol/L for razaxaban and apixaban. The greatest effect was seen when the thromboplastin reagent was diluted 1:2.25 with 100 mmol/L CaCl2 (thus selected for routine use). The optimized assay showed an interassay precision of 1.5 to 9.3 percentage coefficient of variation (%CV) for razaxaban and 3.1 to 4.6 %CV for apixaban. We conclude that the modified PT assay is likely to be suitable as a pharmacodynamic marker for activity at therapeutic concentrations of factor Xa inhibitors.

  11. Amelioration of oxidative stress by anthraquinones in various in vitro assays

    Directory of Open Access Journals (Sweden)

    Manish Kumar


    Full Text Available Objective: The use of natural phytoconstituents for food and as nutritional supplements is an easiest way to be healthier. Anthraquinone pigments have been traditionally used for various purposes viz. food colorants, textile staining, color paints and medicines. Rubia cordifolia L. is a perennial, herbaceous climbing plant belonging to family Rubiaceae. This plant contain substantial amounts of anthraquinones, especially in the roots. The present study deals with the bioactivity evaluation of phytoconstituents viz. alizarin and purpurin from Rubia cordifolia. Methods: The DNA protective and antioxidant potential of alizarin and purpurin was evaluated using different in vitro assays viz. DNA protection assay, ABTS assay, DPPH assay, Ferric ion reduction potential and Phosphomolybdenum assay. Results: Alizarin and purpurin exhibited good free radical scavenging activity in various assays. In DNA protection assay, alizarin showed more DNA protection against hydroxyl radicals generated by Fenton ’s reagent in comparison to purpurin. Conclusions: Being potent antioxidants, these natural coloring compounds can be boon to the food industry as nutraceuticals. Further, these phytochemicals can be explored for their anticancer activity and may serve as potent cancer chemopreventive molecules.

  12. Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. (United States)

    Guillaud-Saumur, Thibaud; Nevez, Gilles; Bazire, Amélie; Virmaux, Michèle; Papon, Nicolas; Le Gal, Solène


    We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohen's kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.

  13. Nondestructive boxed transuranic (TRU) waste assay systems (United States)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.


    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  14. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

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    Jennifer R. McCall


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

  15. Spectrophotometric validation of assay method for selected medicinal plant extracts

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    Matthew Arhewoh


    Full Text Available Objective: To develop UV spectrophotometric assay validation methods for some selected medicinal plant extracts.Methods: Dried, powdered leaves of Annona muricata (AM and Andrographis paniculata (AP as well as seeds of Garcinia kola (GK and Hunteria umbellata (HU were separately subjected to maceration using distilled water. Different concentrations of the extracts were scanned spectrophotometrically to obtain wavelengths of maximum absorbance. The different extracts were then subjected to validation studies following international guidelines at the respective wavelengths obtained.Results: The results showed linearity at peak wavelengths of maximum absorbance of 292, 280, 274 and 230 nm for GK, HU, AM and AP, respectively. The calibration curves for the different concentrations of the extract gave R2 values ranging from 0.9831 for AM to 0.9996 for AP the inter-day and intra-day precision study showed that the relative standard deviation (% was ≤ 10% for all the extracts.Conclusion: The aqueous extracts and isolates of these plants can be assayed and monitored using these wavelengths.

  16. Cell- and biomarker-based assays for predicting nephrotoxicity. (United States)

    Huang, Johnny X; Blaskovich, Mark A; Cooper, Matthew A


    Drug-induced nephrotoxicity contributes to the failure rate of investigational drugs during clinical trials. We are still not able to accurately predict drug-induced nephrotoxicity during early drug discovery and development. There is an urgent need for a robust screening system that can identify nephrotoxic compounds before they reach the clinic. This review discusses traditional and emerging kidney injury biomarkers that are used for the determination of nephrotoxicity and for evaluation and diagnosis of other kidney diseases. The potential for in vivo biomarkers to predict renal toxicity in high-throughput in vitro screening assays is discussed. We also compare cell types and highlight novel three-dimensional (3D) culture technologies with potential for in vitro prediction of nephrotoxicity. Traditional cell culture methods and cytotoxicity assays are well established as in vitro tests for nephrotoxicity but the correlation with in vivo results is extremely poor. Recently validated renal biomarkers show promise for early in vivo detection of nephrotoxicity, but have yet to be successfully applied for in vitro prediction of drug-induced nephrotoxicity. Advanced culture technologies 'kidney-on-a-chip' and 3D culture can produce biomarker signatures from relevant kidney cell types that show promise as better predictive systems.

  17. Development of a Kinetic Assay for Late Endosome Movement. (United States)

    Esner, Milan; Meyenhofer, Felix; Kuhn, Michael; Thomas, Melissa; Kalaidzidis, Yannis; Bickle, Marc


    Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

  18. Embryotoxicity assays for leached components from dental restorative materials

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    Mummolo Stefano


    Full Text Available Abstract Background Currently, there are no suitable assays available to evaluate the embryotoxicity of leached components from restorative dental materials. Methods The effect of the medium conditioned by composites and amalgam on mouse blastocysts in vitro was tested. The materials were also subcutaneously implanted, and the effect of the medium supplemented with serum from the host blood was evaluated in the embryotoxicity assay. The embryo implantation rate in the material-transplanted mothers was also evaluated. Results The results show that while the culture in media conditioned by amalgams did not affect blastocyst development, the medium conditioned by composites caused blastocyst degeneration and apoptosis. The development of blastocysts in a medium containing serum obtained from animals after transplantation was, however, without effect. Finally, inconsistent reduction in the implantation rate in transplanted mothers was observed. Conclusions In this study, we provide examples of in vitro and in vivo tests that may be used to evaluate embryotoxicity for dental materials. Our results show that leached components from our composite-material induced embryotoxicity in vitro, however, no toxicity was observed when subcutaneously implanted in vivo. This highlights the necessity of integrated in vitro and in vivo tests for valuable predictive estimation of embryotoxicity for complex materials.

  19. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis. (United States)

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; Garcia, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; Hermoso de Mendoza, Javier


    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

  20. Comparison of enzyme-linked immunosorbent assay test with immunoblot assay in the diagnosis of pemphigus in Indian patients

    Directory of Open Access Journals (Sweden)

    Khandpur Sujay


    Full Text Available Background: The diagnosis of pemphigus vulgaris (PV and pemphigus foliaceous (PF rests upon clinical, histological and immunofluorescence features. Enzyme-linked immunosorbent assay (ELISA test and immunoblot (IB assay have shown variable sensitivity and specificity. Aims: We compared the utility of ELISA and IB in pemphigus patients. Methods: Sixty-six pemphigus cases (PV-54, PF-12 and 72 controls (other vesicobullous disorders and healthy controls were inducted. ELISA for anti-Dsg 3 and 1 antibodies and IB assay were performed. Results: On ELISA, both mean anti-Dsg 1 and 3 titers were raised in PV and PF. Mean anti-Dsg 1 in mucocutaneous PV was significantly higher than in mucosal PV and mean anti-Dsg 3 was significantly raised in PV than in PF. Anti-Dsg 1 and 3 in the control group were negative. Sensitivity and specificity of ELISA in PV was 98.14% and 90.5% while in PF it was 91.6% and 61.1%, respectively.On IB in PV, 36 cases (66.67% showed the 130 kDa and 160 kDa antigen bands, 12 (22.2% only the 130 kDa and six (11.1% only the 160 kDa band. Eight of the nine pure mucosal cases (88.8% showed only the 130 kDa. In PF, only the 160 kDa antigen was detected. These antigens were not identified in the control group. Sensitivity and specificity of IB in PV was 88.9% and 100% and in PF it was 100% and 95.2%, respectively. Conclusion: Both tests could differentiate pemphigus from other dermatoses, including other blistering disorders. ELISA could not make a distinction between PV and PF or between the various clinical phenotypes of PV. IB differentiated between PV and PF and the different clinical variants of PV.

  1. Antibacterial effect of protamine assayed by impedimetry

    DEFF Research Database (Denmark)

    Johansen, Charlotte; Gill, T.; Gram, Lone


    Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained (r = 0 . 99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when esti...... A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50-500 mu g ml(-1)) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer....

  2. Test procedure for boxed waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Wachter, J. [Los Alamos National Lab., NM (United States)


    This document, prepared by Los Alamos National Laboratory`s NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system`s embedded operating and data reduction software.

  3. Bicarbonate alters cellular responses in respiration assays. (United States)

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


    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.

  4. [Human angiogenin: expression, purification, biological assay]. (United States)

    Yang, H; Zhang, Y Q; Yan, Z; Han, W; Yao, L B; Su, C Z


    Angiogenin cDNA was obtained by RT-PCR, and cloned into the fusion expression vector pRSETB. The recombinant Angiogenin protein was fused with His6 at its N-terminal and expressed as inclusion body. The expression level was about 10% of the total bacteria protein. After dissolved in 8 mol/L urea, the recombinant protein was purified by Ni2(+)-NTA chelating resin, according to the high affinity of His6 with Ni2+. The biological assay indicated that purified rhANG could induced the new blood vessel formation of CAM and degraded tRNA in vitro.

  5. Standardization of cytokine flow cytometry assays

    Directory of Open Access Journals (Sweden)

    Cox Josephine


    Full Text Available Abstract Background Cytokine flow cytometry (CFC or intracellular cytokine staining (ICS can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online. Results Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC were shipped to various sites, where ICS assays using cytomegalovirus (CMV pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V. ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24% for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82% for samples with a mean of Conclusion ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more

  6. Assessing HTS performance using BioAssay Ontology: screening and analysis of a bacterial phospho-N-acetylmuramoyl-pentapeptide translocase campaign. (United States)

    Moberg, Andreas; Zander Balderud, Linda; Hansson, Eva; Boyd, Helen


    With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.

  7. Genome-wide polymorphisms show unexpected targets of natural selection


    Pespeni, Melissa H.; Garfield, David A.; Manier, Mollie K; Palumbi, Stephen R.


    Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species...

  8. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华


    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  9. 酵母双杂交诱饵表达载体pGBKT7-cide-3的构建、鉴定以及毒性、自激活效应的检测%Construction, identification and transformation of "bait plasmid" containing human cide-3 gene in yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    闵婕; 李青; 马楠; 康艳霞; 李娜


    Objective;To construct a bait vector containing human cide -3 gene in yeast two -hybrid system in order to screen the human liver cDNA library. Methods; The fragment including all exons of cide -3 gene was amplified by RT-PCR and was inserted into the multiple cloning sites of the shuttle vector pGBKT7. After confirmation with restricted endonuclease digestion and sequence analysis,by using PEG/LiAC method,the plasmid pGBKT7 - cide -3 was transformed into yeast AH109. The plasmid and protein isolated from the positive yeast AH109 clone was tested its expression,toxicity and transcriptional activation. Results:The human cide -3 cDNA was amplified. The restrictive endonuclease digestion and DNA sequencing proved that the plasmid pGBKT7 - cide - 3 was obtained. The yeast AH109 clone transformed with pGBKT7 - cide - 3 was screened out by the SD/ - Trp nutritional media. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion; The bait plasmid pGBKT7 -cide -3 constructed expresses correctly,and can not activate the transcription of reporter gene alone in yeast two - hybrid system.%目的:构建并转化酵母双杂交体系中“诱饵”载体pGBKT7-cide-3以便用于人肝cDNA文库的筛选.方法:用RT-PCR方法从人肝细胞株QZG中扩增cide-3基因全外显子片段,并定向克隆入细菌-酵母穿梭表达质粒pGBKT7中;用PEG/LiAC法将pGBKT7-cide-3转化感受态酵母菌AH109.提取转化酵母菌的质粒DNA和总蛋白进行鉴定.同时检测表达产物对报告基因的激活作用及对酵母株AH109的毒性.结果:成功扩增出人cide-3 cDNA片段,限制性内切酶酶切和DNA测序证实获得正确的重组质粒pGBKT7-cide-3;获得可在色氨酸缺陷培养基(SD/-Trp)上生长含pGBKT7-cide-3的酵母菌克隆.鉴定表明pG-BKT7-cide-3已转化入酵母菌AH109,无自主激活报告基因的能力及对酵母的生长无毒性.结

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  11. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics (United States)

    Joubert, Marisa K.; Deshpande, Meghana; Yang, Jane; Reynolds, Helen; Bryson, Christine; Fogg, Mark; Baker, Matthew P.; Herskovitz, Jonathan; Goletz, Theresa J.; Zhou, Lei; Moxness, Michael; Flynn, Gregory C.; Narhi, Linda O.; Jawa, Vibha


    An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. PMID:27494246

  12. International network for comparison of HIV neutralization assays: the NeutNet report II.

    Directory of Open Access Journals (Sweden)

    Leo Heyndrickx

    Full Text Available BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s can provide measures of protective immunity. An international collaboration (NeutNet involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs and soluble CD4 (Phase I study. METHODS: In the present study (Phase II, polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs (Virus Infectivity Assays, VIA, or Env (gp160-pseudotyped viruses (pseudoviruses, PSV produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014. Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus

  13. International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II (United States)

    Heyndrickx, Leo; Heath, Alan; Sheik-Khalil, Enas; Alcami, Jose; Bongertz, Vera; Jansson, Marianne; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Ramaswamy, Meghna; Sattentau, Quentin; Tolazzi, Monica; Schuitemaker, Hanneke; Willems, Betty; Wrin, Terri; Fenyö, Eva Maria; Scarlatti, Gabriella


    Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus

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

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)


    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  15. Sensitivity of Francisella tularensis to ultrapure water and deoxycholate: implications for bacterial intracellular growth assay in macrophages (United States)

    Chalabaev, Sabina; Anderson, Christine A.; Onderdonk, Andrew B.; Kasper, Dennis L.


    The ability of Francisella tularensis to replicate in macrophages is critical for its pathogenesis, therefore intracellular growth assays are important tools for assessing virulence. We show that two lysis solutions commonly used in these assays, deionized water and deoxycholate in PBS, lead to highly inaccurate measurements of intracellular bacterial survival. PMID:21420447

  16. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A


    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  17. One-step assay for the quantification of T4 DNA ligase. (United States)

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole


    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  18. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    Institute of Scientific and Technical Information of China (English)

    Kwan Yuet Ping; Ibrahim Darah; Yeng Chen; Sreenivasan Sasidharan


    Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

  19. Assessment of genotoxic effects of flumorph by the comet assay in mice organs. (United States)

    Zhang, T; Zhao, Q; Zhang, Y; Ning, J


    The present study investigated the genotoxic effects of flumorph in various organs (brain, liver, spleen, kidney and sperm) of mice. The DNA damage, measured as comet tail length (µm), was determined using the alkaline comet assay. The comet assay is a sensitive assay for the detection of genotoxicity caused by flumorph using mice as a model. Statistically significant increases in comet assay for both dose-dependent and duration-dependent DNA damage were observed in all the organs assessed. The organs exhibited the maximum DNA damage in 96 h at 54 mg/kg body weight. Brain showed maximum DNA damage followed by spleen > kidney > liver > sperm. Our data demonstrated that flumorph had induced systemic genotoxicity in mammals as it caused DNA damage in all tested vital organs, especially in brain and spleen.

  20. Analysis of JC virus DNA replication using a quantitative and high-throughput assay. (United States)

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


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

  1. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis. (United States)

    van Geffen, Mark; Loof, Arnoud; Lap, Paul; Boezeman, Jan; Laros-van Gorkom, Britta A P; Brons, Paul; Verbruggen, Bert; van Kraaij, Marian; van Heerde, Waander L


    Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

  2. Solvent effects and improvements in the deoxyribose degradation assay for hydroxyl radical-scavenging. (United States)

    Li, Xican


    The deoxyribose degradation assay is widely used to evaluate the hydroxyl (OH) radical-scavenging ability of food or medicines. We compared the hydroxyl radical-scavenging activity of 25 antioxidant samples prepared in ethanol solution with samples prepared after removing the ethanol (residue). The data suggested that there was an approximately 9-fold difference between assay results for the ethanol solution and residue samples. This indicated a strong alcoholic interference. To further study the mechanism, the scavenging activities of 18 organic solvents (including ethanol) were measured by the deoxyribose assay. Most pure organic solvents (especially alcohols) could effectively scavenge hydroxyl radicals. As hydroxyl radicals have extremely high reactivities, they will quickly react with surrounding solvent molecules. This shows that any organic solvent should be completely evaporated before measurement. The proposed method is regarded as a reliable hydroxyl radical-scavenging assay, suitable for all types of antioxidants.

  3. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

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    Soniya Sharma


    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  4. Response definition criteria for ELISPOT assays revisited. (United States)

    Moodie, Z; Price, L; Gouttefangeas, C; Mander, A; Janetzki, S; Löwer, M; Welters, M J P; Ottensmeier, C; van der Burg, S H; Britten, Cedrik M


    No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.

  5. Standardization of anti-DNA antibody assays. (United States)

    Pisetsky, David S


    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  6. Cryptosporidium cell culture infectivity assay design. (United States)

    King, B J; Keegan, A R; Robinson, B S; Monis, P T


    Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.


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    Full Text Available : INTRODUCTION: HIV most commonly uses CCR5 and/or CXCR4 as a co-receptor to enter its target cells. Several chemokine receptors can function as viral co-receptors, but CCR5 is likely the most physiologically important co receptor during natural infection. C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines. This is the process by which T cells are attracted to specific tissue and organ targets. Most forms of HIV, the virus that causes AIDS, initially use CCR5 to enter and infect host cells. We studied the prevalence of CCR5 tropism HIV1 amongst HIV positive patients attending Mahatma Gandhi hospital Jaipur. STUDY DESIGN: This was epidemiological, cross-sectional, and non-interventional study between March and April 2014 in HIV positive patients in Jaipur. METHODS: Co-receptor tropism assay was done in total nine patients who were HIV positive attending Mahatma Gandhi Hospital Dermatology &Venereology of Mahatma Gandhi Hospital OPD in one month duration time. Total nine patients were studied, seven patients were already on ART(anti-retroviral treatment and two patients were not taking any treatment as their CD4 count were above 500 cells/ULwith low viral load. Co-Receptor tropism assay (genetic assay CCR5 (with the help of Emcure pharmaceuticals was done in all of them. RESULT: Only one patient had FPR below 15 %, rest 8 patients had FPR above 15%.The study showed that the prevalence of CCR5 positivity was 88.8%, whereas CXCR4 prevalence was only 11.1%.

  8. Investigation on photorespiration with a sensitive C-assay. (United States)

    Zelitch, I


    A leaf disk assay for photorespiration has been developed based on the rate of release of recently fixed (14)CO(2) in light in a rapid stream of CO(2)-free air at 30 degrees to 35 degrees . In tobacco leaves (Havana Seed) photorespiration with this assay is 3 to 5 times greater than the (14)CO(2) output in the dark. In maize, photorespiration is only 2% of that in tobacco.The importance of open leaf stomata, rapid flow rates of CO(2)-free air, elevated temperatures, and oxygen in the atmosphere in order to obtain release into the air of a larger portion of the (14)CO(2) evolved within the tissue in the light was established in tobacco. Photorespiration, but not dark respiration, was inhibited by alpha-hydroxy-2-pyridinemethanesulfonic acid, an inhibitor of glycolate oxidase, and by 3-(4-chlorophenyl)-1,1-dimethylurea (CMU), an inhibitor of photosynthetic electron transport, under conditions which did not affect the stomata. These experiments show that the substrates of photorespiration and dark respiration differ and also provide additional support for the role of glycolate as a major substrate of photorespiration. It was also shown that at 35 degrees the quantity of (14)CO(2) released in the assay may represent only 33% of the gross (14)CO(2) evolved in the light, the remainder being recycled within the tissue.It was concluded that maize does not evolve appreciable quantities of CO(2) in the light and that this largely accounts for the greater efficiency of net photosynthesis exhibited by maize. Hence low rates of photorespiration may be expected to be correlated with a high rate of CO(2) uptake at the normal concentrations of CO(2) found in air and at higher light intensities.

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

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    Julia Breger


    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. [Clinical evaluation of a novel HBsAg quantitative assay]. (United States)

    Takagi, Kazumi; Tanaka, Yasuhito; Naganuma, Hatsue; Hiramatsu, Kumiko; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi


    The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations in HBV-infected individuals remains unclear. The aim of this study was to evaluate a novel fully automated Chemiluminescence Enzyme Immunoassay (Sysmex HBsAg quantitative assay) by comparative measurements of the reference serum samples versus two independent commercial assays (Lumipulse f or Architect HBsAg QT). Furthermore, clinical usefulness was assessed for monitoring of the serum HBsAg levels during antiviral therapy. A dilution test using 5 reference-serum samples showed linear correlation curve in range from 0.03 to 2,360 IU/ml. The HBsAg was measured in total of 400 serum samples and 99.8% had consistent results between Sysmex and Lumipulse f. Additionally, a positive linear correlation was observed between Sysmex and Architect. To compare the Architect and Sysmex, both methods were applied to quantify the HBsAg in serum samples with different HBV genotypes/subgenotypes, as well as in serum contained HBV vaccine escape mutants (126S, 145R). Correlation between the methods was observed in results for escape mutants and common genotypes (A, B, C) in Japan. Observed during lamivudine therapy, an increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for all HBV genetic variants common in Japan. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response to lamivudine treatment and diagnosis of the breakthrough hepatitis.

  11. Detection of garlic gamma-irradiated by assay comet

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    Moreno Alvarez, Damaris L.; Miranda, Enrique F. Prieto; Carro, Sandra; Iglesias Enrique, Isora; Matos, Wilberto [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear (CEADEN), Ciudad de La Habana (Cuba)], e-mail:


    The garlic samples were irradiated in a facility with {sup 60}Co sources, at absorbed dose values of 0-0,15 kGy. The detection method utilized for the identification of the irradiated garlic was biological comet assay. The samples were classified post-irradiation several times. The irradiated samples showed high strand breaks of DNA exhibiting comets of several forms, while the not irradiated and lower dose samples showed a behavior like round shape and light comets. Significant differences were found for higher absorbed dose values at 0.06 kGy, this absorbed dose value is corresponding with the applied dose value at this food in order to avoid the germination. (author)

  12. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity. (United States)

    Harding, Ethelynda E.; Kimsey, R. Scott


    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)

  13. Assay Method for 235U in Low-Density Waste

    Institute of Scientific and Technical Information of China (English)


    <正>235U assay method will provide a semi-quantitative assay for any uranium lumps that might exist in low-density, low-Z material waste boxes within a short count time. These materials will consist of

  14. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening. (United States)

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


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

  15. Validation of a P2Y12-receptor specific whole blood platelet aggregation assay. (United States)

    Amann, Michael; Ferenc, Miroslaw; Valina, Christian M; Bömicke, Timo; Stratz, Christian; Leggewie, Stefan; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald


    Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen's κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

  16. Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay. (United States)

    Kazimirova, Alena; Magdolenova, Zuzana; Barancokova, Magdalena; Staruchova, Marta; Volkovova, Katarina; Dusinska, Maria


    The in vitro genotoxicity of PLGA-PEO (poly-lactic-co-glycolic acid-polyethylene oxide copolymer) nanoparticles was assessed in TK6 cells using the comet assay as well as cytokinesis-block micronucleus (CBMN) assay. The cells were exposed to 0.12-75μg/cm² of PLGA-PEO nanoparticles during 2 and 24h for analysis in the comet assay, and to 3-75μg/cm² of these nanoparticles during 4, 24, 48 and 72h, respectively, for analysis in the CBMN assay. Two different protocols for treatment with cytochalasin B were used. We found that PLGA-PEO was neither cytotoxic (measured by relative cell growth activity and cytokinesis-block proliferation index (CBPI)), nor did it induce DNA strand-breaks (detected by the comet assay) or oxidative DNA lesions (measured by the comet assay modified with lesion-specific enzyme formamidopyrimidine-DNA-glycosylase). There were no statistically significant differences in the frequencies of micronucleated binucleated cells (MNBNCs) between untreated and treated cells in either of the conditions used. This suggests that PLGA-PEO did not have potential genotoxicity. However, using two experimental protocols of the micronucleus assay, PLGA-PEO nanoparticles showed a weak but significant increase in the level of MN in mononucleated cells, in cells treated for 48h with PLGA-PEO nanoparticles when cytochalasin B was added for the last 24h (1st protocol), and in cells treated for 24h with PLGA-PEO nanoparticles followed by washing of NPs and addition of cytochalasin B for another 24h (2nd protocol). It remains unclear whether the increase of MNMNC after treatment with PLGA-PEO nanoparticles is the effect of a possible, weak aneugenic potential or early effect of these particles, or due to another reason. These results suggest that aneugenicity in addition to clastogenicity may be considered as an important biomarker when assessing the genotoxic potential of polymeric nanoparticles.

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

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    Satomi Kawaguchi


    Full Text Available 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

  18. Development of a Fibrinogen-Specific Sandwich Enzyme-Linked Immunosorbent Assay Microarray Assay for Distinguishing Between Blood Plasma and Serum Samples

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    Gonzales, Rachel M.; Zhang, Qibin; Zangar, Richard C.; Smith, Richard D.; Metz, Thomas O.


    We have developed a fibrinogen-specific sandwich ELISA microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies, 49D2, HPA001900, and F8512, were evaluated in conjunction with 1D6 as detection antibody, and the data show that 49D2 and, to a lesser extent, F8512 successfully identify previously unknown plasma and serum samples based upon a ~28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high throughput manner prior to proteomics analyses.

  19. Assay optimization: a statistical design of experiments approach. (United States)

    Altekar, Maneesha; Homon, Carol A; Kashem, Mohammed A; Mason, Steven W; Nelson, Richard M; Patnaude, Lori A; Yingling, Jeffrey; Taylor, Paul B


    With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. This article focuses on the use of statistically designed experiments in assay optimization.

  20. Performance Characteristics of Xpert Flu/RSV XC Assay. (United States)

    Popowitch, Elena B; Miller, Melissa B


    The Xpert Flu/RSV XC assay was compared to laboratory-developed tests (LDTs) (n = 207) and the Xpert Flu assay (n = 147) using archived nasopharyngeal swabs. The percentages of positive agreements with LDTs were 97.8% for influenza A, 97.2% for influenza B, and 89.3% for RSV. The sensitivity of influenza detection was improved with the Xpert Flu/RSV XC assay compared to the Xpert Flu assay.

  1. A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

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    Wychowski Czeslaw


    Full Text Available Abstract Background/Aim The role of humoral immunity in hepatitis C virus (HCV infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. Methods The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. Results The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134 for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85 for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles

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

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


    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.

  3. Systems, devices, and methods for agglutination assays using sedimentation (United States)

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


    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.

  4. 21 CFR 866.2350 - Microbiological assay culture medium. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from either...

  5. 21 CFR 864.7470 - Glycosylated hemoglobin assay. (United States)


    ... 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...

  6. 21 CFR 864.7400 - Hemoglobin A2 assay. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2 assay. (a) Identification. A hemoglobin A2 assay is a device used to determine the hemoglobin A2...

  7. 21 CFR 864.7500 - Whole blood hemoglobin assays. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  8. 21 CFR 864.7415 - Abnormal hemoglobin assay. (United States)


    ... 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)....

  9. 21 CFR 866.3402 - Plasmodium species antigen detection assays. (United States)


    ... 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...

  10. 40 CFR 79.64 - In vivo micronucleus assay. (United States)


    ... micronucleus assay. (a) Purpose. The micronucleus assay is an in vivo cytogenetic test which uses erythrocytes...) Test evaluation. (i) Positive results in the micronucleus test provide information on the ability of a..., Mammalian Bone Marrow Cytogenetics Tests: Micronucleus Assay. (2) Cihak, R. “Evaluation of Benzidine by...

  11. 21 CFR 866.3305 - Herpes simplex virus serological assays. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3305 Herpes simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices...

  12. 21 CFR 864.7100 - Red blood cell enzyme assay. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  13. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish (United States)

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne


    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  14. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne


    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  15. A simple colorimetric assay for measuring fructosamine 3 kinase activity. (United States)

    Cikomola, Justin C; Kishabongo, Antoine S; Vandepoele, Karl; Mulder, Marieke De; Katchunga, Philippe B; Laukens, Bram; Schie, Loes Van; Grootaert, Hendrik; Callewaert, Nico; Speeckaert, Marijn M; Delanghe, Joris R


    Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes-induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 °C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing ±1000 µmol/L fructosamine) was used as an FN3K substrate. In the assay, 300 µL of substrate was incubated with 50 µL of serum and 100 µL of MgCl2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 °C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 µmol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2±1.6 U/L (n=143). Reference values for FN3K were neither age- nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n=191), values (14.0±2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3±7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p<0.0001) correlated with HbA1c values, although the correlation between FN3K and HbA1c was weak. The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.

  16. Identification of irradiated refrigerated pork with the DNA comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, M.M. E-mail:; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail:; Delincee, H.; Villavicencio, A.L.C.H. E-mail:


    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells ('Comet Assay') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA 'Comet Assay'. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  17. Identification of irradiated refrigerated pork with the DNA comet assay (United States)

    Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.; Villavicencio, A. L. C. H.


    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells (``Comet Assay'') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sa~o Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6°C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA ``Comet Assay''. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.


    Directory of Open Access Journals (Sweden)

    Elly Munadziroh


    Full Text Available The use of acrylic resin restoration basen on methyl methacrylate material in the dentistry, has already been replaced by composite because of certain poor characteristics. Recently, the restoration materials of methyl methacrylate have been re-developed, and made commercially available with cyanoacrylates added. The objective of this study was to investigate the cytotoxic effects of cyanoacrylate veneer materials by 3-(3,4-dimethylthyazol-2yl-2,5-diphenyl retrazolium biomide (MTT assay methods. Cyanoacrylate veneer specimens were immersed in eppendorf microtubes with cell culture media for 1, 12, 24 or 48 hour. Culture media were immersed and used to investigate the cytotoxic effect to BHK 21 cell lines by MTT assay method. The density of optic formazan was live indicate a cell. All data were statistically analyzed by one-way ANOVA and HSD Tukey. The results show that the cytotoxic effects between the five immersion were similar but higher than control cell. In conclusion Immersion in cyanoacrylate veneer has a cytotoxic effect to BHK 21 cell lines. The lowest percentage of cells a live after immersion was 86,95%.

  19. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance. (United States)

    Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl


    The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.

  20. Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu Qiuwei, E-mail:; Vu, Heather; Liu Liping; Wang, Ting-Chuan; Schaefer, William H. [Merck Research Laboratories (United States)


    Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid {beta}-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.